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Sample records for transmembrane helix structure

  1. NMR-based approach to measure the free energy of transmembrane helix-helix interactions.

    Science.gov (United States)

    Mineev, Konstantin S; Lesovoy, Dmitry M; Usmanova, Dinara R; Goncharuk, Sergey A; Shulepko, Mikhail A; Lyukmanova, Ekaterina N; Kirpichnikov, Mikhail P; Bocharov, Eduard V; Arseniev, Alexander S

    2014-01-01

    Knowledge of the energetic parameters of transmembrane helix-helix interactions is necessary for the establishment of a structure-energy relationship for α-helical membrane domains. A number of techniques have been developed to measure the free energies of dimerization and oligomerization of transmembrane α-helices, and all of these have their advantages and drawbacks. In this study we propose a methodology to determine the magnitudes of the free energy of interactions between transmembrane helices in detergent micelles. The suggested approach employs solution nuclear magnetic resonance (NMR) spectroscopy to determine the population of the oligomeric states of the transmembrane domains and introduces a new formalism to describe the oligomerization equilibrium, which is based on the assumption that both the dimerization of the transmembrane domains and the dissociation of the dimer can occur only upon the collision of detergent micelles. The technique has three major advantages compared with other existing approaches: it may be used to analyze both weak and relatively strong dimerization/oligomerization processes, it works well for the analysis of complex equilibria, e.g. when monomer, dimer and high-order oligomer populations are simultaneously present in the solution, and it can simultaneously yield both structural and energetic characteristics of the helix-helix interaction under study. The proposed methodology was applied to investigate the oligomerization process of transmembrane domains of fibroblast growth factor receptor 3 (FGFR3) and vascular endothelium growth factor receptor 2 (VEGFR2), and allowed the measurement of the free energy of dimerization of both of these objects. In addition the proposed method was able to describe the multi-state oligomerization process of the VEGFR2 transmembrane domain. © 2013 Elsevier B.V. All rights reserved.

  2. Transmembrane Helix-helix Association: Relative Stabilities at Low pH†

    Science.gov (United States)

    Valluru, Neelima; Silva, Frances; Dhage, Manmath; Rodriguez, Gustavo; Alloor, Srinivas R.; Renthal, Robert

    2008-01-01

    We have previously studied the unfolding equilibrium of bacterio-opsin in a single phase solvent, using Förster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterio-opsin perturbed by increasing ethanol concentrations [Nannepaga et al.(2004) Biochemistry 43, 50–59]. We have further investigated this transition and find the unfolding is pH-dependent. We have now measured the apparent pK of acid-induced unfolding of bacterio-opsin in 90% ethanol. When the acceptor is on helix B (Lys 41), the apparent pK for unfolding is 4.75; on the EF connecting loop (Cys 163), 5.15; and on helix G (Cys 222), 5.75. Five-helix proteolytic fragments are less stable. The apparent unfolding pKs are, for residues 72-248 (Cys 163), 5.46; and 1-166 (Lys 41), 7.36. When interpreted in terms of a simple equilibrium model for unfolding, the apparent pKs give relative free energies of unfolding, in the range of −0.54 to −3.5 kcal/mol. The results suggest that the C-terminal helix of bacterio-opsin is less stably folded than the N-terminal helices. We analyzed the pair-wise helix-helix interaction surfaces of bacteriorhodopsin and three other 7-transmembrane helix proteins, based on crystal structures. The results show that the interaction surfaces are smoother and the helix axis separations are closer in the amino-terminal two-thirds of the proteins compared with the carboxyl terminal one-third. However, the F helix is important in stabilizing the folded structure, as shown by the instability of the 1-166 fragment. Considering the high resolution crystal structure of bacteriorhodopsin, there are no obvious helix-helix interactions involving protein side chains which would be destabilized by protonation at the estimated pH of the unfolding transitions. However, a number of helix-bridging water molecules could become protonated, thereby weakening the helix-helix

  3. Transmembrane helix-helix association: relative stabilities at low pH.

    Science.gov (United States)

    Valluru, Neelima; Silva, Frances; Dhage, Manmath; Rodriguez, Gustavo; Alloor, Srinivas R; Renthal, Robert

    2006-04-11

    We have previously studied the unfolding equilibrium of bacterioopsin in a single phase solvent, using Förster mechanism fluorescence resonance energy transfer (FRET) as a probe, from tryptophan donors to a dansyl acceptor. We observed an apparent unfolding transition in bacterioopsin perturbed by increasing ethanol concentrations [Nannepaga et al. (2004) Biochemistry 43, 50-59]. We have further investigated this transition and find that the unfolding is pH-dependent. We have now measured the apparent pK of acid-induced unfolding of bacterioopsin in 90% ethanol. When the acceptor is on helix B (Lys 41), the apparent pK for unfolding is 4.75; on the EF connecting loop (Cys 163), 5.15; and on helix G (Cys 222), 5.75. Five-helix proteolytic fragments are less stable. The apparent unfolding pKs are 5.46 for residues 72-248 (Cys 163) and 7.36 for residues 1-166 (Lys 41). When interpreted in terms of a simple equilibrium model for unfolding, the apparent pKs give relative free energies of unfolding in the range of -0.54 to -3.5 kcal/mol. The results suggest that the C-terminal helix of bacterioopsin is less stably folded than the N-terminal helices. We analyzed the pairwise helix-helix interaction surfaces of bacteriorhodopsin and three other seven-transmembrane-helix proteins on the basis of crystal structures. The results show that the interaction surfaces are smoother and the helix axis separations are closer in the amino-terminal two-thirds of the proteins compared with the carboxyl-terminal one-third. However, the F helix is important in stabilizing the folded structure, as shown by the instability of the 1-166 fragment. Considering the high-resolution crystal structure of bacteriorhodopsin, there are no obvious helix-helix interactions involving protein side chains which would be destabilized by protonation at the estimated pH of the unfolding transitions. However, a number of helix-bridging water molecules could become protonated, thereby weakening the helix-helix

  4. Detergent properties influence the stability of the glycophorin A transmembrane helix dimer in lysophosphatidylcholine micelles.

    Science.gov (United States)

    Stangl, Michael; Veerappan, Anbazhagan; Kroeger, Anja; Vogel, Peter; Schneider, Dirk

    2012-12-19

    Detergents might affect membrane protein structures by promoting intramolecular interactions that are different from those found in native membrane bilayers, and fine-tuning detergent properties can be crucial for obtaining structural information of intact and functional transmembrane proteins. To systematically investigate the influence of the detergent concentration and acyl-chain length on the stability of a transmembrane protein structure, the stability of the human glycophorin A transmembrane helix dimer has been analyzed in lyso-phosphatidylcholine micelles of different acyl-chain length. While our results indicate that the transmembrane protein is destabilized in detergents with increasing chain-length, the diameter of the hydrophobic micelle core was found to be less crucial. Thus, hydrophobic mismatch appears to be less important in detergent micelles than in lipid bilayers and individual detergent molecules appear to be able to stretch within a micelle to match the hydrophobic thickness of the peptide. However, the stability of the GpA TM helix dimer linearly depends on the aggregation number of the lyso-PC detergents, indicating that not only is the chemistry of the detergent headgroup and acyl-chain region central for classifying a detergent as harsh or mild, but the detergent aggregation number might also be important. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  5. Molecular Dynamics Simulation of Membranes and a Transmembrane Helix

    Science.gov (United States)

    Duong, Tap Ha; Mehler, Ernest L.; Weinstein, Harel

    1999-05-01

    Three molecular dynamics (MD) simulations of 1.5-ns length were carried out on fully hydrated patches of dimyristoyl phosphatidylcholine (DMPC) bilayers in the liquid-crystalline phase. The simulations were performed using different ensembles and electrostatic conditions: a microcanonical ensemble or constant pressure-temperature ensemble, with or without truncated electrostatic interactions. Calculated properties of the membrane patches from the three different protocols were compared to available data from experiments. These data include the resulting overall geometrical dimensions, the order characteristics of the lipid hydrocarbon chains, as well as various measures of the conformations of the polar head groups. The comparisons indicate that the simulation carried out within the microcanonical ensemble with truncated electrostatic interactions yielded results closest to the experimental data, provided that the initial equilibration phase preceding the production run was sufficiently long. The effects of embedding a non-ideal helical protein domain in the membrane patch were studied with the same MD protocols. This simulation was carried out for 2.5 ns. The protein domain corresponds to the seventh transmembrane segment (TMS7) of the human serotonin 5HT 2Areceptor. The peptide is composed of two α-helical segments linked by a hinge domain around a perturbing Asn-Pro motif that produces at the end of the simulation a kink angle of nearly 80° between the two helices. Several aspects of the TMS7 structure, such as the bending angle, backbone Φ and Ψ torsion angles, the intramolecular hydrogen bonds, and the overall conformation, were found to be very similar to those determined by NMR for the corresponding transmembrane segment of the tachykinin NK-1 receptor. In general, the simulations were found to yield structural and dynamic characteristics that are in good agreement with experiment. These findings support the application of simulation methods to the study

  6. Oligomerization state of photosynthetic core complexes is correlated with the dimerization affinity of a transmembrane helix.

    Science.gov (United States)

    Hsin, Jen; LaPointe, Loren M; Kazy, Alla; Chipot, Christophe; Senes, Alessandro; Schulten, Klaus

    2011-09-07

    In the Rhodobacter (Rba.) species of photosynthetic purple bacteria, a single transmembrane α-helix, PufX, is found within the core complex, an essential photosynthetic macromolecular assembly that performs the absorption and the initial processing of light energy. Despite its structural simplicity, many unresolved questions surround PufX, the most important of which is its location within the photosynthetic core complex. One proposed placement of PufX is at the center of a core complex dimer, where two PufX helices associate in the membrane and form a homodimer. Inability for PufX of certain Rba. species to form a homodimer is thought to lead to monomeric core complexes. In the present study, we employ a combination of computational and experimental techniques to test the hypothesized homodimerization of PufX. We carry out a systematic investigation to measure the dimerization affinity of PufX from four Rba. species, Rba. blasticus , Rba. capsulatus , Rba. sphaeroides , and Rba. veldkampii , using a molecular dynamics-based free-energy method, as well as experimental TOXCAT assays. We found that the four PufX helices have substantially different dimerization affinities. Both computational and experimental techniques demonstrate that species with dimeric core complexes have PufX that can potentially form a homodimer, whereas the one species with monomeric core complexes has a PufX with little to no dimerization propensity. Our analysis of the helix-helix interface revealed a number of positions that may be important for PufX dimerization and the formation of a hydrogen-bond network between these GxxxG-containing helices. Our results suggest that the different oligomerization states of core complexes in various Rba. species can be attributed, among other factors, to the different propensity of its PufX helix to homodimerize.

  7. Buried lysine, but not arginine, titrates and alters transmembrane helix tilt.

    Science.gov (United States)

    Gleason, Nicholas J; Vostrikov, Vitaly V; Greathouse, Denise V; Koeppe, Roger E

    2013-01-29

    The ionization states of individual amino acid residues of membrane proteins are difficult to decipher or assign directly in the lipid-bilayer membrane environment. We address this issue for lysines and arginines in designed transmembrane helices. For lysines (but not arginines) at two locations within dioleoyl-phosphatidylcholine bilayer membranes, we measure pK(a) values below 7.0. We find that buried charged lysine, in fashion similar to arginine, will modulate helix orientation to maximize its own access to the aqueous interface or, if occluded by aromatic rings, may cause a transmembrane helix to exit the lipid bilayer. Interestingly, the influence of neutral lysine (vis-à-vis leucine) upon helix orientation also depends upon its aqueous access. Our results suggest that changes in the ionization states of particular residues will regulate membrane protein function and furthermore illustrate the subtle complexity of ionization behavior with respect to the detailed lipid and protein environment.

  8. Role of Side-Chain Conformational Entropy in Transmembrane Helix Dimerization of Glycophorin A

    Science.gov (United States)

    Liu, Wei; Crocker, Evan; Siminovitch, David J.; Smith, Steven O.

    2003-01-01

    Dimerization of the transmembrane domain of glycophorin A is mediated by a seven residue motif LIxxGVxxGVxxT through a combination of van der Waals and hydrogen bonding interactions. One of the unusual features of the motif is the large number of β-branched amino acids that may limit the entropic cost of dimerization by restricting side-chain motion in the monomeric transmembrane helix. Deuterium NMR spectroscopy is used to characterize the dynamics of fully deuterated Val80 and Val84, two essential amino acids of the dimerization motif. Deuterium spectra of the glycophorin A transmembrane dimer were obtained using synthetic peptides corresponding to the transmembrane sequence containing either perdeuterated Val80 or Val84. These data were compared with spectra of monomeric glycophorin A peptides deuterated at Val84. In all cases, the deuterium line shapes are characterized by fast methyl group rotation with virtually no motion about the Cα-Cβ bond. This is consistent with restriction of the side chain in both the monomer and dimer due to intrahelical packing interactions involving the β-methyl groups, and indicates that there is no energy cost associated with dimerization due to loss of conformational entropy. In contrast, deuterium NMR spectra of Met81 and Val82, in the lipid interface, reflected greater motional averaging and fast exchange between different side-chain conformers. PMID:12547806

  9. Observation of helix associations for insertion of a retinal molecule and distortions of helix structures in bacteriorhodopsin

    Science.gov (United States)

    Urano, Ryo; Okamoto, Yuko

    2015-12-01

    We applied a newly proposed prediction method for membrane protein structures to bacteriorhodopsin that has distorted transmembrane helices in the native structure. This method uses an implicit membrane model, which restricts sampling space during folding in a membrane region, and includes helix bending. Replica-exchange simulations were performed with seven transmembrane helices only without a retinal molecule. Obtained structures were classified into clusters of similar structures, which correspond to local-minimum free energy states. The two lowest free energy states corresponded to a native-like structure with the correct empty space for retinal and a structure with this empty space filled with a helix. Previous experiments of bacteriorhodopsin suggested that association of transmembrane helices enables them to make a room for insertion of a retinal. Our results are consistent with these results. Moreover, distortions of helices in the native-like structures were successfully reproduced. In the distortions, whereas the locations of kinks for all helices were similar to those of Protein Data Bank's data, the amount of bends was more similar for helices away from the retinal than for those close to the retinal in the native structure. This suggests a hypothesis that the amino-acid sequence specifies the location of kinks in transmembrane helices and that the amount of distortions depends on the interactions with the surrounding molecules such as neighboring helices, lipids, and retinal.

  10. The Impact of the ‘Austrian’ Mutation of the Amyloid Precursor Protein Transmembrane Helix is Communicated to the Hinge Region

    DEFF Research Database (Denmark)

    Stelzer, Walter; Scharnagl, Christina; Leurs, Ulrike

    2016-01-01

    The transmembrane helix of the amyloid precursor protein is subject to proteolytic cleavages by γ-secretase at different sites resulting in Aβ peptides of different length and toxicity. A number of point mutations within this transmembrane helix alter the cleavage pattern thus enhancing production...... destabilizes amide hydrogen bonds in the hinge which connects dimerization and cleavage regions. Weaker intrahelical hydrogen bonds at the hinge may enhance helix bending and thereby affect recognition of the transmembrane substrate by the enzyme and/or presentation of its cleavage sites to the catalytic cleft....

  11. Progesterone modulation of transmembrane helix-helix interactions between the α-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane

    Directory of Open Access Journals (Sweden)

    Askari Amir

    2010-05-01

    Full Text Available Abstract Background Progesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit. Results Preparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020 successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME, can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT. Conclusion We propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.

  12. Glycine Perturbs Local and Global Conformational Flexibility of a Transmembrane Helix

    DEFF Research Database (Denmark)

    Högel, Philipp; Götz, Alexander; Kuhne, Felix

    2018-01-01

    the measured exchange kinetics and reveal, at atomic resolution, a severe packing defect at glycine that enhances local hydration. Furthermore, glycine alters H-bond occupancies and triggers a redistribution of α-helical and 310-helical H-bonds. These effects facilitate local helix bending at the glycine site...

  13. Aromatic Residues in the Fourth Transmembrane-Spanning Helix M4 Are Important for GABAρ Receptor Function.

    Science.gov (United States)

    Cory-Wright, James; Alqazzaz, Mona; Wroe, Francesca; Jeffreys, Jenny; Zhou, Lu; Lummis, Sarah C R

    2018-02-21

    GABAρ receptors are a subfamily of the GABA A receptor family of pentameric ligand-gated ion channels (pLGICs). Each of the five subunits has four transmembrane α-helices (M1-M4), with M4 most distant from the central pore. Aromatic residues in this M4 helix are important for receptor assembly in pLGICs and also may interact with adjacent lipids and/or residues in neighboring α-helices and the extracellular domain to modify or enable channel gating. This study examines the role of M4 receptor aromatic residues in the GABAρ receptor transmembrane domain using site-directed mutagenesis and subsequent expression in HEK293 cells, probing functional parameters using a fluorescent membrane-potential-sensitive dye. The data indicate that many of the aromatic residues in M4 play a role in receptor function, as substitution with other residues can ablate and/or modify functional parameters. Modeling showed that these residues likely interact with residues in the adjacent M1 and M3 α-helices and/or residues in the Cys-loop in the extracellular domain. We suggest that many of these aromatic interactions contribute to an "aromatic zipper", which allows interactions between M4 and the rest of the receptor that are essential for function. Thus, the data support other studies showing that M4 does not play a passive role in "protecting" the other transmembrane helices from the lipid bilayer but is actively involved in the function of the protein.

  14. Nucleic acid helix structure determination from NMR proton chemical shifts

    International Nuclear Information System (INIS)

    Werf, Ramon M. van der; Tessari, Marco; Wijmenga, Sybren S.

    2013-01-01

    We present a method for de novo derivation of the three-dimensional helix structure of nucleic acids using non-exchangeable proton chemical shifts as sole source of experimental restraints. The method is called chemical shift de novo structure derivation protocol employing singular value decomposition (CHEOPS) and uses iterative singular value decomposition to optimize the structure in helix parameter space. The correct performance of CHEOPS and its range of application are established via an extensive set of structure derivations using either simulated or experimental chemical shifts as input. The simulated input data are used to assess in a defined manner the effect of errors or limitations in the input data on the derived structures. We find that the RNA helix parameters can be determined with high accuracy. We finally demonstrate via three deposited RNA structures that experimental proton chemical shifts suffice to derive RNA helix structures with high precision and accuracy. CHEOPS provides, subject to further development, new directions for high-resolution NMR structure determination of nucleic acids.

  15. Transmembrane α-Helix 2 and 7 Are Important for Small Molecule-Mediated Activation of the GLP-1 Receptor

    DEFF Research Database (Denmark)

    Underwood, Christina Rye; Møller Knudsen, Sanne; Schjellerup Wulff, Birgitte

    2011-01-01

    Glucagon-like peptide-1 (GLP-1) activates the GLP-1 receptor (GLP-1R), which belongs to family B of the G-protein-coupled receptors. We previously identified a selective small molecule ligand, compound 2, that acted as a full agonist and allosteric modulator of GLP-1R. In this study......, the structurally related small molecule, compound 3, stimulated cAMP production from GLP-1R, but not from the homologous glucagon receptor (GluR). The receptor selectivity encouraged a chimeric receptor approach to identify domains important for compound 3-mediated activation of GLP-1R. A subsegment of the GLP-1R...... transmembrane domain containing TM2 to TM5 was sufficient to transfer compound 3 responsiveness to GluR. Therefore, divergent residues in this subsegment of GLP-1R and GluR are responsible for the receptor selectivity of compound 3. Functional analyses of other chimeric receptors suggested that the existence...

  16. Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase.

    Science.gov (United States)

    Holm, Rikke; Khandelwal, Jaanki; Einholm, Anja P; Andersen, Jens P; Artigas, Pablo; Vilsen, Bente

    2017-01-10

    Na + ,K + -ATPase and H + ,K + -ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H + ,K + -ATPase avoids binding of Na + at the site corresponding to the Na + -specific site of the Na + ,K + -ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na + ,K + -ATPase with arginine, present in the H + ,K + -ATPase at the corresponding position, converted the normal 3Na + :2K + :1ATP stoichiometry of the Na + ,K + -ATPase to electroneutral 2Na + :2K + :1ATP stoichiometry similar to the electroneutral transport mode of the H + ,K + -ATPase. The electroneutral C932R mutant of the Na + ,K + -ATPase retained a wild-type-like enzyme turnover rate for ATP hydrolysis and rate of cellular K + uptake. Only a relatively minor reduction of apparent Na + affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na + affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine + group of the M8 arginine replaces Na + at the third site, thus preventing Na + binding there, although allowing Na + to bind at the two other sites and become transported. Hence, in the H + ,K + -ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

  17. Generating structured light with phase helix and intensity helix using reflection-enhanced plasmonic metasurface at 2 μm

    Science.gov (United States)

    Zhao, Yifan; Du, Jing; Zhang, Jinrun; Shen, Li; Wang, Jian

    2018-04-01

    Mid-infrared (2-20 μm) light has been attracting great attention in many areas of science and technology. Beyond the extended wavelength range from visible and near-infrared to mid-infrared, shaping spatial structures may add opportunities to grooming applications of mid-infrared photonics. Here, we design and fabricate a reflection-enhanced plasmonic metasurface and demonstrate efficient generation of structured light with the phase helix and intensity helix at 2 μm. This work includes two distinct aspects. First, structured light (phase helix, intensity helix) generation at 2 μm, which is far beyond the ability of conventional spatial light modulators, is enabled by the metasurface with sub-wavelength engineered structures. Second, the self-referenced intensity helix against environmental noise is generated without using a spatially separated light. The demonstrations may open up advanced perspectives to structured light applications at 2 μm, such as phase helix for communications and non-communications (imaging, sensing) and intensity helix for enhanced microscopy and advanced metrology.

  18. Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

    Directory of Open Access Journals (Sweden)

    Cinzia Ambrosi

    Full Text Available Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26 that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P. Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels

  19. Structure of the transmembrane domain of HIV-1 envelope glycoprotein.

    Science.gov (United States)

    Chen, Bing; Chou, James J

    2017-04-01

    HIV-1 envelope spike (Env) is a heavily glycosylated, type I membrane protein that mediates fusion of viral and cell membranes to initiate infection. It is also a primary target of neutralizing antibodies and thus an important candidate for vaccine development. We have recently reported a nuclear magnetic resonance structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in a membrane-like environment. Taking HIV-1 as an example, we discuss here how a TM domain can anchor, stabilize, and modulate a viral envelope spike and how its high-resolution structure can contribute to understanding viral membrane fusion and to immunogen design. © 2016 Federation of European Biochemical Societies.

  20. Structure of Staphylococcal α-Hemolysin, a Heptameric Transmembrane Pore

    Science.gov (United States)

    Song, Langzhou; Hobaugh, Michael R.; Shustak, Christopher; Cheley, Stephen; Bayley, Hagan; Gouaux, J. Eric

    1996-12-01

    The structure of the Staphylococcus aureus α-hemolysin pore has been determined to 1.9 overset{circ}{mathrm A} resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 overset{circ}{mathrm A} in length, that runs along the sevenfold axis and ranges from 14 overset{circ}{mathrm A} to 46 overset{circ}{mathrm A} in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel β barrel, to which each protomer contributes two β strands, each 65 overset{circ}{mathrm A} long. The interior of the β barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 overset{circ}{mathrm A} wide. The structure proves the heptameric subunit stoichiometry of the α-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-soluble protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of β barrel pore-forming toxins.

  1. Low-coupling impedance double-helix structure for use in a ferrite kicker magnet

    International Nuclear Information System (INIS)

    Giordano, S.

    1983-01-01

    In a machine such as the CBA, the ejection ferrite kicker magnet has a very large longitudinal and transverse coupling impedance which could destroy the beam. Using a double-helix structure that surrounds the beam, the beam-induced fields are confined within the helix and, therefore, decoupled from the kicker; but at the same time the helix is transparent to the external fields of the kicker. At first, this may seem paradoxical that the helix is opaque to the fields generated inside the structure by the beam and simultaneously transparent to the external fields generated by the kicker

  2. The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region.

    Science.gov (United States)

    Apellániz, Beatriz; Rujas, Edurne; Serrano, Soraya; Morante, Koldo; Tsumoto, Kouhei; Caaveiro, Jose M M; Jiménez, M Ángeles; Nieva, José L

    2015-05-22

    The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Cannabinoid CB1 receptor recognition of endocannabinoids via the lipid bilayer: molecular dynamics simulations of CB1 transmembrane helix 6 and anandamide in a phospholipid bilayer

    Science.gov (United States)

    Lynch, Diane L.; Reggio, Patricia H.

    2006-08-01

    The phospholipid bilayer plays a central role in the lifecycle of the endogenous cannabinoid, N-arachidonoylethanolamine (anandamide, AEA). Therefore, the orientation and location of AEA in the phospholipid bilayer with respect to key membrane associated proteins, is a central issue in understanding the mechanism of endocannabinoid signaling. In this paper, we report a test of the hypothesis that a βXX β motif (formed by beta branching amino acids, V6.43 and I6.46) on the lipid face of the cannabinoid CB1 receptor in its inactive state may serve as an initial CB1 interaction site for AEA. Eight 6 ns NAMD2 molecular dynamics simulations of AEA were conducted in a model system composed of CB1 transmembrane helix 6 (TMH6) in a 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) bilayer. In addition, eight 6 ns NAMD2 molecular dynamics simulations of a low CB1 affinity (20:2, n-6) analog of AEA were conducted in the same model system. AEA was found to exhibit a higher incidence of V6.43/I6.46 groove insertion than did the (20:2, n-6) analog. In certain cases, AEA established a high energy of interaction with TMH6 by first associating with the V6.43/I6.46 groove and then molding itself to the lipid face of TMH6 to establish a hydrogen bonding interaction with the exposed backbone carbonyl of P6.50. Based upon these results, we propose that the formation of this hydrogen bonded AEA/TMH6 complex may be the initial step in CB1 recognition of AEA in the lipid bilayer.

  4. Structure of bacteriophage [phi]29 head fibers has a supercoiled triple repeating helix-turn-helix motif

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, Ye; Rossmann, Michael G. (Purdue)

    2011-12-22

    The tailed bacteriophage {phi}29 capsid is decorated with 55 fibers attached to quasi-3-fold symmetry positions. Each fiber is a homotrimer of gene product 8.5 (gp8.5) and consists of two major structural parts, a pseudohexagonal base and a protruding fibrous portion that is about 110 {angstrom} in length. The crystal structure of the C-terminal fibrous portion (residues 112-280) has been determined to a resolution of 1.6 {angstrom}. The structure is about 150 {angstrom} long and shows three distinct structural domains designated as head, neck, and stem. The stem region is a unique three-stranded helix-turn-helix supercoil that has not previously been described. When fitted into a cryoelectron microscope reconstruction of the virus, the head structure corresponded to a disconnected density at the distal end of the fiber and the neck structure was located in weak density connecting it to the fiber. Thin section studies of Bacillus subtilis cells infected with fibered or fiberless {phi}29 suggest that the fibers might enhance the attachment of the virions onto the host cell wall.

  5. Modeling the structure of SARS 3a transmembrane protein using a ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences; Volume 127; Issue 12. Modeling the structure of SARS 3a transmembrane protein using a minimum unfavorable contact approach. S Ramakrishna ... Keywords. Membrane protein modeling; ion channel; transmembrane helices; viroporin; molecular dynamics; SARS 3a.

  6. Defining the structural requirements for a helix in 23 S ribosomal RNA that confers erythromycin resistance

    DEFF Research Database (Denmark)

    Douthwaite, S; Powers, T; Lee, J Y

    1989-01-01

    The helix spanning nucleotides 1198 to 1247 (helix 1200-1250) in Escherichia coli 23 S ribosomal RNA (rRNA) is functionally important in protein synthesis, and deletions in this region confer erythromycin resistance. In order to define the structural requirements for resistance, we have dissected....... However, removal of either these or non-conserved nucleotides from helix 1200-1250 measurably reduces the efficiency of 23 S RNA in forming functional ribosomes. We have used chemical probing and a modified primer extension method to investigate erythromycin binding to wild-type and resistant ribosomes...

  7. Modeling the Structure of SARS 3a Transmembrane Protein Using a ...

    Indian Academy of Sciences (India)

    Modeling the structure of SARS 3a Transmembrane protein using a minimum unfavorable contact approach. S RAMAKRISHNA, SILADITYA PADHI and U DEVA PRIYAKUMAR*. Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500 032, India.

  8. Structure and function of the cystic fibrosis transmembrane conductance regulator

    Directory of Open Access Journals (Sweden)

    M.M. Morales

    1999-08-01

    Full Text Available Cystic fibrosis (CF is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR. Mutations in the CFTR gene may result in a defective processing of its protein and alter the function and regulation of this channel. Mutations are associated with different symptoms, including pancreatic insufficiency, bile duct obstruction, infertility in males, high sweat Cl-, intestinal obstruction, nasal polyp formation, chronic sinusitis, mucus dehydration, and chronic Pseudomonas aeruginosa and Staphylococcus aureus lung infection, responsible for 90% of the mortality of CF patients. The gene responsible for the cellular defect in CF was cloned in 1989 and its protein product CFTR is activated by an increase of intracellular cAMP. The CFTR contains two membrane domains, each with six transmembrane domain segments, two nucleotide-binding domains (NBDs, and a cytoplasmic domain. In this review we discuss the studies that have correlated the role of each CFTR domain in the protein function as a chloride channel and as a regulator of the outwardly rectifying Cl- channels (ORCCs.

  9. Structure and Mechanism of Proton Transport Through the Transmembrane Tetrameric M2 Protein Bundle of the Influenza A Virus

    Energy Technology Data Exchange (ETDEWEB)

    R Acharya; V Carnevale; G Fiorin; B Levine; A Polishchuk; V Balannick; I Samish; R Lamb; L Pinto; et al.

    2011-12-31

    The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2{sup +} and 3{sup +} with a pK{sub a} near 6. A 1.65 {angstrom} resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

  10. RNA secondary structure prediction based on SHAPE data in helix regions.

    Science.gov (United States)

    Lotfi, Mohadeseh; Zare-Mirakabad, Fatemeh; Montaseri, Soheila

    2015-09-07

    RNA molecules play important and fundamental roles in biological processes. Frequently, the functional form of single-stranded RNA molecules requires a specific tertiary structure. Classically, RNA structure determination has mostly been accomplished by X-Ray crystallography or Nuclear Magnetic Resonance approaches. These experimental methods are time consuming and expensive. In the past two decades, some computational methods and algorithms have been developed for RNA secondary structure prediction. In these algorithms, minimum free energy is known as the best criterion. However, the results of algorithms show that minimum free energy is not a sufficient criterion to predict RNA secondary structure. These algorithms need some additional knowledge about the structure, which has to be added in the methods. Recently, the information obtained from some experimental data, called SHAPE, can greatly improve the consistency between the native and predicted RNA secondary structure. In this paper, we investigate the influence of SHAPE data on four types of RNA substructures, helices, loops, base pairs from the start and end of helices and two base pairs from the start and end of helices. The results show that SHAPE data in helix regions can improve the prediction. We represent a new method to apply SHAPE data in helix regions for finding RNA secondary structure. Finally, we compare the results of the method on a set of RNAs to predict minimum free energy structure based on considering all SHAPE data and only SHAPE data in helix regions as pseudo free energy and without SHAPE data (without any pseudo free energy). The results show that RNA secondary structure prediction based on considering only SHAPE data in helix regions is more successful than not considering SHAPE data and it provides competitive results in comparison with considering all SHAPE data. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Structure of a novel winged-helix like domain from human NFRKB protein.

    Directory of Open Access Journals (Sweden)

    Abhinav Kumar

    Full Text Available The human nuclear factor related to kappa-B-binding protein (NFRKB is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370-495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.

  12. Modeling the structure of SARS 3a transmembrane protein using a ...

    Indian Academy of Sciences (India)

    Abstract. 3a is an accessory protein from SARS coronavirus that is known to play a significant role in the proliferation of the virus by forming tetrameric ion channels. Although the monomeric units are known to consist of three transmembrane (TM) domains, there are no solved structures available for the complete monomer.

  13. Assessing the Structure and Stability of Transmembrane Oligomeric Intermediates of an α-Helical Toxin.

    Science.gov (United States)

    Desikan, Rajat; Maiti, Prabal K; Ayappa, K Ganapathy

    2017-10-24

    Protein membrane interactions play an important role in our understanding of diverse phenomena ranging from membrane-assisted protein aggregation to oligomerization and folding. Pore-forming toxins (PFTs) are the primary vehicle for infection by several strains of bacteria. These proteins which are expressed in a water-soluble form (monomers) bind to the target membrane and conformationally transform (protomers) and self-assemble to form a multimer transmembrane pore complex through a process of oligomerization. On the basis of the structure of the transmembrane domains, PFTs are broadly classified into β or α toxins. In contrast to β-PFTs, the paucity of available crystal structures coupled with the amphipathic nature of the transmembrane domains has hindered our understanding of α-PFT pore formation. In this article, we use molecular dynamics (MD) simulations to examine the process of pore formation of the bacterial α-PFT, cytolysin A from Escherichia coli (ClyA) in lipid bilayer membranes. Using atomistic MD simulations ranging from 50 to 500 ns, we show that transmembrane oligomeric intermediates or "arcs" form stable proteolipidic complexes consisting of protein arcs with toroidal lipids lining the free edges. By creating initial conditions where the lipids are contained within the arcs, we study the dynamics of spontaneous lipid evacuation and toroidal edge formation. This process occurs on the time scale of tens of nanoseconds, suggesting that once protomers oligomerize, transmembrane arcs are rapidly stabilized to form functional water channels capable of leakage. Using umbrella sampling with a coarse-grained molecular model, we obtain the free energy of insertion of a single protomer into the membrane. A single inserted protomer has a stabilization free energy of -52.9 ± 1.2 kJ/mol and forms a stable transmembrane water channel capable of leakage. Our simulations reveal that arcs are stable and viable intermediates that can occur during the pore

  14. Solution structure of LC4 transmembrane segment of CCR5.

    Directory of Open Access Journals (Sweden)

    Kazuhide Miyamoto

    Full Text Available CC-chemokine receptor 5 (CCR5 is a specific co-receptor allowing the entry of human immunodeficiency virus type 1 (HIV-1. The LC4 region in CCR5 is required for HIV-1 entry into the cells. In this study, the solution structure of LC4 in SDS micelles was elucidated by using standard 1H two-dimensional NMR spectroscopy, circular dichroism, and fluorescence quenching. The LC4 structure adopts two helical structures, whereas the C-terminal part remains unstructured. The positions in which LC4 binds to the HIV-1 inhibitory peptide LC5 were determined by docking calculations in addition to NMR data. The poses showed the importance of the hydrophobic interface of the assembled structures. The solution structure of LC4 elucidated in the present work provides a structural basis for further studies on the HIV-1 inhibitory function of the LC4 region.

  15. Solution structure of LC4 transmembrane segment of CCR5.

    Science.gov (United States)

    Miyamoto, Kazuhide; Togiya, Kayo

    2011-01-01

    CC-chemokine receptor 5 (CCR5) is a specific co-receptor allowing the entry of human immunodeficiency virus type 1 (HIV-1). The LC4 region in CCR5 is required for HIV-1 entry into the cells. In this study, the solution structure of LC4 in SDS micelles was elucidated by using standard 1H two-dimensional NMR spectroscopy, circular dichroism, and fluorescence quenching. The LC4 structure adopts two helical structures, whereas the C-terminal part remains unstructured. The positions in which LC4 binds to the HIV-1 inhibitory peptide LC5 were determined by docking calculations in addition to NMR data. The poses showed the importance of the hydrophobic interface of the assembled structures. The solution structure of LC4 elucidated in the present work provides a structural basis for further studies on the HIV-1 inhibitory function of the LC4 region.

  16. Solution Structure of LC4 Transmembrane Segment of CCR5

    OpenAIRE

    Miyamoto, Kazuhide; Togiya, Kayo

    2011-01-01

    CC-chemokine receptor 5 (CCR5) is a specific co-receptor allowing the entry of human immunodeficiency virus type 1 (HIV-1). The LC4 region in CCR5 is required for HIV-1 entry into the cells. In this study, the solution structure of LC4 in SDS micelles was elucidated by using standard 1H two-dimensional NMR spectroscopy, circular dichroism, and fluorescdence quenching. The LC4 structure adopts two helical structures, whereas the C-terminal part remains unstructured. The positions in which LC4 ...

  17. Structural insights into triglyceride storage mediated by fat storage-inducing transmembrane (FIT protein 2.

    Directory of Open Access Journals (Sweden)

    David A Gross

    2010-05-01

    Full Text Available Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2 belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9AAA in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation.

  18. TMDIM: an improved algorithm for the structure prediction of transmembrane domains of bitopic dimers

    Science.gov (United States)

    Cao, Han; Ng, Marcus C. K.; Jusoh, Siti Azma; Tai, Hio Kuan; Siu, Shirley W. I.

    2017-09-01

    α-Helical transmembrane proteins are the most important drug targets in rational drug development. However, solving the experimental structures of these proteins remains difficult, therefore computational methods to accurately and efficiently predict the structures are in great demand. We present an improved structure prediction method TMDIM based on Park et al. (Proteins 57:577-585, 2004) for predicting bitopic transmembrane protein dimers. Three major algorithmic improvements are introduction of the packing type classification, the multiple-condition decoy filtering, and the cluster-based candidate selection. In a test of predicting nine known bitopic dimers, approximately 78% of our predictions achieved a successful fit (RMSD MySQL and Apache, with all major browsers supported.

  19. Structure of the membrane anchor of pestivirus glycoprotein E(rns, a long tilted amphipathic helix.

    Directory of Open Access Journals (Sweden)

    Daniel Aberle

    2014-02-01

    Full Text Available E(rns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the E(rns membrane contact, processing and secretion.

  20. Structure of the Membrane Anchor of Pestivirus Glycoprotein Erns, a Long Tilted Amphipathic Helix

    Science.gov (United States)

    Aberle, Daniel; Muhle-Goll, Claudia; Bürck, Jochen; Wolf, Moritz; Reißer, Sabine; Luy, Burkhard; Wenzel, Wolfgang; Ulrich, Anne S.; Meyers, Gregor

    2014-01-01

    Erns is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the Erns membrane contact, processing and secretion. PMID:24586172

  1. Structure of the membrane anchor of pestivirus glycoprotein E(rns), a long tilted amphipathic helix.

    Science.gov (United States)

    Aberle, Daniel; Muhle-Goll, Claudia; Bürck, Jochen; Wolf, Moritz; Reißer, Sabine; Luy, Burkhard; Wenzel, Wolfgang; Ulrich, Anne S; Meyers, Gregor

    2014-02-01

    E(rns) is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the E(rns) membrane contact, processing and secretion.

  2. Biased and Constitutive Signaling in the CC-Chemokine Receptor CCR5 by manipulating the Interface between Transmembrane Helix 6 and 7

    DEFF Research Database (Denmark)

    Steen, Anne; Thiele, Stefanie; Guo, Dong

    2013-01-01

    The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biased...... signaling. Thus, beta-arrestin recruitment was eliminated, whereas constitutive activity was observed in Gi-mediated signaling. Furthermore, the CCR5 antagonist aplaviroc was converted to a full agonist (a so-called efficacy switch). Computational modeling revealed that the position of the 7TM receptor......-conserved Trp in TM6 (Trp-248 in position VI:13/6.48, part of the CWXP motif) was influenced by the G286F mutation, causing Trp-248 to change orientation away from TM7. The essential role of Trp-248 in CCR5 activation was supported by complete inactivity of W248A-CCR5 despite maintaining chemokine binding...

  3. Computer simulations and modeling-assisted ToxR screening in deciphering 3D structures of transmembrane α-helical dimers: ephrin receptor A1

    International Nuclear Information System (INIS)

    Volynsky, P E; Mineeva, E A; Goncharuk, M V; Ermolyuk, Ya S; Arseniev, A S; Efremov, R G

    2010-01-01

    Membrane-spanning segments of numerous proteins (e.g. receptor tyrosine kinases) represent a novel class of pharmacologically important targets, whose activity can be modulated by specially designed artificial peptides, the so-called interceptors. Rational construction of such peptides requires understanding of the main factors driving peptide–peptide association in lipid membranes. Here we present a new method for rapid prediction of the spatial structure of transmembrane (TM) helix–helix complexes. It is based on computer simulations in membrane-like media and subsequent refinement/validation of the results using experimental studies of TM helix dimerization in a bacterial membrane by means of the ToxR system. The approach was applied to TM fragments of the ephrin receptor A1 (EphA1). A set of spatial structures of the dimer was proposed based on Monte Carlo simulations in an implicit membrane followed by molecular dynamics relaxation in an explicit lipid bilayer. The resulting models were employed for rational design of wild-type and mutant genetic constructions for ToxR assays. The computational and the experimental data are self-consistent and provide an unambiguous spatial model of the TM dimer of EphA1. The results of this work can be further used to develop new biologically active 'peptide interceptors' specifically targeting membrane domains of proteins

  4. Role of the transmembrane domain of FXYD7 in structural and functional interactions with Na,K-ATPase.

    Science.gov (United States)

    Li, Ciming; Crambert, Gilles; Thuillard, Delphine; Roy, Sophie; Schaer, Danièle; Geering, Käthi

    2005-12-30

    Members of the FXYD family are tissue-specific regulators of the Na,K-ATPase. Here, we have investigated the contribution of amino acids in the transmembrane (TM) domain of FXYD7 to the interaction with Na,K-ATPase. Twenty amino acids of the TM domain were replaced individually by tryptophan, and combined mutations and alanine insertion mutants were constructed. Wild type and mutant FXYD7 were expressed in Xenopus oocytes with Na,K-ATPase. Mutational effects on the stable association with Na,K-ATPase and on the functional regulation of Na,K-ATPase were determined by co-immunoprecipitation and two-electrode voltage clamp techniques, respectively. Most residues important for the structural and functional interaction of FXYD7 are clustered in a face of the TM helix containing the two conserved glycine residues, but others are scattered over two-thirds of the FXYD TM helix. Ile-35, Ile-43, and Ile-44 are only involved in the stable association with Na,K-ATPase. Glu-26, Met-30, and Ile-44 are important for the functional effect and/or the efficient association of FXYD7 with Na,K-ATPase, consistent with the prediction that these amino acids contact TM domain 9 of the alpha subunit (Li, C., Grosdidier, A., Crambert, G., Horisberger, J.-D., Michielin, O., and Geering, K. (2004) J. Biol. Chem. 279, 38895-38902). Several amino acids that are not implicated in the efficient association of FXYD7 with the Na,K-ATPase are specifically involved in the functional effect of FXYD7. Leu-32 and Phe-37 influence the apparent affinity for external K+, whereas Val-28 and Ile-42 are implicated in the apparent affinity for both external K+ and external Na+. These amino acids act in a synergistic way. These results highlight the important structural and functional role of the TM domain of FXYD7 and delineate the determinants that mediate the complex interactions of FXYD7 with Na,K-ATPase.

  5. A benchmark server using high resolution protein structure data, and benchmark results for membrane helix predictions.

    Science.gov (United States)

    Rath, Emma M; Tessier, Dominique; Campbell, Alexander A; Lee, Hong Ching; Werner, Tim; Salam, Noeris K; Lee, Lawrence K; Church, W Bret

    2013-03-27

    Helical membrane proteins are vital for the interaction of cells with their environment. Predicting the location of membrane helices in protein amino acid sequences provides substantial understanding of their structure and function and identifies membrane proteins in sequenced genomes. Currently there is no comprehensive benchmark tool for evaluating prediction methods, and there is no publication comparing all available prediction tools. Current benchmark literature is outdated, as recently determined membrane protein structures are not included. Current literature is also limited to global assessments, as specialised benchmarks for predicting specific classes of membrane proteins were not previously carried out. We present a benchmark server at http://sydney.edu.au/pharmacy/sbio/software/TMH_benchmark.shtml that uses recent high resolution protein structural data to provide a comprehensive assessment of the accuracy of existing membrane helix prediction methods. The server further allows a user to compare uploaded predictions generated by novel methods, permitting the comparison of these novel methods against all existing methods compared by the server. Benchmark metrics include sensitivity and specificity of predictions for membrane helix location and orientation, and many others. The server allows for customised evaluations such as assessing prediction method performances for specific helical membrane protein subtypes.We report results for custom benchmarks which illustrate how the server may be used for specialised benchmarks. Which prediction method is the best performing method depends on which measure is being benchmarked. The OCTOPUS membrane helix prediction method is consistently one of the highest performing methods across all measures in the benchmarks that we performed. The benchmark server allows general and specialised assessment of existing and novel membrane helix prediction methods. Users can employ this benchmark server to determine the most

  6. The close-packed triple helix as a possible new structural motif for collagen

    DEFF Research Database (Denmark)

    Bohr, Jakob; Olsen, Kasper

    2010-01-01

    The one-dimensional problem of selecting the triple helix with the highest volume fraction is solved and hence the condition for a helix to be close-packed is obtained. The close-packed triple helix is shown to have a pitch angle of v CP = 43.3°. Contrary to the conventional notion, we suggest th...

  7. Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors

    DEFF Research Database (Denmark)

    Rosenkilde, M M; Smit, M J; Waldhoer, M

    2008-01-01

    A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments-most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue targeting...... pathogenesis is still poorly understood. Here we focus on the current knowledge of structure, function and trafficking patterns of virally encoded chemokine receptors and further address the putative roles of these receptors in virus survival and host -cell and/or -immune system modulation. Finally, we...

  8. Exploiting hydrophobicity for efficient production of transmembrane helices for structure determination by NMR spectroscopy

    DEFF Research Database (Denmark)

    Bugge, Katrine Østergaard; Steinocher, Helena; Brooks, Andrew J.

    2015-01-01

    -labeled protein. In this work, we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations...... of the strategy using seven membrane protein variants that differ in their overall hydrophobicity and length and show a recovery for suitable variants of >70%. The developed production scheme is cost-efficient and easy to implement and has the potential to facilitate an increase in the number of structures...

  9. The soluble loop BC region guides, but not dictates, the assembly of the transmembrane cytochrome b6.

    Directory of Open Access Journals (Sweden)

    Lydia Tome-Stangl

    Full Text Available Studying folding and assembly of naturally occurring α-helical transmembrane proteins can inspire the design of membrane proteins with defined functions. Thus far, most studies have focused on the role of membrane-integrated protein regions. However, to fully understand folding pathways and stabilization of α-helical membrane proteins, it is vital to also include the role of soluble loops. We have analyzed the impact of interhelical loops on folding, assembly and stability of the heme-containing four-helix bundle transmembrane protein cytochrome b6 that is involved in charge transfer across biomembranes. Cytochrome b6 consists of two transmembrane helical hairpins that sandwich two heme molecules. Our analyses strongly suggest that the loop connecting the helical hairpins is not crucial for positioning the two protein "halves" for proper folding and assembly of the holo-protein. Furthermore, proteolytic removal of any of the remaining two loops, which connect the two transmembrane helices of a hairpin structure, appears to also not crucially effect folding and assembly. Overall, the transmembrane four-helix bundle appears to be mainly stabilized via interhelical interactions in the transmembrane regions, while the soluble loop regions guide assembly and stabilize the holo-protein. The results of this study might steer future strategies aiming at designing heme-binding four-helix bundle structures, involved in transmembrane charge transfer reactions.

  10. Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain.

    Science.gov (United States)

    Yao, Hongwei; Lee, Myungwoon; Liao, Shu-Yu; Hong, Mei

    2016-12-13

    The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no 13 C- 13 C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may

  11. Structural Organization of a Full-Length Gp130/LIF-R Cytokine Receptor Transmembrane Complex

    Energy Technology Data Exchange (ETDEWEB)

    Skiniotis, G.; Lupardus, P.J.; Martick, M.; Walz, T.; Garcia, K.C.

    2009-05-26

    gp130 is a shared receptor for at least nine cytokines, and can signal either as a homodimer, or as a heterodimer with Leukemia Inhibitory Factor Receptor (LIF-R). Here we biophysically and structurally characterize the full-length, transmembrane form of a quaternary cytokine receptor complex consisting of gp130, LIF-R, the cytokine Ciliary Neurotrophic Factor (CNTF), and its alpha receptor (CNTF-R{alpha}). Thermodynamic analysis indicates that, unlike the cooperative assembly of the symmetric gp130/Interleukin-6/IL-6R{alpha} hexameric complex, CNTF/CNTF-R{alpha} heterodimerizes gp130 and LIF-R via non-cooperative energetics to form an asymmetric 1:1:1:1 complex. Single particle electron microscopic (EM) analysis of the full-length gp130/LIF-R/CNTF-R{alpha}/CNTF quaternary complex elucidates an asymmetric structural arrangement, in which the receptor extracellular and transmembrane segments join as a continuous, rigid unit, poised to sensitively transduce ligand engagement to the membrane-proximal intracellular signaling regions. These studies also enumerate the organizing principles for assembly of the 'tall' class of gp130-family cytokine receptor complexes including LIF, IL-27, IL-12, and others.

  12. Structural Changes Fundamental to Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Anion Channel Pore.

    Science.gov (United States)

    Linsdell, Paul

    2017-01-01

    Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial cell anion channel. Potentiator drugs used in the treatment of cystic fibrosis act on the channel to increase overall channel function, by increasing the stability of its open state and/or decreasing the stability of its closed state. The structure of the channel in either the open state or the closed state is not currently known. However, changes in the conformation of the protein as it transitions between these two states have been studied using functional investigation and molecular modeling techniques. This review summarizes our current understanding of the architecture of the transmembrane channel pore that controls the movement of chloride and other small anions, both in the open state and in the closed state. Evidence for different kinds of changes in the conformation of the pore as it transitions between open and closed states is described, as well as the mechanisms by which these conformational changes might be controlled to regulate normal channel gating. The ways that key conformational changes might be targeted by small compounds to influence overall CFTR activity are also discussed. Understanding the changes in pore structure that might be manipulated by such small compounds is key to the development of novel therapeutic strategies for the treatment of cystic fibrosis.

  13. Differential transmembrane domain GXXXG motif pairing impacts major histocompatibility complex (MHC) class II structure.

    Science.gov (United States)

    Dixon, Ann M; Drake, Lisa; Hughes, Kelly T; Sargent, Elizabeth; Hunt, Danielle; Harton, Jonathan A; Drake, James R

    2014-04-25

    Major histocompatibility complex (MHC) class II molecules exhibit conformational heterogeneity, which influences their ability to stimulate CD4 T cells and drive immune responses. Previous studies suggest a role for the transmembrane domain of the class II αβ heterodimer in determining molecular structure and function. Our previous studies identified an MHC class II conformer that is marked by the Ia.2 epitope. These Ia.2(+) class II conformers are lipid raft-associated and able to drive both tyrosine kinase signaling and efficient antigen presentation to CD4 T cells. Here, we establish that the Ia.2(+) I-A(k) conformer is formed early in the class II biosynthetic pathway and that differential pairing of highly conserved transmembrane domain GXXXG dimerization motifs is responsible for formation of Ia.2(+) versus Ia.2(-) I-A(k) class II conformers and controlling lipid raft partitioning. These findings provide a molecular explanation for the formation of two distinct MHC class II conformers that differ in their inherent ability to signal and drive robust T cell activation, providing new insight into the role of MHC class II in regulating antigen-presenting cell-T cell interactions critical to the initiation and control of multiple aspects of the immune response.

  14. Implementation and application of helix-helix distance and crossing angle restraint potentials.

    Science.gov (United States)

    Lee, Jinhyuk; Im, Wonpil

    2007-02-01

    Based on the definition of helix-helix distance and crossing angle introduced by Chothia et al. (J Mol Biol 1981, 145, 215), we have developed the restraint potentials by which the distance and crossing angle of two selected helices can be maintained around target values during molecular dynamics simulations. A series of assessments show that calculated restraint forces are numerically accurate. Since the restraint forces are only exerted on atoms which define the helical principal axes, each helix can rotate along its helical axis, depending on the helix-helix intermolecular interactions. Such a restraint potential enables us to characterize the helix-helix interactions at atomic details by sampling their conformational space around specific distance and crossing angle with (restraint) force-dependent fluctuations. Its efficacy is illustrated by calculating the potential of mean force as a function of helix-helix distance between two transmembrane helical peptides in an implicit membrane model.

  15. Right- and left-handed three-helix proteins. I. Experimental and simulation analysis of differences in folding and structure.

    Science.gov (United States)

    Glyakina, Anna V; Pereyaslavets, Leonid B; Galzitskaya, Oxana V

    2013-09-01

    Despite the large number of publications on three-helix protein folding, there is no study devoted to the influence of handedness on the rate of three-helix protein folding. From the experimental studies, we make a conclusion that the left-handed three-helix proteins fold faster than the right-handed ones. What may explain this difference? An important question arising in this paper is whether the modeling of protein folding can catch the difference between the protein folding rates of proteins with similar structures but with different folding mechanisms. To answer this question, the folding of eight three-helix proteins (four right-handed and four left-handed), which are similar in size, was modeled using the Monte Carlo and dynamic programming methods. The studies allowed us to determine the orders of folding of the secondary-structure elements in these domains and amino acid residues which are important for the folding. The obtained data are in good correlation with each other and with the experimental data. Structural analysis of these proteins demonstrated that the left-handed domains have a lesser number of contacts per residue and a smaller radius of cross section than the right-handed domains. This may be one of the explanations of the observed fact. The same tendency is observed for the large dataset consisting of 332 three-helix proteins (238 right- and 94 left-handed). From our analysis, we found that the left-handed three-helix proteins have some less-dense packing that should result in faster folding for some proteins as compared to the case of right-handed proteins. Copyright © 2013 Wiley Periodicals, Inc.

  16. Predicting RNA 3D structure using a coarse-grain helix-centered model.

    Science.gov (United States)

    Kerpedjiev, Peter; Höner Zu Siederdissen, Christian; Hofacker, Ivo L

    2015-06-01

    A 3D model of RNA structure can provide information about its function and regulation that is not possible with just the sequence or secondary structure. Current models suffer from low accuracy and long running times and either neglect or presume knowledge of the long-range interactions which stabilize the tertiary structure. Our coarse-grained, helix-based, tertiary structure model operates with only a few degrees of freedom compared with all-atom models while preserving the ability to sample tertiary structures given a secondary structure. It strikes a balance between the precision of an all-atom tertiary structure model and the simplicity and effectiveness of a secondary structure representation. It provides a simplified tool for exploring global arrangements of helices and loops within RNA structures. We provide an example of a novel energy function relying only on the positions of stems and loops. We show that coupling our model to this energy function produces predictions as good as or better than the current state of the art tools. We propose that given the wide range of conformational space that needs to be explored, a coarse-grain approach can explore more conformations in less iterations than an all-atom model coupled to a fine-grain energy function. Finally, we emphasize the overarching theme of providing an ensemble of predicted structures, something which our tool excels at, rather than providing a handful of the lowest energy structures. © 2015 Kerpedjiev et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  17. High-resolution modeling of transmembrane helical protein structures from distant homologues.

    Directory of Open Access Journals (Sweden)

    Kuang-Yui M Chen

    2014-05-01

    Full Text Available Eukaryotic transmembrane helical (TMH proteins perform a wide diversity of critical cellular functions, but remain structurally largely uncharacterized and their high-resolution structure prediction is currently hindered by the lack of close structural homologues. To address this problem, we present a novel and generic method for accurately modeling large TMH protein structures from distant homologues exhibiting distinct loop and TMH conformations. Models of the adenosine A2AR and chemokine CXCR4 receptors were first ranked in GPCR-DOCK blind prediction contests in the receptor structure accuracy category. In a benchmark of 50 TMH protein homolog pairs of diverse topology (from 5 to 12 TMHs, size (from 183 to 420 residues and sequence identity (from 15% to 70%, the method improves most starting templates, and achieves near-atomic accuracy prediction of membrane-embedded regions. Unlike starting templates, the models are of suitable quality for computer-based protein engineering: redesigned models and redesigned X-ray structures exhibit very similar native interactions. The method should prove useful for the atom-level modeling and design of a large fraction of structurally uncharacterized TMH proteins from a wide range of structural homologues.

  18. The position of the Gly-xxx-Gly motif in transmembrane segments modulates dimer affinity.

    Science.gov (United States)

    Johnson, Rachel M; Rath, Arianna; Deber, Charles M

    2006-12-01

    Although the intrinsic low solubility of membrane proteins presents challenges to their high-resolution structure determination, insight into the amino acid sequence features and forces that stabilize their folds has been provided through study of sequence-dependent helix-helix interactions between single transmembrane (TM) helices. While the stability of helix-helix partnerships mediated by the Gly-xxx-Gly (GG4) motif is known to be generally modulated by distal interfacial residues, it has not been established whether the position of this motif, with respect to the ends of a given TM segment, affects dimer affinity. Here we examine the relationship between motif position and affinity in the homodimers of 2 single-spanning membrane protein TM sequences: glycophorin A (GpA) and bacteriophage M13 coat protein (MCP). Using the TOXCAT assay for dimer affinity on a series of GpA and MCP TM segments that have been modified with either 4 Leu residues at each end or with 8 Leu residues at the N-terminal end, we show that in each protein, centrally located GG4 motifs are capable of stronger helix-helix interactions than those proximal to TM helix ends, even when surrounding interfacial residues are maintained. The relative importance of GG4 motifs in stabilizing helix-helix interactions therefore must be considered not only in its specific residue context but also in terms of the location of the interactive surface relative to the N and C termini of alpha-helical TM segments.

  19. Probabilistic grammatical model for helix‐helix contact site classification

    Science.gov (United States)

    2013-01-01

    Background Hidden Markov Models power many state‐of‐the‐art tools in the field of protein bioinformatics. While excelling in their tasks, these methods of protein analysis do not convey directly information on medium‐ and long‐range residue‐residue interactions. This requires an expressive power of at least context‐free grammars. However, application of more powerful grammar formalisms to protein analysis has been surprisingly limited. Results In this work, we present a probabilistic grammatical framework for problem‐specific protein languages and apply it to classification of transmembrane helix‐helix pairs configurations. The core of the model consists of a probabilistic context‐free grammar, automatically inferred by a genetic algorithm from only a generic set of expert‐based rules and positive training samples. The model was applied to produce sequence based descriptors of four classes of transmembrane helix‐helix contact site configurations. The highest performance of the classifiers reached AUCROC of 0.70. The analysis of grammar parse trees revealed the ability of representing structural features of helix‐helix contact sites. Conclusions We demonstrated that our probabilistic context‐free framework for analysis of protein sequences outperforms the state of the art in the task of helix‐helix contact site classification. However, this is achieved without necessarily requiring modeling long range dependencies between interacting residues. A significant feature of our approach is that grammar rules and parse trees are human‐readable. Thus they could provide biologically meaningful information for molecular biologists. PMID:24350601

  20. Assessment of the transmembrane domain structures in GPCR Dock 2013 models.

    Science.gov (United States)

    Wang, Ting; Liu, Haiguang; Duan, Yong

    2018-03-01

    The community-wide blind prediction of G-protein coupled receptor (GPCR) structures and ligand docking has been conducted three times and the quality of the models was primarily assessed by the accuracy of ligand binding modes. The seven transmembrane (TM) helices of the receptors were taken as a whole; thus the model quality within the 7TM domains has not been evaluated. Here we evaluate the 7TM domain structures in the models submitted for the last round of prediction - GPCR Dock 2013. Applying the 7 × 7 RMSD matrix analysis described in our prior work, we show that the models vary widely in prediction accuracy of the 7TM structures, exhibiting diverse structural differences from the targets. For the prediction of the 5-hydroxytryptamine receptors, the top 7TM models are rather close to the targets, which however are not ranked top by ligand-docking. On the other hand, notable deviations of the TMs are found in in the previously identified top docking models that closely resemble other receptors. We further reveal reasons of success and failure in ligand docking for the models. This current assessment not only complements the previous assessment, but also provides important insights into the current status of GPCR modeling and ligand docking. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure.

    Science.gov (United States)

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H; Qin, Yali; Cho, Michael W

    2016-03-01

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; (671)NWFDITNWLWYIK(683)) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Flanking Polyproline Sequences Inhibit [beta]-Sheet Structure in Polyglutamine Segments by Inducing PPII-like Helix Structure

    Energy Technology Data Exchange (ETDEWEB)

    Darnell, Gregory; Orgel, Joseph P.R.O.; Pahl, Reinhard; Meredith, Stephen C. (IIT); (UC)

    2008-06-24

    Polyglutamine (poly(Q)) expansion is associated with protein aggregation into {beta}-sheet amyloid fibrils and neuronal cytotoxicity. In the mutant poly(Q) protein huntingtin, associated with Huntington's disease, both aggregation and cytotoxicity may be abrogated by a polyproline (poly(P)) domain flanking the C terminus of the poly(Q) region. To understand structural changes that may occur with the addition of the poly(P) sequence, we synthesized poly(Q) peptides with 3-15 glutamine residues and a corresponding set of poly(Q) peptides flanked on the C terminus by 11 proline residues (poly(Q)-poly(P)), as occurs in the huntingtin sequence. The shorter soluble poly(Q) peptides (three or six glutamine residues) showed polyproline type II-like (PPII)-like helix conformation when examined by circular dichroism spectroscopy and were monomers as judged by size-exclusion chromatography (SEC), while the longer poly(Q) peptides (nine or 15 glutamine residues) showed a {beta}-sheet conformation by CD and defined oligomers by SEC. Soluble poly(Q)-poly(P) peptides showed PPII-like content but SEC showed poorly defined, overlapping oligomeric peaks, and as judged by CD these peptides retained significant PPII-like structure with increasing poly(Q) length. More importantly, addition of the poly(P) domain increased the threshold for fibril formation to {approx} 15 glutamine residues. X-ray diffraction, electron microscopy, and film CD showed that, while poly(Q) peptides with {ge} 6 glutamine residues formed {beta}-sheet-rich fibrils, only the longest poly(Q)-poly(P) peptide (15 glutamine residues) did so. From these and other observations, we propose that poly(Q) domains exist in a 'tug-of-war' between two conformations, a PPII-like helix and a {beta}-sheet, while the poly(P) domain is conformationally constrained into a proline type II helix (PPII). Addition of poly(P) to the C terminus of a poly(Q) domain induces a PPII-like structure, which opposes the

  3. The structure of the XPF-ssDNA complex underscores the distinct roles of the XPF and ERCC1 helix- hairpin-helix domains in ss/ds DNA recognition

    NARCIS (Netherlands)

    Das, Devashish; Folkers, Gert; van Dijk, Marc; Jaspers, Nicolaas G.J.; Hoeijmakers, Jan H.J.; Kaptein, Robert; Boelens, Rolf

    2012-01-01

    Human XPF/ERCC1 is a structure-specific DNA endonuclease that nicks the damaged DNA strand at the 5' end during nucleotide excision repair. We determined the structure of the complex of the C-terminal domain of XPF with 10 nt ssDNA. A positively charged region within the second helix of the first

  4. Characterisation of the salmon cystic fibrosis transmembrane conductance regulator protein for structural studies

    Directory of Open Access Journals (Sweden)

    Naomi L. Pollock

    2014-11-01

    Full Text Available The cystic fibrosis transmembrane conductance regulator protein (CFTR is a chloride channel highly expressed in the gills of Salmo salar, with a role in osmoregulation. It shares 60% identity with the human CFTR channel, mutations to which can cause the common genetic disorder cystic fibrosis CF. The expression and localisation of salmon CFTR have been investigated, but the isolated protein has not been extensively characterised. Here we present a protocol for the purification of recombinant salmon CFTR, along with biophysical and structural characterisation of the purified protein. Salmon CFTR was overexpressed in Saccharomyces cerevisiae, solubilised in the detergent LPG-14 and chromatographically purified by nickel-affinity and size-exclusion chromatography methods. Prior to size-exclusion chromatography samples of salmon CFTR had low purity, and contained large quantities of aggregated protein. Compared to size-exclusion chromatography profiles of other orthologues of CFTR, which had less evidence of aggregation, salmon CFTR appeared to have lower intrinsic stability than human and platypus CFTR. Nonetheless, repeated size-exclusion chromatography allowed monodisperse salmon CFTR to be isolated, and multi-angle light scattering was used to determine its oligomeric state. The monodispersity of the sample and its oligomeric state were confirmed using cryo-electron microscopy and small-angle X-ray scattering (SAXS. These data were also processed to calculate a low-resolution structure of the salmon CFTR, which showed similar architecture to other ATP-binding cassette proteins.

  5. MemBrain: An Easy-to-Use Online Webserver for Transmembrane Protein Structure Prediction

    Science.gov (United States)

    Yin, Xi; Yang, Jing; Xiao, Feng; Yang, Yang; Shen, Hong-Bin

    2018-03-01

    Membrane proteins are an important kind of proteins embedded in the membranes of cells and play crucial roles in living organisms, such as ion channels, transporters, receptors. Because it is difficult to determinate the membrane protein's structure by wet-lab experiments, accurate and fast amino acid sequence-based computational methods are highly desired. In this paper, we report an online prediction tool called MemBrain, whose input is the amino acid sequence. MemBrain consists of specialized modules for predicting transmembrane helices, residue-residue contacts and relative accessible surface area of α-helical membrane proteins. MemBrain achieves a prediction accuracy of 97.9% of A TMH, 87.1% of A P, 3.2 ± 3.0 of N-score, 3.1 ± 2.8 of C-score. MemBrain-Contact obtains 62%/64.1% prediction accuracy on training and independent dataset on top L/5 contact prediction, respectively. And MemBrain-Rasa achieves Pearson correlation coefficient of 0.733 and its mean absolute error of 13.593. These prediction results provide valuable hints for revealing the structure and function of membrane proteins. MemBrain web server is free for academic use and available at www.csbio.sjtu.edu.cn/bioinf/MemBrain/. [Figure not available: see fulltext.

  6. Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix

    International Nuclear Information System (INIS)

    Pilch, D.S.; Shafer, R.H.; Levenson, C.

    1991-01-01

    The authors have investigated the structure and physical chemistry of the d(C 3 T 4 C 3 )·2[d(G 3 A 4 G 3 )] triple helix by polyacrylamide gel electrophoresis (PAGE), 1 H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl 2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur·pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the puring strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 C, depending on the DNA concentration. This marked enhancement in stability, coupled with the lack of an acidic pH requirement, suggests that pur-pur-pyr triplexes are appealing choices for use in applications involving oligonucleotide targeting of duplex DNA in vitro and in vivo

  7. Modulating immunogenic properties of HIV-1 gp41 membrane-proximal external region by destabilizing six-helix bundle structure

    Energy Technology Data Exchange (ETDEWEB)

    Banerjee, Saikat; Shi, Heliang; Habte, Habtom H.; Qin, Yali; Cho, Michael W., E-mail: mcho@iastate.edu

    2016-03-15

    The C-terminal alpha-helix of gp41 membrane-proximal external region (MPER; {sup 671}NWFDITNWLWYIK{sup 683}) encompassing 4E10/10E8 epitopes is an attractive target for HIV-1 vaccine development. We previously reported that gp41-HR1-54Q, a trimeric protein comprised of the MPER in the context of a stable six-helix bundle (6HB), induced strong immune responses against the helix, but antibodies were directed primarily against the non-neutralizing face of the helix. To better target 4E10/10E8 epitopes, we generated four putative fusion intermediates by introducing double point mutations or deletions in the heptad repeat region 1 (HR1) that destabilize 6HB in varying degrees. One variant, HR1-∆10-54K, elicited antibodies in rabbits that targeted W672, I675 and L679, which are critical for 4E10/10E8 recognition. Overall, the results demonstrated that altering structural parameters of 6HB can influence immunogenic properties of the MPER and antibody targeting. Further exploration of this strategy could allow development of immunogens that could lead to induction of 4E10/10E8-like antibodies. - Highlights: • Four gp41 MPER-based immunogens that resemble fusion intermediates were generated. • C-terminal region of MPER that contains 4E10/10E8 epitopes was highly immunogenic. • Altering 6HB structure can influence immunogenic properties of the MPER. • Induced antibodies targeted multiple residues critical for 4E10/10E8 binding. • Development of immunogens based on fusion intermediates is a promising strategy.

  8. Molecular structure of r/GCG/d/TATACGC/ - A DNA-RNA hybrid helix joined to double helical DNA

    Science.gov (United States)

    Wang, A. H.-J.; Fujii, S.; Rich, A.; Van Boom, J. H.; Van Der Marel, G. A.; Van Boeckel, S. A. A.

    1982-01-01

    The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA-RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.

  9. Evolutionary repair of HIV type 1 gp41 with a kink in the N-terminal helix leads to restoration of the six-helix bundle structure

    NARCIS (Netherlands)

    Sanders, Rogier W.; Busser, Els; Moore, John P.; Lu, Min; Berkhout, Ben

    2004-01-01

    The HIV-1 envelope glycoprotein complex (Env) can be stabilized by the introduction of a disulfide bond between the gp120 and gp41 subunits. The resulting protein is monomeric, but trimerization can be improved by the introduction of a single helix-breaking residue at the conserved Ile559 site in

  10. Crystal structure of hormone-bound atrial natriuretic peptide receptor extracellular domain: rotation mechanism for transmembrane signal transduction.

    Science.gov (United States)

    Ogawa, Haruo; Qiu, Yue; Ogata, Craig M; Misono, Kunio S

    2004-07-02

    A cardiac hormone, atrial natriuretic peptide (ANP), plays a major role in blood pressure and volume regulation. ANP activities are mediated by a single span transmembrane receptor carrying intrinsic guanylate cyclase activity. ANP binding to its extracellular domain stimulates guanylate cyclase activity by an as yet unknown mechanism. Here we report the crystal structure of dimerized extracellular hormone-binding domain in complex with ANP. The structural comparison with the unliganded receptor reveals that hormone binding causes the two receptor monomers to undergo an intermolecular twist with little intramolecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains in the dimer with essentially no change in the interdomain distance. This movement alters the relative orientation of the two domains by a shift equivalent to counterclockwise rotation of each by 24 degrees. These results suggest that transmembrane signaling by the ANP receptor is initiated via a hormone-induced rotation mechanism.

  11. Loop-to-helix transition in the structure of multidrug regulator AcrR at the entrance of the drug-binding cavity

    Energy Technology Data Exchange (ETDEWEB)

    Manjasetty, Babu A.; Halavaty, Andrei S.; Luan, Chi-Hao; Osipiuk, Jerzy; Mulligan, Rory; Kwon, Keehwan; Anderson, Wayne F.; Joachimiak, Andrzej

    2016-04-01

    Multidrug transcription regulator AcrR from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 belongs to the tetracycline repressor family, one of the largest groups of bacterial transcription factors. The crystal structure of dimeric AcrR was determined and refined to 1.56 Å resolution. The tertiary and quaternary structures of AcrR are similar to those of its homologs. The multidrug binding site was identified based on structural alignment with homologous proteins and has a di(hydroxyethyl)ether molecule bound. Residues from helices a4 and a7 shape the entry into this binding site. The structure of AcrR reveals that the extended helical conformation of helix a4 is stabilized by the hydrogen bond between Glu67 (helix a4) and Gln130 (helix a7). Based on the structural comparison with the closest homolog structure, the Escherichia coli AcrR, we propose that this hydrogen bond is responsible for control of the loop-to-helix transition within helix a4. This local conformational switch of helix a4 may be a key step in accessing the multidrug binding site and securing ligands at the binding site. Solution smallmolecule binding studies suggest that AcrR binds ligands with their core chemical structure resembling the tetracyclic ring of cholesterol.

  12. Structure-Based Sequence Alignment of the Transmembrane Domains of All Human GPCRs: Phylogenetic, Structural and Functional Implications

    Science.gov (United States)

    Cvicek, Vaclav; Goddard, William A.; Abrol, Ravinder

    2016-01-01

    The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs

  13. Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors

    NARCIS (Netherlands)

    Rosenkilde, M.M.; Smit, M.J.; Waldhoer, M.

    2008-01-01

    A number of human and animal herpes viruses encode G-protein coupled receptors with seven transmembrane (7TM) segments - most of which are clearly related to human chemokine receptors. It appears, that these receptors are used by the virus for immune evasion, cellular transformation, tissue

  14. Structural studies of Helix aspersa agglutinin complexed with GalNAc: A lectin that serves as a diagnostic tool.

    Science.gov (United States)

    Pietrzyk, Agnieszka J; Bujacz, Anna; Mak, Paweł; Potempa, Barbara; Niedziela, Tomasz

    2015-11-01

    Lectins belong to a differentiated group of proteins known to possess sugar-binding properties. Due to this fact, they are interesting research targets in medical diagnostics. Helix aspersa agglutinin (HAA) is a lectin that recognizes the epitopes containing α-d-N-acetylgalactosamine (GalNAc), which is present at the surface of metastatic cancer cells. Although several reports have already described the use of HAA as a diagnostic tool, this protein was not characterized on the molecular level. Here, we present for the first time the structural information about lectin isolated from mucus of Helix aspersa (garden snail). The amino acid sequence of this agglutinin was determined by Edman degradation and tertiary as well as quaternary structure by X-ray crystallography. The high resolution crystal structure (1.38Å) and MALDI-TOF mass spectrometry analysis provide the detailed information about a large part of the HAA natural glycan chain. The topology of the GalNAc binding cleft and interaction with lectin are very well defined in the structure and fully confirmed by STD HSQC NMR spectroscopy. Together, this provides structural clues regarding HAA specificity and opens possibilities to rational modifications of this important diagnostic tool. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Structural models of the transmembrane region of voltage-gated and other K+ channels in open, closed, and inactivated conformations.

    Science.gov (United States)

    Durell, S R; Hao, Y; Guy, H R

    1998-01-01

    A large collaborative, multidisciplinary effort involving many research laboratories continues which uses indirect methods of molecular biology and membrane biophysics to analyze the three-dimensional structures and functional mechanisms of K+ channels. This work also extends to the distant relatives of these channels, including the voltage-gated Na+ and Ca2+ channels. The role that our group plays in this process is to combine the information gained from experimental studies with molecular modeling techniques to generate atomic-scale structural models of these proteins. The modeling process involves three stages which are summarized as: (I) prediction of the channel sequence transmembrane topology, including the functionality and secondary structure of the segments; (II) prediction of the relative positions of the transmembrane segments, and (III) filling in all atoms of the amino acid residues, with conformations for energetically stabilized interactions. Both physiochemical and evolutionary principles (including sequence homology analysis) are used to guide the development. In addition to testing the steric and energetic feasibilities of different structural hypotheses, the models provide guidance for the design of new experiments. Structural modeling also serves to "fill in the gaps" of experimental data, such as predicting additional residue interactions and conformational changes responsible for functional processes. The modeling process is currently at the stage that experimental studies have definitely confirmed most of our earlier predictions about the transmembrane topology and functionality of different segments. Additionally, this report describes the detailed, three-dimensional models we have developed for the entire transmembrane region and important functional sites of the voltage-gated Shaker K+ channel in the open, closed, and inactivated conformations (including the ion-selective pore and voltage-sensor regions). As part of this effort, we also

  16. Structural studies of the natriuretic peptide receptor: a novel hormone-induced rotation mechanism for transmembrane signal transduction.

    Science.gov (United States)

    Misono, Kunio S; Ogawa, Haruo; Qiu, Yue; Ogata, Craig M

    2005-06-01

    The atrial natriuretic peptide (ANP) receptor is a single-span transmembrane receptor that is coupled to its intrinsic intracellular guanylate cyclase (GCase) catalytic activity. To investigate the mechanisms of hormone binding and signal transduction, we have expressed the extracellular hormone-binding domain of the ANP receptor (ANPR) and characterized its structure and function. The disulfide-bond structure, state of glycosylation, binding-site residues, chloride-dependence of ANP binding, dimerization, and binding stoichiometry have been determined. More recently, the crystal structures of both the apoANPR dimer and ANP-bound complex have been determined. The structural comparison between the two has shown that, upon ANP binding, two ANPR molecules in the dimer undergo an inter-molecular twist with little intra-molecular conformational change. This motion produces a Ferris wheel-like translocation of two juxtamembrane domains with essentially no change in the inter-domain distance. This movement alters the relative orientation of the two domains equivalent to counter-clockwise rotation of each by 24 degrees . These results suggest that transmembrane signaling by the ANP receptor is mediated by a novel hormone-induced rotation mechanism.

  17. Structural Dynamics Of The S4 Voltage-Sensor Helix In Lipid Bilayers Lacking Lipid Phosphates

    Science.gov (United States)

    Andersson, Magnus; Freites, J. Alfredo; Tobias, Douglas J.; White, Stephen H.

    2011-01-01

    Voltage-dependent K+ (Kv) channels require lipid phosphates for functioning. The S4 helix, which carries the gating charges in the voltage-sensing domain (VSD), inserts into membranes while being stabilized by a protein-lipid interface in which lipid phosphates play an essential role. To examine the physical basis of the protein-lipid interface in the absence of lipid phosphates, we performed molecular dynamics (MD) simulations of a KvAP S4 variant (S4mut) in bilayers with and without lipid phosphates. We find that in dioleoyltrimethylammoniumpropane (DOTAP) bilayers lacking lipid phosphates, the gating charges are solvated by anionic counterions and, hence, lack the bilayer support provided by phosphate-containing palmitoyloleoylglycerophosphocholine (POPC) bilayers. The result is a water-permeable bilayer with a significantly smaller deformations around the peptide. Together, these results provide an explanation for the non-functionality of VSDs in terms of a destabilizing protein-lipid interface. PMID:21692541

  18. Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix.

    Science.gov (United States)

    Doose, Sören; Neuweiler, Hannes; Barsch, Hannes; Sauer, Markus

    2007-10-30

    Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Förster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics of poly-l-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET). A single fluorophore and a tryptophan residue, introduced at the termini of polyproline peptides, serve as sensitive probes for distance changes on the subnanometer length scale. Using a combination of ensemble fluorescence and fluorescence correlation spectroscopy, we demonstrate that polyproline samples exhibit static structural heterogeneity with subpopulations of distinct end-to-end distances that do not interconvert on time scales from nano- to milliseconds. By observing prolyl isomerization through changes in PET quenching interactions, we provide experimental evidence that the observed heterogeneity can be explained by interspersed cis isomers. Computer simulations elucidate the influence of trans/cis isomerization on polyproline structures in terms of end-to-end distance and provide a structural justification for the experimentally observed effects. Our results demonstrate that structural heterogeneity inherent in polyprolines, which to date are commonly applied as a molecular ruler, disqualifies them as appropriate tool for an accurate determination of absolute distances at a molecular scale.

  19. Solution structure and DNA-binding properties of the winged helix domain of the meiotic recombination HOP2 protein.

    Science.gov (United States)

    Moktan, Hem; Guiraldelli, Michel F; Eyster, Craig A; Zhao, Weixing; Lee, Chih-Ying; Mather, Timothy; Camerini-Otero, R Daniel; Sung, Patrick; Zhou, Donghua H; Pezza, Roberto J

    2014-05-23

    The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Solution Structure and DNA-binding Properties of the Winged Helix Domain of the Meiotic Recombination HOP2 Protein*

    Science.gov (United States)

    Moktan, Hem; Guiraldelli, Michel F.; Eyster, Craig A.; Zhao, Weixing; Lee, Chih-Ying; Mather, Timothy; Camerini-Otero, R. Daniel; Sung, Patrick; Zhou, Donghua H.; Pezza, Roberto J.

    2014-01-01

    The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination. PMID:24711446

  1. Observation of triple helix motif on electrospun collagen nanofibers and its effect on the physical and structural properties

    Science.gov (United States)

    Bürck, Jochen; Aras, Onur; Bertinetti, Luca; Ilhan, Caner A.; Ermeydan, Mahmut A.; Schneider, Reinhard; Ulrich, Anne S.; Kazanci, Murat

    2018-01-01

    Collagen is a very popular natural biomaterial due to its high biocompatibility and bioactivity. Electrospinning is currently the only technique that allows the fabrication of continuous fibers with diameters down to a few nanometers. In order to regenerate collagen in the forms of nanofibers, it is necessary to dissolve it in suitable solvents. The solvents and electrospinning process cause unfolding of collagen nanofibers. It is proposed that acidic solvents preserve better the natural structure of collagen fibers. In this paper, the structures of collagen nanofibers were examined by using circular dichroism (CD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, differential scanning calorimetry (DSC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) methods in order to test this hypothesis. The increase in PP-II fraction, representing the triple helix structure in collagen, that was observed in CD analysis of HAc derived collagen nanofibers, for the first time was successfully confirmed and illustrated by using SEM and TEM methods. Furthermore, CD revealed the mostly detrimental effect of stabilization conditions such as heat, vacuum and UV treatment on the secondary structure of the collagen nanofibers.

  2. Ectopic expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus.

    Science.gov (United States)

    Paolocci, Francesco; Robbins, Mark P; Madeo, Laura; Arcioni, Sergio; Martens, Stefan; Damiani, Francesco

    2007-01-01

    Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

  3. Detecting pore-lining regions in transmembrane protein sequences

    Directory of Open Access Journals (Sweden)

    Nugent Timothy

    2012-07-01

    Full Text Available Abstract Background Alpha-helical transmembrane channel and transporter proteins play vital roles in a diverse range of essential biological processes and are crucial in facilitating the passage of ions and molecules across the lipid bilayer. However, the experimental difficulties associated with obtaining high quality crystals has led to their significant under-representation in structural databases. Computational methods that can identify structural features from sequence alone are therefore of high importance. Results We present a method capable of automatically identifying pore-lining regions in transmembrane proteins from sequence information alone, which can then be used to determine the pore stoichiometry. By labelling pore-lining residues in crystal structures using geometric criteria, we have trained a support vector machine classifier to predict the likelihood of a transmembrane helix being involved in pore formation. Results from testing this approach under stringent cross-validation indicate that prediction accuracy of 72% is possible, while a support vector regression model is able to predict the number of subunits participating in the pore with 62% accuracy. Conclusion To our knowledge, this is the first tool capable of identifying pore-lining regions in proteins and we present the results of applying it to a data set of sequences with available crystal structures. Our method provides a way to characterise pores in transmembrane proteins and may even provide a starting point for discovering novel routes of therapeutic intervention in a number of important diseases. This software is freely available as source code from: http://bioinf.cs.ucl.ac.uk/downloads/memsat-svm/.

  4. Structural basis of typhod: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishna, A.; Saxena, A; Mok, H; Swaminathan, K

    2009-01-01

    The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

  5. Structural basis of typhoid: Salmonella typhi type IVb pilin (PiLS) and cystic fibrosis transmembrane conductance regulator interaction

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishna, A.M.; Saxena, A.; Mok, H. Y.-K.; Swaminathan, K.

    2009-11-01

    The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein ({Delta}PilS), which makes the pilus, was determined at 1.9 {angstrom} resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of {Delta}PilS and a target CFTR peptide, determined at 1.8 {angstrom}, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

  6. Structural Basis of Typhoid: Salmonella typhi Type IVb pilin (PilS) and Cystic Fibrosis Transmembrane Conductance Regulatory Interaction

    Energy Technology Data Exchange (ETDEWEB)

    Balakrishna, A.; Saxena, A; Mok, H; Swaminathan, K

    2009-01-01

    The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (PilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of PilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

  7. Structure of the Newcastle disease virus hemagglutinin-neuraminidase (HN) ectodomain reveals a four-helix bundle stalk

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Ping; Swanson, Kurt A.; Leser, George P.; Paterson, Reay G.; Lamb, Robert A.; Jardetzky, Theodore S. (Stanford-MED); (NWU)

    2014-10-02

    The paramyxovirus hemagglutinin-neuraminidase (HN) protein plays multiple roles in viral entry and egress, including binding to sialic acid receptors, activating the fusion (F) protein to activate membrane fusion and viral entry, and cleaving sialic acid from carbohydrate chains. HN is an oligomeric integral membrane protein consisting of an N-terminal transmembrane domain, a stalk region, and an enzymatically active neuraminidase (NA) domain. Structures of the HN NA domains have been solved previously; however, the structure of the stalk region has remained elusive. The stalk region contains specificity determinants for F interactions and activation, underlying the requirement for homotypic F and HN interactions in viral entry. Mutations of the Newcastle disease virus HN stalk region have been shown to affect both F activation and NA activities, but a structural basis for understanding these dual affects on HN functions has been lacking. Here, we report the structure of the Newcastle disease virus HN ectodomain, revealing dimers of NA domain dimers flanking the N-terminal stalk domain. The stalk forms a parallel tetrameric coiled-coil bundle (4HB) that allows classification of extensive mutational data, providing insight into the functional roles of the stalk region. Mutations that affect both F activation and NA activities map predominantly to the 4HB hydrophobic core, whereas mutations that affect only F-protein activation map primarily to the 4HB surface. Two of four NA domains interact with the 4HB stalk, and residues at this interface in both the stalk and NA domain have been implicated in HN function.

  8. Structural and Biochemical Analysis of DNA Helix Invasion by the Bacterial 8-Oxoguanine DNA Glycosylase MutM*

    Science.gov (United States)

    Sung, Rou-Jia; Zhang, Michael; Qi, Yan; Verdine, Gregory L.

    2013-01-01

    MutM is a bacterial DNA glycosylase that serves as the first line of defense against the highly mutagenic 8-oxoguanine (oxoG) lesion, catalyzing glycosidic bond cleavage of oxoG to initiate base excision DNA repair. Previous work has shown that MutM actively interrogates DNA for the presence of an intrahelical oxoG lesion. This interrogation process involves significant buckling and bending of the DNA to promote extrusion of oxoG from the duplex. Structural snapshots have revealed several different highly conserved residues that are prominently inserted into the duplex in the vicinity of the target oxoG before and after base extrusion has occurred. However, the roles of these helix-invading residues during the lesion recognition and base extrusion process remain unclear. In this study, we set out to probe the function of residues Phe114 and Met77 in oxoG recognition and repair. Here we report a detailed biochemical and structural characterization of MutM variants containing either a F114A or M77A mutation, both of which showed significant decreases in the efficiency of oxoG repair. These data reveal that Met77 plays an important role in stabilizing the lesion-extruded conformation of the DNA. Phe114, on the other hand, appears to destabilize the intrahelical state of the oxoG lesion, primarily by buckling the target base pair. We report the observation of a completely unexpected interaction state, in which the target base pair is ruptured but remains fully intrahelical; this structure vividly illustrates the disruptive influence of MutM on the target base pair. PMID:23404556

  9. Designability landscape reveals sequence features that define axial helix rotation in four-helical homo-oligomeric antiparallel coiled-coil structures.

    Science.gov (United States)

    Szczepaniak, Krzysztof; Lach, Grzegorz; Bujnicki, Janusz M; Dunin-Horkawicz, Stanislaw

    2014-11-01

    Coiled coils are widespread protein domains comprising α-helices wound around each other in a regular fashion. Owing to their regularity, coiled-coil structures can be fully described by parametric equations. This in turn makes them an excellent model for studying sequence-structure relationships in proteins. Here, we used computational design to identify sequence features that determine the degree of helix axial rotation in four-helical homo-oligomeric antiparallel coiled coils. We designed 135,000 artificial sequences for a repertoire of backbone models representing all theoretically possible axial rotation states. Analysis of the designed sequences revealed features that precisely define the rotation of the helices. Based on these features we implemented a bioinformatic tool, which given a coiled-coil sequence, predicts the rotation of the helices in its structure. Moreover, we showed that another structural parameter, helix axial shift, is coupled to helix axial rotation and that dependence between these two parameters narrows the number of possible axial rotation states. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. An Algorithm for Protein Helix Assignment Using Helix Geometry.

    Science.gov (United States)

    Cao, Chen; Xu, Shutan; Wang, Lincong

    2015-01-01

    Helices are one of the most common and were among the earliest recognized secondary structure elements in proteins. The assignment of helices in a protein underlies the analysis of its structure and function. Though the mathematical expression for a helical curve is simple, no previous assignment programs have used a genuine helical curve as a model for helix assignment. In this paper we present a two-step assignment algorithm. The first step searches for a series of bona fide helical curves each one best fits the coordinates of four successive backbone Cα atoms. The second step uses the best fit helical curves as input to make helix assignment. The application to the protein structures in the PDB (protein data bank) proves that the algorithm is able to assign accurately not only regular α-helix but also 310 and π helices as well as their left-handed versions. One salient feature of the algorithm is that the assigned helices are structurally more uniform than those by the previous programs. The structural uniformity should be useful for protein structure classification and prediction while the accurate assignment of a helix to a particular type underlies structure-function relationship in proteins.

  11. Triple-helix DNA structural studies using a Love wave acoustic biosensor.

    Science.gov (United States)

    Papadakis, George; Tsortos, Achilleas; Gizeli, Electra

    2009-12-15

    The development of sensors for detecting the conformation of surface-attached molecules is an emerging field with significance in the pharmaceutical industry and in drug design. In this work, triplex-forming oligos (TFOs), a separate class of non-natural DNA bending agents that can affect the mechanical properties of DNA through the formation of triple-helical structures of specific conformation and/or flexibility, are used as a model system in combination with an acoustic biosensor to determine molecular geometrical features. In practice, the degree of bending of a specific DNA target caused by a particular TFO was evaluated by measuring the ratio of acoustic energy change over phase change observed during the binding of pre-formed triplex DNA molecules to the device surface. The DNA bending angle derived via acoustic measurements is in excellent agreement with previously reported values using molecular biology techniques. The reported acoustic technique appears quite appealing for the biophysical study of DNA molecules providing rapid qualitative and quantitative information, at the same time holding promise to be developed as a high-throughput method for the evaluation of DNA conformational changes.

  12. Solution structure of the Arabidopsis thaliana telomeric repeat-binding protein DNA binding domain: a new fold with an additional C-terminal helix.

    Science.gov (United States)

    Sue, Shih-Che; Hsiao, Hsin-Hao; Chung, Ben C-P; Cheng, Ying-Hsien; Hsueh, Kuang-Lung; Chen, Chung Mong; Ho, Chia Hsing; Huang, Tai-Huang

    2006-02-10

    The double-stranded telomeric repeat-binding protein (TRP) AtTRP1 is isolated from Arabidopsis thaliana. Using gel retardation assays, we defined the C-terminal 97 amino acid residues, Gln464 to Val560 (AtTRP1(464-560)), as the minimal structured telomeric repeat-binding domain. This region contains a typical Myb DNA-binding motif and a C-terminal extension of 40 amino acid residues. The monomeric AtTRP1(464-560) binds to a 13-mer DNA duplex containing a single repeat of an A.thaliana telomeric DNA sequence (GGTTTAG) in a 1:1 complex, with a K(D) approximately 10(-6)-10(-7) M. Nuclear magnetic resonance (NMR) examination revealed that the solution structure of AtTRP1(464-560) is a novel four-helix tetrahedron rather than the three-helix bundle structure found in typical Myb motifs and other TRPs. Binding of the 13-mer DNA duplex to AtTRP1(464-560) induced significant chemical shift perturbations of protein amide resonances, which suggests that helix 3 (H3) and the flexible loop connecting H3 and H4 are essential for telomeric DNA sequence recognition. Furthermore, similar to that in hTRF1, the N-terminal arm likely contributes to or stabilizes DNA binding. Sequence comparisons suggested that the four-helix structure and the involvement of the loop residues in DNA binding may be features unique to plant TRPs.

  13. Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

    Energy Technology Data Exchange (ETDEWEB)

    Storrs, R.W.

    1992-08-01

    Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by [sup 31]P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 [Angstrom] of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an [alpha]-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

  14. Structural studies of polypeptides: Mechanism of immunoglobin catalysis and helix propagation in hybrid sequence, disulfide containing peptides

    Energy Technology Data Exchange (ETDEWEB)

    Storrs, Richard Wood [Univ. of California, Berkeley, CA (United States)

    1992-08-01

    Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by 31P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 Å of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an α-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.

  15. HMM_RA: An Improved Method for Alpha-Helical Transmembrane Protein Topology Prediction

    Directory of Open Access Journals (Sweden)

    Changhui Yan

    2008-01-01

    Full Text Available α-helical transmembrane (TM proteins play important and diverse functional roles in cells. The ability to predict the topology of these proteins is important for identifying functional sites and inferring function of membrane proteins. This paper presents a Hidden Markov Model (referred to as HMM_RA that can predict the topology of α-helical transmembrane proteins with improved performance. HMM_RA adopts the same structure as the HMMTOP method, which has five modules: inside loop, inside helix tail, membrane helix, outside helix tail and outside loop. Each module consists of one or multiple states. HMM_RA allows using reduced alphabets to encode protein sequences. Thus, each state of HMM_RA is associated with n emission probabilities, where n is the size of the reduced alphabet set. Direct comparisons using two standard data sets show that HMM_RA consistently outperforms HMMTOP and TMHMM in topology prediction. Specifically, on a high-quality data set of 83 proteins, HMM_RA outperforms HMMTOP by up to 7.6% in topology accuracy and 6.4% in α-helices location accuracy. On the same data set, HMM_RA outperforms TMHMM by up to 6.4% in topology accuracy and 2.9% in location accuracy. Comparison also shows that HMM_RA achieves comparable performance as Phobius, a recently published method.

  16. Effect of secondary structure on the potential of mean force for poly-L-lysine in the alpha-Helix and beta-sheet conformations

    Energy Technology Data Exchange (ETDEWEB)

    Grigsby, J.J.; Blanch, H.W.; Prausnitz, J.M.

    2001-10-30

    Because poly-L-lysine (PLL) can exist in the {alpha}-helix or {beta}-sheet conformation depending on solution preparation and solution conditions, PLL is a suitable candidate to probe the dependence of protein interactions on secondary structure. The osmotic second virial coefficient and weight-average molecular weight are reported from low-angle laser-light scattering measurements for PLL as a function of NaCl concentration, pH, and {alpha}-helix or {beta}-sheet content. Interactions between PLL molecules become more attractive as salt concentration increases due to screening of PLL charge by salt ions and at low salt concentration become more attractive as pH increases due to decreased net charge on PLL. The experimental results show that interactions are stronger for the {beta}-sheet conformation than for the {alpha}-helix conformation. A spherically-symmetric model for the potential of mean force is used to account for specific interactions not described by DLVO theory and to show how differences in secondary structure affect PLL interactions.

  17. Recombinant expression in E. coli of human FGFR2 with its transmembrane and extracellular domains

    Directory of Open Access Journals (Sweden)

    Adam Bajinting

    2017-06-01

    Full Text Available Fibroblast growth factor receptors (FGFRs are a family of receptor tyrosine kinases containing three domains: an extracellular receptor domain, a single transmembrane helix, and an intracellular tyrosine kinase domain. FGFRs are activated by fibroblast growth factors (FGFs as part of complex signal transduction cascades regulating angiogenesis, skeletal formation, cell differentiation, proliferation, cell survival, and cancer. We have developed the first recombinant expression system in E. coli to produce a construct of human FGFR2 containing its transmembrane and extracellular receptor domains. We demonstrate that the expressed construct is functional in binding heparin and dimerizing. Size exclusion chromatography demonstrates that the purified FGFR2 does not form a complex with FGF1 or adopts an inactive dimer conformation. Progress towards the successful recombinant production of intact FGFRs will facilitate further biochemical experiments and structure determination that will provide insight into how extracellular FGF binding activates intracellular kinase activity.

  18. Structure and function of transmembrane segment XII in osmosensor and osmoprotectant transporter ProP of Escherichia coli.

    Science.gov (United States)

    Liu, Feng; Culham, Doreen E; Vernikovska, Yaroslava I; Keates, Robert A B; Boggs, Joan M; Wood, Janet M

    2007-05-15

    Escherichia coli transporter ProP acts as both an osmosensor and an osmoregulator. As medium osmolality rises, ProP is activated and mediates H+-coupled uptake of osmolytes like proline. A homology model of ProP with 12-transmembrane (TM) helices and cytoplasmic termini was created, and the protein's topology was substantiated experimentally. Residues 468-497, at the end of the C-terminal domain and linked to TM XII, form an intermolecular, homodimeric alpha-helical coiled-coil that tunes the transporter's response to osmolality. We aim to further define the structure and function of ProP residues Q415-E440, predicted to include TM XII. Each residue was replaced with cysteine (Cys) in a histidine-tagged, Cys-less ProP variant (ProP*). Cys at positions 415-418 and 438-440 were most reactive with Oregon Green Maleimide (OGM), suggesting that residues 419 through 437 are in the membrane. Except for V429-I433, reactivity of those Cys varied with helical periodicity. Cys predicted to face the interior of ProP were more reactive than Cys predicted to face the lipid. The former may be exposed to hydrated polar residues in the protein interior, particularly on the periplasmic side. Intermolecular cross-links formed when ProP* variants with Cys at positions 419, 420, 422, and 439 were treated with DTME. Thus TM XII can participate, along its entire length, in the dimer interface of ProP. Cys substitution E440C rendered ProP* inactive. All other variants retained more than 30% of the proline uptake activity of ProP* at high osmolality. Most variants with Cys substitutions in the periplasmic half of TM XII activated at lower osmolalities than ProP*. Variants with Cys substitutions on one face of the cytoplasmic half of TM XII required a higher osmolality to activate. They included elements of a GXXXG motif that are predicted to form the interface of TM XII with TM VII. These studies define the position of ProP TM XII within the membrane, further support the predicted

  19. Spatial attributes of the four-helix bundle group of bacteriocins – The high-resolution structure of BacSp222 in solution

    KAUST Repository

    Nowakowski, Michał

    2017-11-01

    BacSp222 is a multifunctional bacteriocin produced by Staphylococcus pseudintermedius strain 222, an opportunistic pathogen of domestic animals. At micromolar concentrations, BacSp222 kills Gram-positive bacteria and is cytotoxic toward mammalian cells, while at nanomolar doses, it acts as an immunomodulatory factor, enhancing nitric oxide release in macrophage-like cell lines. The bacteriocin is a cationic, N-terminally formylated, 50-amino-acid-long linear peptide that is rich in tryptophan residues.In this study, the solution structure of BacSp222 was determined and compared to the currently known structures of similar bacteriocins. BacSp222 was isolated from a liquid culture medium in a uniformly 13C- and 15N-labeled form, and NMR data were collected. The structure was calculated based on NMR-derived constraints and consists of a rigid and tightly packed globular bundle of four alpha-helices separated by three short turns.Although the amino acid sequence of BacSp222 has no significant similarity to any known peptide or protein, a 3D structure similarity search indicates a close relation to other four-helix bundle-motif bacteriocins, such as aureocin A53, lacticin Q and enterocins 7A/7B. Assuming similar functions, biology, structure and physicochemical properties, we propose to distinguish the four-helix bundle bacteriocins as a new Type A in subclass IId of bacteriocins, containing linear, non-pediocin-like peptides.

  20. Three-dimensional structures of the mammalian multidrug resistance P-glycoprotein demonstrate major conformational changes in the transmembrane domains upon nucleotide binding.

    Science.gov (United States)

    Rosenberg, Mark F; Kamis, Alhaji Bukar; Callaghan, Richard; Higgins, Christopher F; Ford, Robert C

    2003-03-07

    P-glycoprotein is an ATP-binding cassette transporter that is associated with multidrug resistance and the failure of chemotherapy in human patients. We have previously shown, based on two-dimensional projection maps, that P-glycoprotein undergoes conformational changes upon binding of nucleotide to the intracellular nucleotide binding domains. Here we present the three-dimensional structures of P-glycoprotein in the presence and absence of nucleotide, at a resolution limit of approximately 2 nm, determined by electron crystallography of negatively stained crystals. The data reveal a major reorganization of the transmembrane domains throughout the entire depth of the membrane upon binding of nucleotide. In the absence of nucleotide, the two transmembrane domains form a single barrel 5-6 nm in diameter and about 5 nm deep with a central pore that is open to the extracellular surface and spans much of the membrane depth. Upon binding nucleotide, the transmembrane domains reorganize into three compact domains that are each 2-3 nm in diameter and 5-6 nm deep. This reorganization opens the central pore along its length in a manner that could allow access of hydrophobic drugs (transport substrates) directly from the lipid bilayer to the central pore of the transporter.

  1. Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix.

    Science.gov (United States)

    Smith, Nicola L; Taylor, Edward J; Lindsay, Anna-Marie; Charnock, Simon J; Turkenburg, Johan P; Dodson, Eleanor J; Davies, Gideon J; Black, Gary W

    2005-12-06

    Streptococcus pyogenes (group A Streptococcus) causes severe invasive infections including scarlet fever, pharyngitis (streptococcal sore throat), skin infections, necrotizing fasciitis (flesh-eating disease), septicemia, erysipelas, cellulitis, acute rheumatic fever, and toxic shock. The conversion from nonpathogenic to toxigenic strains of S. pyogenes is frequently mediated by bacteriophage infection. One of the key bacteriophage-encoded virulence factors is a putative "hyaluronidase," HylP1, a phage tail-fiber protein responsible for the digestion of the S. pyogenes hyaluronan capsule during phage infection. Here we demonstrate that HylP1 is a hyaluronate lyase. The 3D structure, at 1.8-angstroms resolution, reveals an unusual triple-stranded beta-helical structure and provides insight into the structural basis for phage tail assembly and the role of phage tail proteins in virulence. Unlike the triple-stranded beta-helix assemblies of the bacteriophage T4 injection machinery and the tailspike endosialidase of the Escherichia coli K1 bacteriophage K1F, HylP1 possesses three copies of the active center on the triple-helical fiber itself without the need for an accessory catalytic domain. The triple-stranded beta-helix is not simply a structural scaffold, as previously envisaged; it is harnessed to provide a 200-angstroms-long substrate-binding groove for the optimal reduction in hyaluronan viscosity to aid phage penetration of the capsule.

  2. Nucleic acid binding properties of a helix stabilising nucleoid protein from the thermoacidophilic archaeon Sulfolobus acidocaldarius that condenses DNA into compact structures.

    Science.gov (United States)

    Celestina, F; Suryanarayana, T

    1995-12-01

    Helix stabilising nucleoid protein (HSNP-C') from an acidothermophilic archaeon Sulfolobus acidocaldarius has been characterised with respect to interaction with nucleic acids by gel retardation assay, binding to nucleic acid columns, fluorescence titrations and electron microscopy. The protein exists in solution as very large multimeric aggregates as indicated by cross-linking studies. The protein binds strongly and co-operatively to double stranded DNA. Electron microscopy of the complexes of the protein with DNA shows compact structures suggesting that the protein condenses DNA.

  3. Helix-Hopes on Finite Hyperfields

    Directory of Open Access Journals (Sweden)

    Thomas Vougiouklis

    2016-12-01

    Full Text Available Hyperstructure theory can overcome restrictions which ordinary algebraic structures have. A hyperproduct on non-square ordinary matrices can be defined by using the so called helix-hyperoperations. We study the helix-hyperstructures on the representations using ordinary fields. The related theory can be faced by defining the hyperproduct on the set of non square matrices. The main tools of the Hyperstructure Theory are the fundamental relations which connect the largest class of hyperstructures, the Hv-structures, with the corresponding classical ones. We focus on finite dimensional helix-hyperstructures and on small Hv-fields, as well.

  4. Structure, dynamics, and elasticity of free 16S rRNA helix 44 studied by molecular dynamics simulations

    Czech Academy of Sciences Publication Activity Database

    Réblová, Kamila; Lankaš, F.; Rázga, Filip; Krasovská, Maryna V.; Koča, J.; Šponer, Jiří

    2006-01-01

    Roč. 82, č. 5 (2006), s. 504-520 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA203/05/0388; GA ČR(CZ) GA203/05/0009; GA ČR(CZ) GD204/03/H016; GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z50040507 Keywords : molecular dynamics * helix 44 * 16S rRNA Subject RIV: BO - Biophysics Impact factor: 2.480, year: 2006

  5. RT-173: Helix, 2017 Helix Technical Report

    Science.gov (United States)

    2018-01-16

    genders across Helix dataset...few exceptions. Each individual, again, has several characteristics, including gender , whether they are a systems engineer or a...Experience Less than 9 years Equal to or greater than 9 years Equal to or greater than 9 years Positions Title’s - 0 years titled as

  6. Probing and improving student's understanding of protein α-helix structure using targeted assessment and classroom interventions in collaboration with a faculty community of practice.

    Science.gov (United States)

    Loertscher, Jennifer; Villafañe, Sachel M; Lewis, Jennifer E; Minderhout, Vicky

    2014-01-01

    The increasing availability of concept inventories and other assessment tools in the molecular life sciences provides instructors with myriad avenues to probe student understanding. For example, although molecular visualization is central to the study of biochemistry, a growing body of evidence suggests that students have substantial limitations in their ability to recognize and interpret basic features of biological macromolecules. In this study, a pre/posttest administered to students at diverse institutions nationwide revealed a robust incorrect idea about the location of the amino acid side chains in the protein α-helix structure. Because this incorrect idea was present even after a semester of biochemistry instruction at a range of institutions, an intervention was necessary. A community of expert biochemistry instructors collaborated to design two active learning classroom activities that systematically examine α-helix structure and function. Several participating faculty used one or both of the activities in their classrooms and some improvement of student understanding of this concept was observed. This study provides a model of how a community of instructors can work together using assessment data to inform targeted changes in instruction with the goal of improving student understanding of fundamental concepts. Copyright © 2014 by The International Union of Biochemistry and Molecular Biology.

  7. Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor

    Science.gov (United States)

    Sarker, Muzaddid; de Antueno, Roberto; Langelaan, David N.; Parmar, Hiren B.; Shin, Kyungsoo; Rainey, Jan K.; Duncan, Roy

    2015-01-01

    Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS) in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST) protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS) but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation. PMID:26061049

  8. Reovirus FAST Proteins Drive Pore Formation and Syncytiogenesis Using a Novel Helix-Loop-Helix Fusion-Inducing Lipid Packing Sensor.

    Directory of Open Access Journals (Sweden)

    Jolene Read

    2015-06-01

    Full Text Available Pore formation is the most energy-demanding step during virus-induced membrane fusion, where high curvature of the fusion pore rim increases the spacing between lipid headgroups, exposing the hydrophobic interior of the membrane to water. How protein fusogens breach this thermodynamic barrier to pore formation is unclear. We identified a novel fusion-inducing lipid packing sensor (FLiPS in the cytosolic endodomain of the baboon reovirus p15 fusion-associated small transmembrane (FAST protein that is essential for pore formation during cell-cell fusion and syncytiogenesis. NMR spectroscopy and mutational studies indicate the dependence of this FLiPS on a hydrophobic helix-loop-helix structure. Biochemical and biophysical assays reveal the p15 FLiPS preferentially partitions into membranes with high positive curvature, and this partitioning is impeded by bis-ANS, a small molecule that inserts into hydrophobic defects in membranes. Most notably, the p15 FLiPS can be functionally replaced by heterologous amphipathic lipid packing sensors (ALPS but not by other membrane-interactive amphipathic helices. Furthermore, a previously unrecognized amphipathic helix in the cytosolic domain of the reptilian reovirus p14 FAST protein can functionally replace the p15 FLiPS, and is itself replaceable by a heterologous ALPS motif. Anchored near the cytoplasmic leaflet by the FAST protein transmembrane domain, the FLiPS is perfectly positioned to insert into hydrophobic defects that begin to appear in the highly curved rim of nascent fusion pores, thereby lowering the energy barrier to stable pore formation.

  9. Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function.

    Science.gov (United States)

    Therien, J P Daniel; Baenziger, John E

    2017-03-27

    Although transmembrane helix-helix interactions must be strong enough to drive folding, they must still permit the inter-helix movements associated with conformational change. Interactions between the outermost M4 and adjacent M1 and M3 α-helices of pentameric ligand-gated ion channels have been implicated in folding and function. Here, we evaluate the role of different physical interactions at this interface in the function of two prokaryotic homologs, GLIC and ELIC. Strikingly, disruption of most interactions in GLIC lead to either a reduction or a complete loss of expression and/or function, while analogous disruptions in ELIC often lead to gains in function. Structural comparisons suggest that GLIC and ELIC represent distinct transmembrane domain archetypes. One archetype, exemplified by GLIC, the glycine and GABA receptors and the glutamate activated chloride channel, has extensive aromatic contacts that govern M4-M1/M3 interactions and that are essential for expression and function. The other archetype, exemplified by ELIC and both the nicotinic acetylcholine and serotonin receptors, has relatively few aromatic contacts that are detrimental to function. These archetypes likely have evolved different mechanisms to balance the need for strong M4 "binding" to M1/M3 to promote folding/expression, and the need for weaker interactions that allow for greater conformational flexibility.

  10. Protein Secondary Structures (α-helix and β-sheet) at a Cellular Level and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    International Nuclear Information System (INIS)

    Yu, P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the α-helix and β-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of β-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution (∼10 μm). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of α-helixes and β-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of α-helixes (from 47.1% to 36.1%: S-FTIR absorption intensity), increased the

  11. Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

    Energy Technology Data Exchange (ETDEWEB)

    Yu,P.

    2007-01-01

    Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the {alpha}-helix and {beta}-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of {beta}-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present study were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution ({approx}10 {mu}m). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of {alpha}-helixes and {beta}-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of {alpha}-helixes (from 47.1% to 36.1%: S

  12. Structure of the unique SEFIR domain from human interleukin 17 receptor A reveals a composite ligand-binding site containing a conserved α-helix for Act1 binding and IL-17 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bing [Oklahoma State University, Stillwater, OK 74078 (United States); Liu, Caini; Qian, Wen [Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195 (United States); Han, Yue [Oklahoma State University, Stillwater, OK 74078 (United States); Li, Xiaoxia, E-mail: lix@ccf.org [Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195 (United States); Deng, Junpeng, E-mail: lix@ccf.org [Oklahoma State University, Stillwater, OK 74078 (United States)

    2014-05-01

    Crystal structure of the SEFIR domain from human IL-17 receptor A provides new insights into IL-17 signaling. Interleukin 17 (IL-17) cytokines play a crucial role in mediating inflammatory and autoimmune diseases. A unique intracellular signaling domain termed SEFIR is found within all IL-17 receptors (IL-17Rs) as well as the key adaptor protein Act1. SEFIR-mediated protein–protein interaction is a crucial step in IL-17 cytokine signaling. Here, the 2.3 Å resolution crystal structure of the SEFIR domain of IL-17RA, the most commonly shared receptor for IL-17 cytokine signaling, is reported. The structure includes the complete SEFIR domain and an additional α-helical C-terminal extension, which pack tightly together to form a compact unit. Structural comparison between the SEFIR domains of IL-17RA and IL-17RB reveals substantial differences in protein topology and folding. The uniquely long insertion between strand βC and helix αC in IL-17RA SEFIR is mostly well ordered, displaying a helix (αCC′{sub ins}) and a flexible loop (CC′). The DD′ loop in the IL-17RA SEFIR structure is much shorter; it rotates nearly 90° with respect to the counterpart in the IL-17RB SEFIR structure and shifts about 12 Å to accommodate the αCC′{sub ins} helix without forming any knots. Helix αC was identified as critical for its interaction with Act1 and IL-17-stimulated gene expression. The data suggest that the heterotypic SEFIR–SEFIR association via helix αC is a conserved and signature mechanism specific for IL-17 signaling. The structure also suggests that the downstream motif of IL-17RA SEFIR together with helix αC could provide a composite ligand-binding surface for recruiting Act1 during IL-17 signaling.

  13. SU-E-T-241: Monte Carlo Simulation Study About the Prediction of Proton-Induced DNA Strand Breakage On the Double Helix Structure

    Energy Technology Data Exchange (ETDEWEB)

    Shin, J; Park, S; Jeong, J; Jeong, C [National Cancer Center, Goyang, Gyeonggi-do (Korea, Republic of); Lim, Y; Lee, S [National Cancer Center in Korea, Goyang, Gyeonggi-do (Korea, Republic of); SHIN, D [National Cancer Center, Goyangsi, Gyeonggi-do (Korea, Republic of); Incerti, S [Universite Bordeaux 1, CNRS.IN2P3, Centres d’Etudes Nucleaires de Bordeau, Gradignan, Gradignan (France)

    2014-06-01

    Purpose: In particle therapy and radiobiology, the investigation of mechanisms leading to the death of target cancer cells induced by ionising radiation is an active field of research. Recently, several studies based on Monte Carlo simulation codes have been initiated in order to simulate physical interactions of ionising particles at cellular scale and in DNA. Geant4-DNA is the one of them; it is an extension of the general purpose Geant4 Monte Carlo simulation toolkit for the simulation of physical interactions at sub-micrometre scale. In this study, we present Geant4-DNA Monte Carlo simulations for the prediction of DNA strand breakage using a geometrical modelling of DNA structure. Methods: For the simulation of DNA strand breakage, we developed a specific DNA geometrical structure. This structure consists of DNA components, such as the deoxynucleotide pairs, the DNA double helix, the nucleosomes and the chromatin fibre. Each component is made of water because the cross sections models currently available in Geant4-DNA for protons apply to liquid water only. Also, at the macroscopic-scale, protons were generated with various energies available for proton therapy at the National Cancer Center, obtained using validated proton beam simulations developed in previous studies. These multi-scale simulations were combined for the validation of Geant4-DNA in radiobiology. Results: In the double helix structure, the deposited energy in a strand allowed to determine direct DNA damage from physical interaction. In other words, the amount of dose and frequency of damage in microscopic geometries was related to direct radiobiological effect. Conclusion: In this report, we calculated the frequency of DNA strand breakage using Geant4- DNA physics processes for liquid water. This study is now on-going in order to develop geometries which use realistic DNA material, instead of liquid water. This will be tested as soon as cross sections for DNA material become available in Geant4

  14. The crystal structure of GXGD membrane protease FlaK

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Jian; Xue, Yi; Lee, Sangwon; Ha, Ya (Yale-MED)

    2011-09-20

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  15. The Crystal Structure of GXGD Membrane Protease FlaK

    Energy Technology Data Exchange (ETDEWEB)

    J Hu; Y Xue; S Lee; Y Ha

    2011-12-31

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  16. Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala.

    OpenAIRE

    Yang, W. Z.; Ko, T. P.; Corselli, L.; Johnson, R. C.; Yuan, H. S.

    1998-01-01

    The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves...

  17. PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation.

    Science.gov (United States)

    Pedersen, Lise R L; Nielsen, Søren B; Hansted, Jon G; Petersen, Torben E; Otzen, Daniel E; Sørensen, Esben S

    2012-01-01

    The milk protein proteose peptone component 3 (PP3), also called lactophorin, is a small phosphoglycoprotein that is expressed exclusively in lactating mammary tissue. The C-terminal part of the protein contains an amphipathic helix, which, upon proteolytic liberation, shows antibacterial activity. Previous studies indicate that PP3 forms multimeric structures and inhibits lipolysis in milk. PP3 is the principal component of the proteose peptone fraction of milk. This fraction is obtained by heating and acidifying skimmed milk, and in the dairy industry milk products are also typically exposed to treatments such as pasteurization, which potentially could result in irreversible denaturation and inactivation of bioactive components. We show here, by the use of CD, that PP3 undergoes reversible thermal denaturation and that the α-helical structure of PP3 remains stable even at gastric pH levels. This suggests that the secondary structure survives treatment during the purification and possibly some of the industrial processing of milk. Finally, asymmetric flow field-flow fractionation and multi-angle light scattering reveal that PP3 forms a rather stable tetrameric complex, which dissociates and unfolds in guanidinium chloride. The cooperative unfolding of PP3 was completely removed by the surfactant n-dodecyl-β-d-maltoside and by oleic acid. We interpret this to mean that the PP3 monomers associate through hydrophobic interactions via the hydrophobic surface of the amphipathic helix. These observations suggest that PP3 tetramers act as reservoirs of PP3 molecules, which in the monomeric state may stabilize the milk fat globule. © 2011 The Authors Journal compilation © 2011 FEBS.

  18. Helix mimetics: Recent developments.

    Science.gov (United States)

    Wilson, Andrew J

    2015-10-01

    The development of protein-protein interaction (PPIs) inhibitors represents a challenging goal in chemical biology and drug discovery. PPIs are problematic targets because they involve large surfaces with less well defined features and recognition motifs that are less amenable to conventional experimental and computational ligand discovery methodologies. α-Helix mediated PPIs represent a sub group with a clearly defined interface and thus may be more amenable to the development of generic ligand discovery methods. Indeed, this is borne out in numerous studies using peptides covalently constrained into a helical conformation resulting in improvement of myriad biophysical and cellular properties. It is however desirable to have small molecule alternatives: a helix mimetic (proteomimetic) is a generic small molecule scaffold that projects functional groups in a similar spatial orientation so as to mimic the presentation of key amino acid side chains from the helix that mediates the PPI. The first true example of a helix mimetic was described over a decade ago however this approach has not yet been elaborated to the extent that it receives similar levels of attention to constrained peptides. This review explores recent significant developments in the area of small molecule α-helix mimetics and provides a critical overview of success stories, potential limitations of the approach, and areas for future development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Designing cooperatively folded abiotic uni- and multimolecular helix bundles

    Science.gov (United States)

    de, Soumen; Chi, Bo; Granier, Thierry; Qi, Ting; Maurizot, Victor; Huc, Ivan

    2018-01-01

    Abiotic foldamers, that is foldamers that have backbones chemically remote from peptidic and nucleotidic skeletons, may give access to shapes and functions different to those of peptides and nucleotides. However, design methodologies towards abiotic tertiary and quaternary structures are yet to be developed. Here we report rationally designed interactional patterns to guide the folding and assembly of abiotic helix bundles. Computational design facilitated the introduction of hydrogen-bonding functionalities at defined locations on the aromatic amide backbones that promote cooperative folding into helix-turn-helix motifs in organic solvents. The hydrogen-bond-directed aggregation of helices not linked by a turn unit produced several thermodynamically and kinetically stable homochiral dimeric and trimeric bundles with structures that are distinct from the designed helix-turn-helix. Relative helix orientation within the bundles may be changed from parallel to tilted on subtle solvent variations. Altogether, these results prefigure the richness and uniqueness of abiotic tertiary structure behaviour.

  20. Transmembrane protein topology prediction using support vector machines

    Directory of Open Access Journals (Sweden)

    Nugent Timothy

    2009-05-01

    Full Text Available Abstract Background Alpha-helical transmembrane (TM proteins are involved in a wide range of important biological processes such as cell signaling, transport of membrane-impermeable molecules, cell-cell communication, cell recognition and cell adhesion. Many are also prime drug targets, and it has been estimated that more than half of all drugs currently on the market target membrane proteins. However, due to the experimental difficulties involved in obtaining high quality crystals, this class of protein is severely under-represented in structural databases. In the absence of structural data, sequence-based prediction methods allow TM protein topology to be investigated. Results We present a support vector machine-based (SVM TM protein topology predictor that integrates both signal peptide and re-entrant helix prediction, benchmarked with full cross-validation on a novel data set of 131 sequences with known crystal structures. The method achieves topology prediction accuracy of 89%, while signal peptides and re-entrant helices are predicted with 93% and 44% accuracy respectively. An additional SVM trained to discriminate between globular and TM proteins detected zero false positives, with a low false negative rate of 0.4%. We present the results of applying these tools to a number of complete genomes. Source code, data sets and a web server are freely available from http://bioinf.cs.ucl.ac.uk/psipred/. Conclusion The high accuracy of TM topology prediction which includes detection of both signal peptides and re-entrant helices, combined with the ability to effectively discriminate between TM and globular proteins, make this method ideally suited to whole genome annotation of alpha-helical transmembrane proteins.

  1. A monodisperse transmembrane α-helical peptide barrel

    Science.gov (United States)

    Mahendran, Kozhinjampara R.; Niitsu, Ai; Kong, Lingbing; Thomson, Andrew R.; Sessions, Richard B.; Woolfson, Derek N.; Bayley, Hagan

    2017-05-01

    The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid α-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such α-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing.

  2. Structural properties of a peptide derived from H+-V-ATPase subunit a

    NARCIS (Netherlands)

    Vermeer, L.S.; Reat, V.; Hemminga, M.A.; Milon, A.

    2009-01-01

    The 3D structure of a peptide derived from the putative transmembrane segment 7 (TM7) of subunit a from H+-V-ATPase from Saccharomyces cerevisiae has been determined by solution state NMR in SDS. A stable helix is formed from L736 up to and including Q745, the lumenal half of the putative TM7. The

  3. Computational Approaches for Revealing the Structure of Membrane Transporters: Case Study on Bilitranslocase

    Directory of Open Access Journals (Sweden)

    Katja Venko

    2017-01-01

    Full Text Available The structural and functional details of transmembrane proteins are vastly underexplored, mostly due to experimental difficulties regarding their solubility and stability. Currently, the majority of transmembrane protein structures are still unknown and this present a huge experimental and computational challenge. Nowadays, thanks to X-ray crystallography or NMR spectroscopy over 3000 structures of membrane proteins have been solved, among them only a few hundred unique ones. Due to the vast biological and pharmaceutical interest in the elucidation of the structure and the functional mechanisms of transmembrane proteins, several computational methods have been developed to overcome the experimental gap. If combined with experimental data the computational information enables rapid, low cost and successful predictions of the molecular structure of unsolved proteins. The reliability of the predictions depends on the availability and accuracy of experimental data associated with structural information. In this review, the following methods are proposed for in silico structure elucidation: sequence-dependent predictions of transmembrane regions, predictions of transmembrane helix–helix interactions, helix arrangements in membrane models, and testing their stability with molecular dynamics simulations. We also demonstrate the usage of the computational methods listed above by proposing a model for the molecular structure of the transmembrane protein bilitranslocase. Bilitranslocase is bilirubin membrane transporter, which shares similar tissue distribution and functional properties with some of the members of the Organic Anion Transporter family and is the only member classified in the Bilirubin Transporter Family. Regarding its unique properties, bilitranslocase is a potentially interesting drug target.

  4. Structural details (kinks and non-alpha conformations) in transmembrane helices are intrahelically determined and can be predicted by sequence pattern descriptors.

    Science.gov (United States)

    Rigoutsos, Isidore; Riek, Peter; Graham, Robert M; Novotny, Jiri

    2003-08-01

    One of the promising methods of protein structure prediction involves the use of amino acid sequence-derived patterns. Here we report on the creation of non-degenerate motif descriptors derived through data mining of training sets of residues taken from the transmembrane-spanning segments of polytopic proteins. These residues correspond to short regions in which there is a deviation from the regular alpha-helical character (i.e. pi-helices, 3(10)-helices and kinks). A 'search engine' derived from these motif descriptors correctly identifies, and discriminates amongst instances of the above 'non-canonical' helical motifs contained in the SwissProt/TrEMBL database of protein primary structures. Our results suggest that deviations from alpha-helicity are encoded locally in sequence patterns only about 7-9 residues long and can be determined in silico directly from the amino acid sequence. Delineation of such variations in helical habit is critical to understanding the complex structure-function relationships of polytopic proteins and for drug discovery. The success of our current methodology foretells development of similar prediction tools capable of identifying other structural motifs from sequence alone. The method described here has been implemented and is available on the World Wide Web at http://cbcsrv.watson.ibm.com/Ttkw.html.

  5. Comparing an Atomic Model or Structure to a Corresponding Cryo-electron Microscopy Image at the Central Axis of a Helix

    Science.gov (United States)

    Zeil, Stephanie; Kovacs, Julio; Wriggers, Willy

    2017-01-01

    Abstract Three-dimensional density maps of biological specimens from cryo-electron microscopy (cryo-EM) can be interpreted in the form of atomic models that are modeled into the density, or they can be compared to known atomic structures. When the central axis of a helix is detectable in a cryo-EM density map, it is possible to quantify the agreement between this central axis and a central axis calculated from the atomic model or structure. We propose a novel arc-length association method to compare the two axes reliably. This method was applied to 79 helices in simulated density maps and six case studies using cryo-EM maps at 6.4–7.7 Å resolution. The arc-length association method is then compared to three existing measures that evaluate the separation of two helical axes: a two-way distance between point sets, the length difference between two axes, and the individual amino acid detection accuracy. The results show that our proposed method sensitively distinguishes lateral and longitudinal discrepancies between the two axes, which makes the method particularly suitable for the systematic investigation of cryo-EM map–model pairs. PMID:27936925

  6. Comparing an Atomic Model or Structure to a Corresponding Cryo-electron Microscopy Image at the Central Axis of a Helix.

    Science.gov (United States)

    Zeil, Stephanie; Kovacs, Julio; Wriggers, Willy; He, Jing

    2017-01-01

    Three-dimensional density maps of biological specimens from cryo-electron microscopy (cryo-EM) can be interpreted in the form of atomic models that are modeled into the density, or they can be compared to known atomic structures. When the central axis of a helix is detectable in a cryo-EM density map, it is possible to quantify the agreement between this central axis and a central axis calculated from the atomic model or structure. We propose a novel arc-length association method to compare the two axes reliably. This method was applied to 79 helices in simulated density maps and six case studies using cryo-EM maps at 6.4-7.7 Å resolution. The arc-length association method is then compared to three existing measures that evaluate the separation of two helical axes: a two-way distance between point sets, the length difference between two axes, and the individual amino acid detection accuracy. The results show that our proposed method sensitively distinguishes lateral and longitudinal discrepancies between the two axes, which makes the method particularly suitable for the systematic investigation of cryo-EM map-model pairs.

  7. Structural details (kinks and non-α conformations) in transmembrane helices are intrahelically determined and can be predicted by sequence pattern descriptors

    Science.gov (United States)

    Rigoutsos, Isidore; Riek, Peter; Graham, Robert M.; Novotny, Jiri

    2003-01-01

    One of the promising methods of protein structure prediction involves the use of amino acid sequence-derived patterns. Here we report on the creation of non-degenerate motif descriptors derived through data mining of training sets of residues taken from the transmembrane-spanning segments of polytopic proteins. These residues correspond to short regions in which there is a deviation from the regular α-helical character (i.e. π-helices, 310-helices and kinks). A ‘search engine’ derived from these motif descriptors correctly identifies, and discriminates amongst instances of the above ‘non-canonical’ helical motifs contained in the SwissProt/TrEMBL database of protein primary structures. Our results suggest that deviations from α-helicity are encoded locally in sequence patterns only about 7–9 residues long and can be determined in silico directly from the amino acid sequence. Delineation of such variations in helical habit is critical to understanding the complex structure–function relationships of polytopic proteins and for drug discovery. The success of our current methodology foretells development of similar prediction tools capable of identifying other structural motifs from sequence alone. The method described here has been implemented and is available on the World Wide Web at http://cbcsrv.watson.ibm.com/Ttkw.html. PMID:12888523

  8. Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

    Energy Technology Data Exchange (ETDEWEB)

    Bahl, C.; Morisseau, C; Bomberger, J; Stanton, B; Hammock, B; O& apos; Toole, G; Madden, D

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other {alpha}/{beta} hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-{angstrom} resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across the family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of {alpha}/{beta} hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.

  9. Lasing thresholds of helical photonic structures with different positions of a single light-amplifying helix turn

    Energy Technology Data Exchange (ETDEWEB)

    Blinov, L M; Palto, S P [A.V. Shubnikov Institute of Crystallography, Russian Academy of Sciences, Moscow, Russian Federaion (Russian Federation)

    2013-09-30

    Numerical simulation is used to assess the lasing threshold of helical structures of cholesteric liquid crystals (CLCs) in which only one turn amplifies light. This turn is located either in the centre of symmetric structures of various sizes or in an arbitrary place in asymmetric structures of preset size. In all cases, we find singularities in light amplification by a one-dimensional CLC structure for the most important band-edge modes (m1, m2 and m3) and plot the threshold gain coefficient k{sub th} against the position of the amplifying turn. For the symmetric structures, the lasing threshold of the m1 mode is shown to vary linearly with the inverse of the square of the cavity length. Moreover, modes with a lower density of photonic states (DOS) in the cavity may have a lower lasing threshold. This can be accounted for by the dependence of the density of photonic states on the position of the amplifying turn and, accordingly, by the nonuniform electromagnetic field intensity distribution along the cavity for different modes. In the asymmetric structures, the same field energy distribution is responsible for a correlation between k{sub th} and DOS curves. (lasers)

  10. The Role of Conformational Entropy in the Determination of Structural-Kinetic Relationships for Helix-Coil Transitions

    Directory of Open Access Journals (Sweden)

    Joseph F. Rudzinski

    2018-02-01

    Full Text Available Coarse-grained molecular simulation models can provide significant insight into the complex behavior of protein systems, but suffer from an inherently distorted description of dynamical properties. We recently demonstrated that, for a heptapeptide of alanine residues, the structural and kinetic properties of a simulation model are linked in a rather simple way, given a certain level of physics present in the model. In this work, we extend these findings to a longer peptide, for which the representation of configuration space in terms of a full enumeration of sequences of helical/coil states along the peptide backbone is impractical. We verify the structural-kinetic relationships by scanning the parameter space of a simple native-biased model and then employ a distinct transferable model to validate and generalize the conclusions. Our results further demonstrate the validity of the previous findings, while clarifying the role of conformational entropy in the determination of the structural-kinetic relationships. More specifically, while the global, long timescale kinetic properties of a particular class of models with varying energetic parameters but approximately fixed conformational entropy are determined by the overarching structural features of the ensemble, a shift in these kinetic observables occurs for models with a distinct representation of steric interactions. At the same time, the relationship between structure and more local, faster kinetic properties is not affected by varying the conformational entropy of the model.

  11. Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

    Science.gov (United States)

    Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.

    2011-01-01

    The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452

  12. Noncanonical structures and their thermodynamics of DNA and RNA under molecular crowding: beyond the Watson-Crick double helix.

    Science.gov (United States)

    Sugimoto, Naoki

    2014-01-01

    How does molecular crowding affect the stability of nucleic acid structures inside cells? Water is the major solvent component in living cells, and the properties of water in the highly crowded media inside cells differ from that in buffered solution. As it is difficult to measure the thermodynamic behavior of nucleic acids in cells directly and quantitatively, we recently developed a cell-mimicking system using cosolutes as crowding reagents. The influences of molecular crowding on the structures and thermodynamics of various nucleic acid sequences have been reported. In this chapter, we discuss how the structures and thermodynamic properties of nucleic acids differ under various conditions such as highly crowded environments, compartment environments, and in the presence of ionic liquids, and the major determinants of the crowding effects on nucleic acids are discussed. The effects of molecular crowding on the activities of ribozymes and riboswitches on noncanonical structures of DNA- and RNA-like quadruplexes that play important roles in transcription and translation are also described. © 2014 Elsevier Inc. All rights reserved.

  13. Tryptophan scanning mutagenesis reveals distortions in the helical structure of the δM4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.

    Science.gov (United States)

    Caballero-Rivera, Daniel; Cruz-Nieves, Omar A; Oyola-Cintrón, Jessica; Torres-Nunez, David A; Otero-Cruz, Jose D; Lasalde-Dominicci, José A

    2012-01-01

    The lipid-protein interface is an important domain of the nicotinic acetylcholine receptor (nAChR) that has recently garnered increased relevance. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, there is still a need to gain insight into the mechanism by which lipid-protein interactions regulate the function and conformational transitions of the nAChR. In this study, we extended the tryptophan scanning mutagenesis (TrpScanM) approach to dissect secondary structure and monitor the conformational changes experienced by the δM4 transmembrane domain (TMD) of the Torpedo californica nAChR, and to identify which positions on this domain are potentially linked to the regulation of ion channel kinetics. The difference in oscillation patterns between the closed- and open-channel states suggests a substantial conformational change along this domain as a consequence of channel activation. Furthermore, TrpScanM revealed distortions along the helical structure of this TMD that are not present on current models of the nAChR. Our results show that a Thr-Pro motif at positions 462-463 markedly bends the helical structure of the TMD, consistent with the recent crystallographic structure of the GluCl Cys-loop receptor which reveals a highly bent TMD4 in each subunit. This Thr-Pro motif acts as a molecular hinge that delineates two gating blocks in the δM4 TMD. These results suggest a model in which a hinge-bending motion that tilts the helical structure is combined with a spring-like motion during transition between the closed- and open-channel states of the δM4 TMD.

  14. Structure of Leishmania donovani coronin coiled coil domain reveals an antiparallel 4 helix bundle with inherent asymmetry.

    Science.gov (United States)

    Nayak, Ashok Ranjan; Karade, Sharanbasappa Shrimant; Srivastava, Vijay Kumar; Rana, Ajay Kumar; Gupta, C M; Sahasrabuddhe, Amogh A; Pratap, J Venkatesh

    2016-07-01

    Coiled coils are ubiquitous structural motifs that serve as a platform for protein-protein interactions and play a central role in myriad physiological processes. Though the formation of a coiled coil requires only the presence of suitably spaced hydrophobic residues, sequence specificities have also been associated with specific oligomeric states. RhXXhE is one such sequence motif, associated with parallel trimers, found in coronins and other proteins. Coronin, present in all eukaryotes, is an actin-associated protein involved in regulating actin turnover. Most eukaryotic coronins possess the RhXXhE trimerization motif. However, a unique feature of parasitic kinetoplastid coronin is that the positions of R and E are swapped within their coiled coil domain, but were still expected to form trimers. To understand the role of swapped motif in oligomeric specificity, we determined the X-ray crystal structure of Leishmania donovani coronin coiled coil domain (LdCoroCC) at 2.2Å, which surprisingly, reveals an anti-parallel tetramer assembly. Small angle X-ray scattering studies and chemical crosslinking confirm the tetramer in solution and is consistent with the oligomerization observed in the full length protein. Structural analyses reveal that LdCoroCC possesses an inherent asymmetry, in that one of the helices of the bundle is axially shifted with respect to the other three. The analysis also identifies steric reasons that cause this asymmetry. The bundle adapts an extended a-d-e core packing, the e residue being polar (with an exception) which results in a thermostable bundle with polar and apolar interfaces, unlike the existing a-d-e core antiparallel homotetramers with apolar core. Functional implications of the anti-parallel association in kinetoplastids are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Improving succinylation prediction accuracy by incorporating the secondary structure via helix, strand and coil, and evolutionary information from profile bigrams

    Science.gov (United States)

    Lal, Sunil Pranit; Taherzadeh, Ghazaleh; Sattar, Abdul; Tsunoda, Tatsuhiko; Sharma, Alok

    2018-01-01

    Post-translational modification refers to the biological mechanism involved in the enzymatic modification of proteins after being translated in the ribosome. This mechanism comprises a wide range of structural modifications, which bring dramatic variations to the biological function of proteins. One of the recently discovered modifications is succinylation. Although succinylation can be detected through mass spectrometry, its current experimental detection turns out to be a timely process unable to meet the exponential growth of sequenced proteins. Therefore, the implementation of fast and accurate computational methods has emerged as a feasible solution. This paper proposes a novel classification approach, which effectively incorporates the secondary structure and evolutionary information of proteins through profile bigrams for succinylation prediction. The proposed predictor, abbreviated as SSEvol-Suc, made use of the above features for training an AdaBoost classifier and consequently predicting succinylated lysine residues. When SSEvol-Suc was compared with four benchmark predictors, it outperformed them in metrics such as sensitivity (0.909), accuracy (0.875) and Matthews correlation coefficient (0.75). PMID:29432431

  16. Accurate computational design of multipass transmembrane proteins.

    Science.gov (United States)

    Lu, Peilong; Min, Duyoung; DiMaio, Frank; Wei, Kathy Y; Vahey, Michael D; Boyken, Scott E; Chen, Zibo; Fallas, Jorge A; Ueda, George; Sheffler, William; Mulligan, Vikram Khipple; Xu, Wenqing; Bowie, James U; Baker, David

    2018-03-02

    The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  17. Membrane topology of the sodium ion-dependent citrate carrier of Klebsiella pneumoniae - Evidence for a new structural class of secondary transporters

    NARCIS (Netherlands)

    Geest, Marleen van; Lolkema, Juke S.

    1996-01-01

    The predicted secondary structure model of the sodium ion-dependent citrate carrier of Klebsiella pneumoniae (CitS) presents the la-transmembrane helix motif observed for many secondary transporters, Biochemical evidence presented in this paper is not consistent with this model. N-terminal and

  18. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer (SG); (Columbia); (JHU)

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  19. On the helix equation

    Directory of Open Access Journals (Sweden)

    Taouil Hajer

    2012-08-01

    Full Text Available This paper is devoted to the helices processes, i.e. the solutions H : ℝ × Ω → ℝd, (t, ω ↦ H(t, ω of the helix equation egin{eqnarray} H(0,o=0; quad H(s+t,o= H(s,Phi(t,o +H(t,oonumber end{eqnarray} H ( 0 ,ω = 0 ;   H ( s + t,ω = H ( s, Φ ( t,ω + H ( t,ω where Φ : ℝ × Ω → Ω, (t, ω ↦ Φ(t, ω is a dynamical system on a measurable space (Ω, ℱ. More precisely, we investigate dominated solutions and non differentiable solutions of the helix equation. For the last case, the Wiener helix plays a fundamental role. Moreover, some relations with the cocycle equation defined by Φ, are investigated. Ce papier est consacré aux hélices, c’est-à-dire les solutions H : ℝ × Ω → ℝd, (t, ω ↦ H(t, ω de l’équation fonctionnelle egin{eqnarray} H(0,o=0; quad H(s+t,o= H(s,Phi(t,o +H(t,o onumber end{eqnarray} H ( 0 ,ω = 0 ;   H ( s + t,ω = H ( s, Φ ( t,ω + H ( t,ω où Φ : ℝ × Ω → Ω, (t, ω ↦ Φ(t, ω est un système dynamique défini sur un espace mesurable (Ω, ℱ. Plus présisément, nous déterminons d’abord les hélices dominées puis nous caractérisons les hélices non différentiables. Dans ce dernier cas, l’hélice de Wiener joue un rôle important. Nous précisons aussi quelques relations des hélices avec les cocycles définis par Φ.

  20. Transmembrane Signaling Proteoglycans

    DEFF Research Database (Denmark)

    Couchman, John R

    2010-01-01

    and their glycosaminoglycan chains is matched by diverse functions. However, all assume roles as coreceptors, often working alongside high-affinity growth factor receptors or adhesion receptors such as integrins. Other common themes are an ability to signal through their cytoplasmic domains, often to the actin cytoskeleton......, and linkage to PDZ protein networks. Many transmembrane proteoglycans associate on the cell surface with metzincin proteases and can be shed by them. Work with model systems in vivo and in vitro reveal roles in growth, adhesion, migration, and metabolism. Furthermore, a wide range of phenotypes for the core...

  1. Controlling chirality with helix inversion in cholesteric liquid crystals

    NARCIS (Netherlands)

    Katsonis, Nathalie Hélène; Lacaze, E.; Ferrarini, A.

    2012-01-01

    The helical organization of cholesteric liquid crystals is omnipresent in living matter. Achieving control over the structure of the cholesteric helix consequently holds great potential for developing stimuli-responsive materials matching the level of sophistication of biological systems. In

  2. Structure of the human CD97 gene: exon shuffling has generated a new type of seven-span transmembrane molecule related to the secretin receptor superfamily

    NARCIS (Netherlands)

    Hamann, J. [=Jörg; Hartmann, E.; van Lier, R. A.

    1996-01-01

    Recent cDNA cloning of EMR1 and CD97 suggests the existence of a new group of seven-span transmembrane (7-TM) molecules, likely encoded by a gene cluster on the short arm of chromosome 19. The membrane-spanning region of both molecules is homologous to the secretin receptor (SecR) superfamily, a

  3. SuperBiHelix method for predicting the pleiotropic ensemble of G-protein-coupled receptor conformations.

    Science.gov (United States)

    Bray, Jenelle K; Abrol, Ravinder; Goddard, William A; Trzaskowski, Bartosz; Scott, Caitlin E

    2014-01-07

    There is overwhelming evidence that G-protein-coupled receptors (GPCRs) exhibit several distinct low-energy conformations, each of which might favor binding to different ligands and/or lead to different downstream functions. Understanding the function of such proteins requires knowledge of the ensemble of low-energy configurations that might play a role in this pleiotropic functionality. We earlier reported the BiHelix method for efficiently sampling the (12)(7) = 35 million conformations resulting from 30° rotations about the axis (η) of all seven transmembrane helices (TMHs), showing that the experimental structure is reliably selected as the best conformation from this ensemble. However, various GPCRs differ sufficiently in the tilts of the TMHs that this method need not predict the optimum conformation starting from any other template. In this paper, we introduce the SuperBiHelix method in which the tilt angles (θ, ϕ) are optimized simultaneously with rotations (η) efficiently enough that it is practical and sufficient to sample (5 × 3 × 5)(7) = 13 trillion configurations. This method can correctly identify the optimum structure of a GPCR starting with the template from a different GPCR. We have validated this method by predicting known crystal structure conformations starting from the template of a different protein structure. We find that the SuperBiHelix conformational ensemble includes the higher energy conformations associated with the active protein in addition to those associated with the more stable inactive protein. This methodology was then applied to design and experimentally confirm structures of three mutants of the CB1 cannabinoid receptor associated with different functions.

  4. Energetics and Structure Prediction of the Network of Homo- and Hetero-Oligomers Formed by the Transmembrane Domains of the ErbReceptor Family of Proteins

    Science.gov (United States)

    2006-06-01

    higher-order oligomer) can bind to the cholera toxin promoter and activate transcription of a reporter gene. Using this assay, Lemmon and colleagues...identical to the transmembrane sequences of the chicken Klg and Hydra Lemon orthologues (9), and a pattern of residues with helical periodicity is...Steele, R. E. (2000) Lemon encodes an unusual receptor protein-tyrosine kinase expressed during gametogenesis in Hydra , DeV. Biol. 224, 286-298. 10

  5. A Classification of Basic Helix-Loop-Helix Transcription Factors of Soybean

    Directory of Open Access Journals (Sweden)

    Karen A. Hudson

    2015-01-01

    Full Text Available The complete genome sequence of soybean allows an unprecedented opportunity for the discovery of the genes controlling important traits. In particular, the potential functions of regulatory genes are a priority for analysis. The basic helix-loop-helix (bHLH family of transcription factors is known to be involved in controlling a wide range of systems critical for crop adaptation and quality, including photosynthesis, light signalling, pigment biosynthesis, and seed pod development. Using a hidden Markov model search algorithm, 319 genes with basic helix-loop-helix transcription factor domains were identified within the soybean genome sequence. These were classified with respect to their predicted DNA binding potential, intron/exon structure, and the phylogeny of the bHLH domain. Evidence is presented that the vast majority (281 of these 319 soybean bHLH genes are expressed at the mRNA level. Of these soybean bHLH genes, 67% were found to exist in two or more homeologous copies. This dataset provides a framework for future studies on bHLH gene function in soybean. The challenge for future research remains to define functions for the bHLH factors encoded in the soybean genome, which may allow greater flexibility for genetic selection of growth and environmental adaptation in this widely grown crop.

  6. Double helix vortex breakdown in a turbulent swirling annular jet flow

    NARCIS (Netherlands)

    Vanierschot, M.; Perçin, M.; van Oudheusden, B.W.

    2018-01-01

    In this paper, we report on the structure and dynamics of double helix vortex breakdown in a turbulent annular swirling jet. Double helix breakdown has been reported previously for the laminar flow regime, but this structure has rarely been observed in turbulent flow. The flow field is

  7. An unusual helix turn helix motif in the catalytic core of HIV-1 integrase binds viral DNA and LEDGF.

    Directory of Open Access Journals (Sweden)

    Hayate Merad

    Full Text Available BACKGROUND: Integrase (IN of the type 1 human immunodeficiency virus (HIV-1 catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149-186 in the crystal structure of the catalytic core (CC of the IN-Phe185Lys variant that consists of the alpha(4 and alpha(5 helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. PRINCIPAL FINDINGS: We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the alpha(4 and alpha(5 helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat ends of virus DNA with a K(d (dissociation constant in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix, it is the N terminal helix (alpha(4 which has the role of DNA recognition helix, while the C terminal helix (alpha(5 would rather contribute to the motif stabilization by interactions with the alpha(4 helix. CONCLUSION: The motif, termed HTHi (i, for inverted emerges as a central piece of the IN structure and function. It could therefore represent an attractive target in the search for inhibitors working at the DNA-IN, IN-IN and IN-LEDGF interfaces.

  8. NMR studies of the fifth transmembrane segment of Na+,K+-ATPase reveals a non-helical ion-binding region

    DEFF Research Database (Denmark)

    Underhaug, Jarl; Jakobsen, Louise Odgaard; Esmann, Mikael

    2006-01-01

    The structure of a synthetic peptide corresponding to the fifth membrane-spanning segment (M5) in Na(+),K(+)-ATPase in sodium dodecyl sulfate (SDS) micelles was determined using liquid-state nuclear magnetic resonance (NMR) spectroscopy. The spectra reveal that this peptide is substantially less...... transmembrane element of the Ca(2+)-ATPase. Furthermore, this region spans the residues implicated in Na(+) and K(+) transport, where they are likely to offer the flexibility needed to coordinate Na(+) as well as K(+) during active transport....... alpha-helical than the corresponding M5 peptide of Ca(2+)-ATPase. A well-defined alpha-helix is shown in the C-terminal half of the peptide. Apart from a short helical stretch at the N-terminus, the N-terminal half contains a non-helical region with two proline residues and sequence similarity to a non-structured...

  9. Connecting two proteins using a fusion alpha helix stabilized by a chemical cross linker

    Science.gov (United States)

    Jeong, Woo Hyeon; Lee, Haerim; Song, Dong Hyun; Eom, Jae-Hoon; Kim, Sun Chang; Lee, Hee-Seung; Lee, Hayyoung; Lee, Jie-Oh

    2016-03-01

    Building a sophisticated protein nano-assembly requires a method for linking protein components in a predictable and stable structure. Most of the cross linkers available have flexible spacers. Because of this, the linked hybrids have significant structural flexibility and the relative structure between their two components is largely unpredictable. Here we describe a method of connecting two proteins via a `fusion α helix' formed by joining two pre-existing helices into a single extended helix. Because simple ligation of two helices does not guarantee the formation of a continuous helix, we used EY-CBS, a synthetic cross linker that has been shown to react selectively with cysteines in α-helices, to stabilize the connecting helix. Formation and stabilization of the fusion helix was confirmed by determining the crystal structures of the fusion proteins with and without bound EY-CBS. Our method should be widely applicable for linking protein building blocks to generate predictable structures.

  10. Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.

    Science.gov (United States)

    Caballero-Rivera, Daniel; Cruz-Nieves, Omar A; Oyola-Cintrón, Jessica; Torres-Núñez, David A; Otero-Cruz, Jose D; Lasalde-Dominicci, José A

    2011-01-01

    The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the δM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the δM3 transmembrane domain were characterized by two-electrode voltage clamp and (125)I-labeled α-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300-301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid-exposed transmembrane domain and the nAChR gating machinery.

  11. Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor

    Science.gov (United States)

    Caballero-Rivera, Daniel; Cruz-Nieves, Omar A; Oyola-Cintrón, Jessica; Torres-Núñez, David A; Otero-Cruz, José D

    2011-01-01

    The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the δM3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the δM3 transmembrane domain were characterized by two-electrode voltage clamp and 125I-labeled α-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300–301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid-exposed transmembrane domain and the nAChR gating machinery. PMID:21785268

  12. Induction of helical structure in a heptapeptide with a metal cross-link: modification of the Lifson-Roig helix-coil theory to account for covalent cross-links.

    Science.gov (United States)

    Kise, Kenneth J; Bowler, Bruce E

    2002-12-31

    A short peptide, acetyl-AHAAAHA-carboxamide, has been synthesized and the histidines cross-linked with a cis-tetraammineruthenium(III) moiety. In the absence of the Ru(III) cross-link, the heptapeptide is essentially structureless, as judged by circular dichroism, NMR chemical shift, and NMR-monitored hydrogen deuterium exchange data. The presence of the cis-Ru(III) cross-link is confirmed by mass spectral data and the characteristic pH dependence of the UV-vis spectrum of the cis-(bis-(imidazole))ruthenium(III) unit. Circular dichroism data indicate that the Ru(III) cross-linked heptapeptide is approximately 37% helical at 0 degrees C. The NMR spectrum of the cross-linked peptide has been fully assigned using TOCSY and ROESY experiments. ROE interactions and J-coupling data provide evidence for helical structure. NMR-monitored hydrogen-deuterium exchange data for the Ru(III)-cross-linked peptide, resolved at the level of the individual amides, give larger protection factors at the ends than in the center of the helix. Steric and polarization effects of the Ru(III) cross-link are proposed to cause this unusual apparent protection pattern. A modification to the Lifson-Roig helix-coil model to account for the effect of the i,i+4 Ru(III) cross-link on the helix-coil transition of a peptide is presented. The model provides an excellent fit to the temperature dependence of the circular dichroism spectrum of the Ru(III)-cross-linked peptide. The modified model indicates that the effect of the cross-link on the nucleation parameter, v(2), is modest (about 7-fold) for residues bounded by the cross-link. Significant increases in the propagation parameter, w, occur for residues within the cross-link. The modification to the Lifson-Roig model accounts for the effect of a Ru(III) cross-link on the circular dichroism spectrum of a previously reported 17 residue peptide.

  13. Modeling Transmembrane Domain Dimers/Trimers of Plexin Receptors: Implications for Mechanisms of Signal Transmission across the Membrane

    Science.gov (United States)

    Zhang, Liqun; Polyansky, Anton; Buck, Matthias

    2015-01-01

    Single-pass transmembrane (TM) receptors transmit signals across lipid bilayers by helix association or by configurational changes within preformed dimers. The structure determination for such TM regions is challenging and has mostly been accomplished by NMR spectroscopy. Recently, the computational prediction of TM dimer structures is becoming recognized for providing models, including alternate conformational states, which are important for receptor regulation. Here we pursued a strategy to predict helix oligomers that is based on packing considerations (using the PREDDIMER webserver) and is followed by a refinement of structures, utilizing microsecond all-atom molecular dynamics simulations. We applied this method to plexin TM receptors, a family of 9 human proteins, involved in the regulation of cell guidance and motility. The predicted models show that, overall, the preferences identified by PREDDIMER are preserved in the unrestrained simulations and that TM structures are likely to be diverse across the plexin family. Plexin-B1 and –B3 TM helices are regular and tend to associate, whereas plexin-A1, -A2, –A3, -A4, -C1 and –D1 contain sequence elements, such as poly-Glycine or aromatic residues that distort helix conformation and association. Plexin-B2 does not form stable dimers due to the presence of TM prolines. No experimental structural information on the TM region is available for these proteins, except for plexin-C1 dimeric and plexin-B1 – trimeric structures inferred from X-ray crystal structures of the intracellular regions. Plexin-B1 TM trimers utilize Ser and Thr sidechains for interhelical contacts. We also modeled the juxta-membrane (JM) region of plexin-C1 and plexin-B1 and show that it synergizes with the TM structures. The structure and dynamics of the JM region and TM-JM junction provide determinants for the distance and distribution of the intracellular domains, and for their binding partners relative to the membrane. The structures

  14. PDBTM: Protein Data Bank of transmembrane proteins after 8 years.

    Science.gov (United States)

    Kozma, Dániel; Simon, István; Tusnády, Gábor E

    2013-01-01

    The PDBTM database (available at http://pdbtm.enzim.hu), the first comprehensive and up-to-date transmembrane protein selection of the Protein Data Bank, was launched in 2004. The database was created and has been continuously updated by the TMDET algorithm that is able to distinguish between transmembrane and non-transmembrane proteins using their 3D atomic coordinates only. The TMDET algorithm can locate the spatial positions of transmembrane proteins in lipid bilayer as well. During the last 8 years not only the size of the PDBTM database has been steadily growing from ∼400 to 1700 entries but also new structural elements have been identified, in addition to the well-known α-helical bundle and β-barrel structures. Numerous 'exotic' transmembrane protein structures have been solved since the first release, which has made it necessary to define these new structural elements, such as membrane loops or interfacial helices in the database. This article reports the new features of the PDBTM database that have been added since its first release, and our current efforts to keep the database up-to-date and easy to use so that it may continue to serve as a fundamental resource for the scientific community.

  15. When proteome meets genome: the alpha helix and the beta strand ...

    Indian Academy of Sciences (India)

    The avoidance of the -helix and the -strand by splice junctions suggests the existence of a selection pressure against their disruption, perhaps underscoring the investment made by nature in building these intricate secondary structures. A corollary is that the helix and the strand are the smallest integral architectural units ...

  16. Crystal structure of YwpF from Staphylococcus aureus reveals its architecture comprised of a β-barrel core domain resembling type VI secretion system proteins and a two-helix pair.

    Science.gov (United States)

    Lee, Sang Jae; Lee, Kyu-Yeon; Lee, Ki-Young; Kim, Dong-Gyun; Kim, Soon-Jong; Lee, Bong-Jin

    2015-04-01

    The ywpF gene (SAV2097) of the Staphylococcus aureus strain Mu50 encodes the YwpF protein, which may play a role in antibiotic resistance. Here, we report the first crystal structure of the YwpF superfamily from S. aureus at 2.5-Å resolution. The YwpF structure consists of two regions: an N-terminal core β-barrel domain that shows structural similarity to type VI secretion system (T6SS) proteins (e.g., Hcp1, Hcp3, and EvpC) and a C-terminal two-helix pair. Although the monomer structure of S. aureus YwpF resembles those of T6SS proteins, the dimer/tetramer model of S. aureus YwpF is distinct from the functionally important hexameric ring of T6SS proteins. We therefore suggest that the S. aureus YwpF may have a different function compared to T6SS proteins. © 2015 Wiley Periodicals, Inc.

  17. Evaluation of sequence variability in HIV-1 gp41 C-peptide helix-grafted proteins.

    Science.gov (United States)

    Tennyson, Rachel L; Walker, Susanne N; Ikeda, Terumasa; Harris, Reuben S; McNaughton, Brian R

    2017-08-01

    Many therapeutically-relevant protein-protein interactions (PPIs) have been reported that feature a helix and helix-binding cleft at the interface. Given this, different approaches to disrupting such PPIs have been developed. While short peptides (<15 amino acids) typically do not fold into a stable helix, researchers have reported chemical approaches to constraining helix structure. However, these approaches rely on laborious, and often expensive, chemical synthesis and purification. Our premise is that protein-based solutions that stabilize a therapeutically-relevant helix offer a number of advantages. In contrast to chemically constrained helical peptides, or minimal/miniature proteins, which must be synthesized (at great expense and labor), a protein can be expressed in a cellular system (like all current protein therapeutics). If selected properly, the protein scaffold can stabilize the therapeutically-relevant helix. We recently reported a protein engineering strategy, which we call "helix-grafted display", and applied it to the challenge of suppressing HIV entry. We have reported helix-grafted display proteins that inhibit formation of an intramolecular PPI involving HIV gp41 C-peptide helix, and HIV gp41 N-peptide trimer, which contain C-peptide helix-binding clefts. Here, we used yeast display to screen a library of grafted C-peptide helices for N-peptide trimer recognition. Using 'hits' from yeast display library screening, we evaluated the effect helix mutations have on structure, expression, stability, function (target recognition), and suppression of HIV entry. Copyright © 2017. Published by Elsevier Ltd.

  18. Transmembrane helix connectivity in Orai1 controls two gates for calcium-dependent transcription

    Czech Academy of Sciences Publication Activity Database

    Frischauf, I.; Litviňuková, M.; Schober, R.; Zayats, Vasilina; Svobodová, B.; Bonhenry, Daniel; Lunz, V.; Cappello, S.; Tociu, L.; Řeha, David; Stallinger, A.; Hochreiter, A.; Pammer, T.; Butorac, C.; Muik, M.; Groschner, K.; Bogeski, I.; Ettrich, Horst Rüdiger; Romanin, Ch.; Schindl, R.

    2017-01-01

    Roč. 10, č. 507 (2017), č. článku eaao0358. ISSN 1937-9145 R&D Projects: GA ČR(CZ) GA13-21053S; GA MŠk(CZ) LM2015055; GA MŠk(CZ) LTC17069 Institutional support: RVO:61388971 Keywords : COMPREHENSIVE MOLECULAR CHARACTERIZATION * FAST CA2+-DEPENDENT INACTIVATION * ACTIVATES CRAC CHANNELS Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology

  19. Multiple helix ecosystems for sustainable competitiveness

    CERN Document Server

    Ferreira, João; Farinha, Luís; Fernandes, Nuno

    2016-01-01

    This book discusses the main issues, challenges, opportunities, and trends involving the interactions between academia, industry, government and society. Specifically, it aims to explore how these interactions enhance the ways in which companies deliver products and services in order to achieve sustainable competitiveness in the marketplace. Sustainable competitiveness has been widely discussed by academics and practitioners, considering the importance of protecting the environment while sustaining the economic goals of organizations. The Quintuple Helix innovation model is a framework for facilitating knowledge, innovation and sustainable competitive advantage. It embeds the Triple and the Quadruple Helix models by adding a fifth helix, the “natural environment.” The Triple Helix model focuses on the university-industry-government triad, while the Quadruple adds civil society (the media- and culture-driven public) as a fourth helix. The Quintuple Helix model facilitates research, public policy, and pract...

  20. HELIX: The High Energy Light Isotope Experiment

    Science.gov (United States)

    Wakely, Scott

    This is the lead proposal for a new suborbital program, HELIX (High-Energy Light Isotope eXperiment), designed to make measurements of the isotopic composition of light cosmic-ray nuclei from ~200 MeV/nuc to ~10 GeV/nuc. Past measurements of this kind have provided profound insights into the nature and origin of cosmic rays, revealing, for instance, information on acceleration and confinement time scales, and exposing some conspicuous discrepancies between solar and cosmic-ray abundances. The most detailed information currently available comes from the ACE/CRIS mission, but is restricted to energies below a few 100 MeV/nuc. HELIX aims at extending this energy range by over an order of magnitude, where, in most cases, no measurements of any kind exist, and where relativistic time dilation affects the apparent lifetime of radioactive clock nuclei. The HELIX measurements will provide essential information for understanding the propagation history of cosmic rays in the galaxy. This is crucial for properly interpreting several intriguing anomalies reported in recent cosmic-ray measurements, pertaining to the energy spectra of protons, helium, and heavier nuclei, and to the anomalous rise in the positron fraction at higher energy. HELIX employs a high-precision magnet spectrometer to provide measurements which are not achievable by any current or planned instrument. The superconducting magnet originally used for the HEAT payload in five successful high-altitude flights will be combined with state-of-the-art detectors to measure the charge, time-of-flight, magnetic rigidity, and velocity of cosmic-ray particles with high precision. The instrumentation includes plastic scintillators, silicon-strip detectors repurposed from Fermilab's CDF detector, a high-performance gas drift chamber, and a ring-imaging Cherenkov counter employing aerogel radiators and silicon photomultipliers. To reduce cost and technical risk, the HELIX program will be structured in two stages. The first

  1. Structure of the TPR domain of AIP: lack of client protein interaction with the C-terminal α-7 helix of the TPR domain of AIP is sufficient for pituitary adenoma predisposition.

    Directory of Open Access Journals (Sweden)

    Rhodri M L Morgan

    Full Text Available Mutations of the aryl hydrocarbon receptor interacting protein (AIP have been associated with familial isolated pituitary adenomas predisposing to young-onset acromegaly and gigantism. The precise tumorigenic mechanism is not well understood as AIP interacts with a large number of independent proteins as well as three chaperone systems, HSP90, HSP70 and TOMM20. We have determined the structure of the TPR domain of AIP at high resolution, which has allowed a detailed analysis of how disease-associated mutations impact on the structural integrity of the TPR domain. A subset of C-terminal α-7 helix (Cα-7h mutations, R304* (nonsense mutation, R304Q, Q307* and R325Q, a known site for AhR and PDE4A5 client-protein interaction, occur beyond those that interact with the conserved MEEVD and EDDVE sequences of HSP90 and TOMM20. These C-terminal AIP mutations appear to only disrupt client-protein binding to the Cα-7h, while chaperone binding remains unaffected, suggesting that failure of client-protein interaction with the Cα-7h is sufficient to predispose to pituitary adenoma. We have also identified a molecular switch in the AIP TPR-domain that allows recognition of both the conserved HSP90 motif, MEEVD, and the equivalent sequence (EDDVE of TOMM20.

  2. Structural elucidation of transmembrane domain zero (TMD0) of EcdL: A multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporter protein revealed by atomistic simulation.

    Science.gov (United States)

    Bera, Krishnendu; Rani, Priyanka; Kishor, Gaurav; Agarwal, Shikha; Kumar, Antresh; Singh, Durg Vijay

    2017-09-20

    ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 μs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.

  3. Solid-state NMR spectroscopy of the HIV gp41 membrane fusion protein supports intermolecular antiparallel β sheet fusion peptide structure in the final six-helix bundle state.

    Science.gov (United States)

    Sackett, Kelly; Nethercott, Matthew J; Zheng, Zhaoxiong; Weliky, David P

    2014-03-06

    The HIV gp41 protein catalyzes fusion between viral and target cell membranes. Although the ~20-residue N-terminal fusion peptide (FP) region is critical for fusion, the structure of this region is not well characterized in large gp41 constructs that model the gp41 state at different times during fusion. This paper describes solid-state NMR (SSNMR) studies of FP structure in a membrane-associated construct (FP-Hairpin), which likely models the final fusion state thought to be thermostable trimers with six-helix bundle structure in the region C-terminal of the FP. The SSNMR data show that there are populations of FP-Hairpin with either α helical or β sheet FP conformation. For the β sheet population, measurements of intermolecular (13)C-(13)C proximities in the FP are consistent with a significant fraction of intermolecular antiparallel β sheet FP structure with adjacent strand crossing near L7 and F8. There appears to be negligible in-register parallel structure. These findings support assembly of membrane-associated gp41 trimers through interleaving of N-terminal FPs from different trimers. Similar SSNMR data are obtained for FP-Hairpin and a construct containing the 70 N-terminal residues of gp41 (N70), which is a model for part of the putative pre-hairpin intermediate state of gp41. FP assembly may therefore occur at an early fusion stage. On a more fundamental level, similar SSNMR data are obtained for FP-Hairpin and a construct containing the 34 N-terminal gp41 residues (FP34) and support the hypothesis that the FP is an autonomous folding domain. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Double helix vortex breakdown in a turbulent swirling annular jet flow

    Science.gov (United States)

    Vanierschot, M.; Percin, M.; van Oudheusden, B. W.

    2018-03-01

    In this paper, we report on the structure and dynamics of double helix vortex breakdown in a turbulent annular swirling jet. Double helix breakdown has been reported previously for the laminar flow regime, but this structure has rarely been observed in turbulent flow. The flow field is investigated experimentally by means of time-resolved tomographic particle image velocimetry. Notwithstanding the axisymmetric nature of the time-averaged flow, analysis of the instantaneous three-dimensional (3D) vortical structures shows the existence of a vortex core along the central axis which breaks up into a double helix downstream. The winding sense of this double helix is opposite to the swirl direction (m =-2 ) and it is wrapped around a central vortex breakdown bubble. This structure is quite different from double helix breakdown found in laminar flows where the helix is formed in the wake of the bubble and not upstream. The double helix precesses around the central axis of the jet with a precessing frequency corresponding to a Strouhal number of 0.27.

  5. Folding and insertion thermodynamics of the transmembrane WALP peptide.

    Science.gov (United States)

    Bereau, Tristan; Bennett, W F Drew; Pfaendtner, Jim; Deserno, Markus; Karttunen, Mikko

    2015-12-28

    The anchor of most integral membrane proteins consists of one or several helices spanning the lipid bilayer. The WALP peptide, GWW(LA)n (L)WWA, is a common model helix to study the fundamentals of protein insertion and folding, as well as helix-helix association in the membrane. Its structural properties have been illuminated in a large number of experimental and simulation studies. In this combined coarse-grained and atomistic simulation study, we probe the thermodynamics of a single WALP peptide, focusing on both the insertion across the water-membrane interface, as well as folding in both water and a membrane. The potential of mean force characterizing the peptide's insertion into the membrane shows qualitatively similar behavior across peptides and three force fields. However, the Martini force field exhibits a pronounced secondary minimum for an adsorbed interfacial state, which may even become the global minimum-in contrast to both atomistic simulations and the alternative PLUM force field. Even though the two coarse-grained models reproduce the free energy of insertion of individual amino acids side chains, they both underestimate its corresponding value for the full peptide (as compared with atomistic simulations), hinting at cooperative physics beyond the residue level. Folding of WALP in the two environments indicates the helix as the most stable structure, though with different relative stabilities and chain-length dependence.

  6. Kevlar: Transitioning Helix from Research to Practice

    Science.gov (United States)

    2015-04-01

    KEVLAR : TRANSITIONING HELIX FROM RESEARCH TO PRACTICE UNIVERSITY OF VIRGINIA APRIL 2015 FINAL TECHNICAL REPORT...DATES COVERED (From - To) FEB 2013 – NOV 2014 4. TITLE AND SUBTITLE KEVLAR : TRANSITIONING HELIX FROM RESEARCH TO PRACTICE 5a. CONTRACT NUMBER...possible exploitation. Our technology, called Kevlar , includes key security technologies are protective transformations and targeted recovery. The

  7. Kevlar: Transitioning Helix for Research to Practice

    Science.gov (United States)

    2016-03-01

    KEVLAR : TRANSITIONING HELIX FROM RESEARCH TO PRACTICE UNIVERSITY OF VIRGINIA MARCH 2016 FINAL TECHNICAL REPORT...REPORT 3. DATES COVERED (From - To) FEB 2015 – SEP 2015 4. TITLE AND SUBTITLE KEVLAR : TRANSITIONING HELIX FROM RESEARCH TO PRACTICE 5a. CONTRACT...subject to possible exploitation. Our technology, called Kevlar , includes key security technologies are protective transformations and targeted

  8. Double Helix Nodal Line Superconductor

    Science.gov (United States)

    Sun, Xiao-Qi; Lian, Biao; Zhang, Shou-Cheng

    2017-10-01

    Time-reversal invariant superconductors in three dimensions may contain nodal lines in the Brillouin zone, which behave as Wilson loops of 3D momentum-space Chern-Simons theory of the Berry connection. Here we study the conditions of realizing linked nodal lines (Wilson loops), which yield a topological contribution to the thermal magnetoelectric coefficient that is given by the Chern-Simons action. We find the essential conditions are the existence of torus or higher genus Fermi surfaces and spiral spin textures. We construct such a model with two torus Fermi surfaces, where a generic spin-dependent interaction leads to double-helix-like linked nodal lines as the superconductivity is developed.

  9. RENNSH: a novel α-helix identification approach for intermediate resolution electron density maps.

    Science.gov (United States)

    Ma, Lingyu; Reisert, Marco; Burkhardt, Hans

    2012-01-01

    Accurate identification of protein secondary structures is beneficial to understand three-dimensional structures of biological macromolecules. In this paper, a novel refined classification framework is proposed, which treats alpha-helix identification as a machine learning problem by representing each voxel in the density map with its Spherical Harmonic Descriptors (SHD). An energy function is defined to provide statistical analysis of its identification performance, which can be applied to all the α-helix identification approaches. Comparing with other existing α-helix identification methods for intermediate resolution electron density maps, the experimental results demonstrate that our approach gives the best identification accuracy and is more robust to the noise.

  10. Design and synthesis of DNA four-helix bundles

    International Nuclear Information System (INIS)

    Rangnekar, Abhijit; Gothelf, Kurt V; LaBean, Thomas H

    2011-01-01

    The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

  11. Design and synthesis of DNA four-helix bundles

    Energy Technology Data Exchange (ETDEWEB)

    Rangnekar, Abhijit; Gothelf, Kurt V [Department of Chemistry, Centre for DNA Nanotechnology (CDNA) and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C (Denmark); LaBean, Thomas H, E-mail: kvg@chem.au.dk, E-mail: thl@cs.duke.edu [Department of Chemistry, Duke University, Durham, NC 27708 (United States)

    2011-06-10

    The field of DNA nanotechnology has evolved significantly in the past decade. Researchers have succeeded in synthesizing tile-based structures and using them to form periodic lattices in one, two and three dimensions. Origami-based structures have also been used to create nanoscale structures in two and three dimensions. Design and construction of DNA bundles with fixed circumference has added a new dimension to the field. Here we report the design and synthesis of a DNA four-helix bundle. It was found to be extremely rigid and stable. When several such bundles were assembled using appropriate sticky-ends, they formed micrometre-long filaments. However, when creation of two-dimensional sheet-like arrays of the four-helix bundles was attempted, nanoscale rings were observed instead. The exact reason behind the nanoring formation is yet to be ascertained, but it provides an exciting prospect for making programmable circular nanostructures using DNA.

  12. Transmembrane protein sorting driven by membrane curvature

    Science.gov (United States)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  13. Structures of OppA and PstS from Yersinia pestis indicate variability of interactions with transmembrane domains

    DEFF Research Database (Denmark)

    Tanabe, Mikio; Mirza, Osman; Bertrand, Thomas

    2007-01-01

    -infective development. Here, the crystallization of five proteins (OppA, PstS, PiuA, YrbD and CysP) from Yersinia pestis, the causative agent of plague, are reported that diffracted to resolution limits ranging from 1.6 to 5 A. The first crystal structures of ABC system components from Y. pestis, OppA and Pst...

  14. The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition.

    Science.gov (United States)

    Meijberg, Wim; Booth, Paula J

    2002-06-07

    The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane alpha-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-l-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane alpha-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces. (c) 2002 Elsevier Science Ltd.

  15. Genome-wide identification of basic helix-loop-helix and NF-1 motifs underlying GR binding sites in male rat hippocampus

    DEFF Research Database (Denmark)

    Pooley, John R.; Flynn, Ben P.; Grøntved, Lars

    2017-01-01

    in hippocampal GR function. Our findings imply a dosedependent and context-independent action of GRs in the hippocampus. Alterations in the expression or activity of NF-1/basic helix-loop-helix factors may play an as yet undetermined role in glucocorticoid-related disease susceptibility and outcome by altering......Glucocorticoids regulate hippocampal function in part by modulating gene expression through the glucocorticoid receptor (GR). GR binding is highly cell type specific, directed to accessible chromatin regions established during tissue differentiation. Distinct classes of GR binding sites...... linked to structural and organizational roles, an absence of major tethering partners for GRs, and little or no evidence for binding at negative glucocorticoid response elements. A basic helix-loop-helix motif closely resembling a NeuroD1 or Olig2 binding site was found underlying a subset of GR binding...

  16. pH-dependent activities and structural stability of loop-2-anchoring helix of RadA recombinase from Methanococcus voltae.

    Science.gov (United States)

    Rao, D E C S; Luo, Yu

    2014-07-01

    RadA is an archaeal orthologue of human recombinase Rad51. This superfamily of recombinases, which also includes eukaryal meiosis-specific DMC1 and remotely related bacterial RecA, form filaments on single-stranded DNA in the presence of ATP and promote a strand exchange reaction between the single-stranded DNA and a homologous double stranded DNA. Due to its feasibility of getting crystals and similarity (> 40% sequence identity) to eukaryal homologues, we have studied RadA from Methanococcus voltae (MvRadA) as a structural model for understanding the molecular mechanism of homologous strand exchange. Here we show this protein's ATPase and strand exchange activities are minimal at pH 6.0. Interestingly, MvRadA's pH dependence is similar to the properties of human Rad51 but dissimilar to that of the well-studied E. coli RecA. A structure subsequently determined at pH 6.0 reveals features indicative of an ATPase- inactive form with a disordered L2 loop. Comparison with a previously determined ATPase-active form at pH 7.5 implies that the stability of the ATPase-active conformation is reduced at the acidic pH. We interpret these results as further suggesting an ordered disposition of the DNA-binding L2 region, similar to what has been observed in the previously observed ATPase-active conformation, is required for promoting hydrolysis of ATP and strand exchange between singleand double-stranded DNA. His-276 in the mobile L2 region was observed to be partially responsible for the pH-dependent activities of MvRadA.

  17. First principles design of a core bioenergetic transmembrane electron-transfer protein.

    Science.gov (United States)

    Goparaju, Geetha; Fry, Bryan A; Chobot, Sarah E; Wiedman, Gregory; Moser, Christopher C; Leslie Dutton, P; Discher, Bohdana M

    2016-05-01

    Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. First principles design of a core bioenergetic transmembrane electron-transfer protein

    Energy Technology Data Exchange (ETDEWEB)

    Goparaju, Geetha; Fry, Bryan A.; Chobot, Sarah E.; Wiedman, Gregory; Moser, Christopher C.; Leslie Dutton, P.; Discher, Bohdana M.

    2016-05-01

    Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

  19. A genome-wide survey on basic helix-loop-helix transcription factors in giant panda.

    Directory of Open Access Journals (Sweden)

    Chunwang Dang

    Full Text Available The giant panda (Ailuropoda melanoleuca is a critically endangered mammalian species. Studies on functions of regulatory proteins involved in developmental processes would facilitate understanding of specific behavior in giant panda. The basic helix-loop-helix (bHLH proteins play essential roles in a wide range of developmental processes in higher organisms. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, mouse and human. Our present study identified 107 bHLH family members being encoded in giant panda genome. Phylogenetic analyses revealed that they belong to 44 bHLH families with 46, 25, 15, 4, 11 and 3 members in group A, B, C, D, E and F, respectively, while the remaining 3 members were assigned into "orphan". Compared to mouse, the giant panda does not encode seven bHLH proteins namely Beta3a, Mesp2, Sclerax, S-Myc, Hes5 (or Hes6, EBF4 and Orphan 1. These results provide useful background information for future studies on structure and function of bHLH proteins in the regulation of giant panda development.

  20. The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

    International Nuclear Information System (INIS)

    Hollier, Mark J.; Dimmock, Nigel J.

    2005-01-01

    In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, β-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell

  1. An approach to membrane protein structure without crystals

    Science.gov (United States)

    Sorgen, Paul L.; Hu, Yonglin; Guan, Lan; Kaback, H. Ronald; Girvin, Mark E.

    2002-01-01

    The lactose permease of Escherichia coli catalyzes coupled translocation of galactosides and H+ across the cell membrane. It is the best-characterized member of the Major Facilitator Superfamily, a related group of membrane proteins with 12 transmembrane domains that mediate transport of various substrates across cell membranes. Despite decades of effort and their functional importance in all kingdoms of life, no high-resolution structures have been solved for any member of this family. However, extensive biochemical, genetic, and biophysical studies on lactose permease have established its transmembrane topology, secondary structure, and numerous interhelical contacts. Here we demonstrate that this information is sufficient to calculate a structural model at the level of helix packing or better. PMID:12391320

  2. Superconducting Helix loaded cavity for heavy ion acceleration

    International Nuclear Information System (INIS)

    Ramstein, G.; Cauvin, B.; Fouan, J.P.

    1987-01-01

    Half-wave helix loaded structures have been designed to increase the mass range of heavy ions which will be accelerated by the CEN Saclay superconducting accelerator. In this paper we shall list the main characteristics of these resonators; especially, the quality factor and the energy gain per charge measured with a 12 C beam. The important advantages of these new structures are the broadness of the transit time factor curve which allows us to accelerate low velocity beams with a sufficient efficiency, and the simplicity of mechanical design which requires only two helix welds. We shall also give the performances achieved by the series of resonators which will be mounted into two of the six machine cryostats. The new superconducting accelerator will provide the first beams at the end of this year [fr

  3. Identification of amino acids essential for estrone-3-sulfate transport within transmembrane domain 2 of organic anion transporting polypeptide 1B1.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available As an important structure in membrane proteins, transmembrane domains have been found to be crucial for properly targeting the protein to cell membrane as well as carrying out transport functions in transporters. Computer analysis of OATP sequences revealed transmembrane domain 2 (TM2 is among those transmembrane domains that have high amino acid identities within different family members. In the present study, we identify four amino acids (Asp70, Phe73, Glu74, and Gly76 that are essential for the transport function of OATP1B1, an OATP member that is specifically expressed in the human liver. A substitution of these four amino acids with alanine resulted in significantly reduced transport activity. Further mutagenesis showed the charged property of Asp70 and Glu74 is critical for proper function of the transporter protein. Comparison of the kinetic parameters indicated that Asp70 is likely to interact with the substrate while Glu74 may be involved in stabilizing the binding site through formation of a salt-bridge. The aromatic ring structure of Phe73 seems to play an important role because substitution of Phe73 with tyrosine, another amino acid with a similar structure, led to partially restored transport function. On the other hand, replacement of Gly76 with either alanine or valine could not recover the function of the transporter. Considering the nature of a transmembrane helix, we proposed that Gly76 may be important for maintaining the proper structure of the protein. Interestingly, when subjected to transport function analysis of higher concentration of esteone-3-sulfate (50 µM that corresponds to the low affinity binding site of OATP1B1, mutants of Phe73, Glu74, and Gly76 all showed a transport function that is comparable to that of the wild-type, suggesting these amino acids may have less impact on the low affinity component of esteone-3-sulfate within OATP1B1, while Asp 70 seems to be involved in the interaction of both sites.

  4. Manifestation of cystic fibrosis transmembrane regulator (CFTR) in hepatic ductal structures and renal tubules of female rats with experimental cholestasis of pregnancy.

    Science.gov (United States)

    Aleksandrova, M I; Kushnareva, N S; Smirnova, O V

    2014-03-01

    Studies by the immunohistochemical method with semiquantitative analysis of images showed that hyperprolactinemia stimulated CFTR protein manifestation in the bile ducts of female rats, which was clearly expressed in experimental cholestasis of pregnancy. The expression of CFTR in the renal tubules was reduced in hyperprolactinemia under conditions of normal liver function and in cholestasis of pregnancy. Significant positive correlations between CFTR, prolactin receptor, and multiple drug resistance protein 3 were detected in the bile ducts, but not in the renal tubules. Presumably, prolactin has a direct effect on CFTR expression in the bile ducts and indirect effect in the renal tubules. Changes in CFTR protein manifestation in the hepatic ductal structures and renal tubules in experimental pregnancy cholestasis could aggravate the disease.

  5. Modeling structure of G protein-coupled receptors in huan genome

    KAUST Repository

    Zhang, Yang

    2016-01-26

    G protein-coupled receptors (or GPCRs) are integral transmembrane proteins responsible to various cellular signal transductions. Human GPCR proteins are encoded by 5% of human genes but account for the targets of 40% of the FDA approved drugs. Due to difficulties in crystallization, experimental structure determination remains extremely difficult for human GPCRs, which have been a major barrier in modern structure-based drug discovery. We proposed a new hybrid protocol, GPCR-I-TASSER, to construct GPCR structure models by integrating experimental mutagenesis data with ab initio transmembrane-helix assembly simulations, assisted by the predicted transmembrane-helix interaction networks. The method was tested in recent community-wide GPCRDock experiments and constructed models with a root mean square deviation 1.26 Å for Dopamine-3 and 2.08 Å for Chemokine-4 receptors in the transmembrane domain regions, which were significantly closer to the native than the best templates available in the PDB. GPCR-I-TASSER has been applied to model all 1,026 putative GPCRs in the human genome, where 923 are found to have correct folds based on the confidence score analysis and mutagenesis data comparison. The successfully modeled GPCRs contain many pharmaceutically important families that do not have previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin and Neuropeptide Y receptors. All the human GPCR models have been made publicly available through the GPCR-HGmod database at http://zhanglab.ccmb.med.umich.edu/GPCR-HGmod/ The results demonstrate new progress on genome-wide structure modeling of transmembrane proteins which should bring useful impact on the effort of GPCR-targeted drug discovery.

  6. Teaching helix and problems connected with helix using GeoGebra

    Science.gov (United States)

    Bímová, Daniela

    2017-12-01

    The contribution presents the dynamic applets created in GeoGebra that show the origin and main properties of a helix and it also presents some constructive problems connected with the helix. There are created the step by step algorithms of some constructions in the chosen applets. Three-dimensional applets include illustrative helix samples and spatial animations that help students better see problems concerning the helix spatially. There is mentioned the website in the contribution on which there is situated GeoGebra book dedicated to the topic "Helix" and containing the mentioned applets. The created applets and materials of the GeoGebra book "Helix" help in teaching and studying the course Constructive Geometry determined for the students of the Faculty of Mechanical Engineering of the Technical University of Liberec.

  7. Equilibrium crossing exhibited by an ethynylhelicene (M)-nonamer during random-coil-to-double-helix thermal transition in solution.

    Science.gov (United States)

    Miyagawa, Masamichi; Yagi, Atsushi; Shigeno, Masanori; Yamaguchi, Masahiko

    2014-11-28

    The structural change between the random-coil and the double-helix of an ethynylhelicene (M)-nonamer during heating crosses equilibrium. This is a phenomenon where a chemical reaction crosses equilibrium and returns to equilibrium. It is due to an accelerated rate of formation of the double-helix by self-catalysis and an equilibrium shift.

  8. The Origins of Transmembrane Ion Channels

    Science.gov (United States)

    Pohorille, Andrew; Wilson, Michael A.

    2012-01-01

    Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

  9. Biophysical Aspects of Transmembrane Signaling

    CERN Document Server

    Damjanovich, Sandor

    2005-01-01

    Transmembrane signaling is one of the most significant cell biological events in the life and death of cells in general and lymphocytes in particular. Until recently biochemists and biophysicists were not accustomed to thinking of these processes from the side of a high number of complex biochemical events and an equally high number of physical changes at molecular and cellular levels at the same time. Both types of researchers were convinced that their findings are the most decisive, having higher importance than the findings of the other scientist population. Both casts were wrong. Life, even at cellular level, has a number of interacting physical and biochemical mechanisms, which finally build up the creation of an "excited" cell that will respond to particular signals from the outer or inner world. This book handles both aspects of the signalling events, and in some cases tries to unify our concepts and help understand the signals that govern the life and death of our cells. Not only the understanding, bu...

  10. Approaches to ab initio molecular replacement of α-helical transmembrane proteins

    OpenAIRE

    Thomas, Jens M. H.; Simkovic, Felix; Keegan, Ronan; Mayans, Olga; Zhang, Chengxin; Zhang, Yang; Rigden, Daniel J.

    2017-01-01

    α-Helical transmembrane proteins are a ubiquitous and important class of proteins, but present difficulties for crystallographic structure solution. Here, the effectiveness of the AMPLE molecular replacement pipeline in solving α-helical transmembrane-protein structures is assessed using a small library of eight ideal helices, as well as search models derived from ab initio models generated both with and without evolutionary contact information. The ideal helices prove to be surprisingly effe...

  11. Mapping side chain interactions at protein helix termini.

    Science.gov (United States)

    Newell, Nicholas E

    2015-07-25

    Interactions that involve one or more amino acid side chains near the ends of protein helices stabilize helix termini and shape the geometry of the adjacent loops, making a substantial contribution to overall protein structure. Previous work has identified key helix-terminal motifs, such as Asx/ST N-caps, the capping box, and hydrophobic and electrostatic interactions, but important questions remain, including: 1) What loop backbone geometries are favoured by each motif? 2) To what extent are multi-amino acid motifs likely to represent genuine cooperative interactions? 3) Can new motifs be identified in a large, recent dataset using the latest bioinformatics tools? Three analytical tools are applied here to answer these questions. First, helix-terminal structures are partitioned by loop backbone geometry using a new 3D clustering algorithm. Next, Cascade Detection, a motif detection algorithm recently published by the author, is applied to each cluster to determine which sequence motifs are overrepresented in each geometry. Finally, the results for each motif are presented in a CapMap, a 3D conformational heatmap that displays the distribution of the motif's overrepresentation across loop geometries, enabling the rapid isolation and characterization of the associated side chain interaction. This work identifies a library of geometry-specific side chain interactions that provides a new, detailed picture of loop structure near the helix terminus. Highlights include determinations of the favoured loop geometries for the Asx/ST N-cap motifs, capping boxes, "big" boxes, and other hydrophobic, electrostatic, H-bond, and pi stacking interactions, many of which have not been described before. This work demonstrates that the combination of structural clustering and motif detection in the sequence space can efficiently identify side chain motifs and map them to the loop geometries which they support. Protein designers should find this study useful, because it identifies side

  12. Crystal structure of a copper-transporting PIB-type ATPase

    DEFF Research Database (Denmark)

    Gourdon, Pontus Emanuel; Liu, Xiang-Yu; Skjørringe, Tina

    2011-01-01

    Heavy-metal homeostasis and detoxification is crucial for cell viability. P-type ATPases of the class IB (PIB) are essential in these processes, actively extruding heavy metals from the cytoplasm of cells. Here we present the structure of a PIB-ATPase, a Legionella pneumophila CopA Cu......(+)-ATPase, in a copper-free form, as determined by X-ray crystallography at 3.2 Å resolution. The structure indicates a three-stage copper transport pathway involving several conserved residues. A PIB-specific transmembrane helix kinks at a double-glycine motif displaying an amphipathic helix that lines a putative...... copper entry point at the intracellular interface. Comparisons to Ca(2+)-ATPase suggest an ATPase-coupled copper release mechanism from the binding sites in the membrane via an extracellular exit site. The structure also provides a framework to analyse missense mutations in the human ATP7A and ATP7B...

  13. Mutations in the SH1 helix alter the thermal properties of myosin II.

    Science.gov (United States)

    Shibata, Kotomi; Koyama, Tsubasa; Inde, Shohei; Iwai, Sosuke; Chaen, Shigeru

    2017-01-01

    The myosin II SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain and possibly plays a key role in the arrangement of the converter/lever arm. Several point mutations within the SH1 helix in human myosin IIs have been shown to cause diseases. To reveal whether these SH1 helix mutations affect not only motile activities but also thermal properties of myosin II, here we introduced the E683K or R686C point mutation into the SH1 helix in Dictyostelium myosin II. Thermal inactivation as well as thermal aggregation rates of these mutant proteins demonstrated that these mutations decreased the thermal stability of myosin II. Temperature dependence of sliding velocities of actin filaments showed that these mutations also reduced the activation energy of a rate-limiting process involved in actin movement. Given that these mutations are likely to alter coupling between the subdomains, and thus their thermal fluctuations, we propose that the SH1 helix is a key structural element that determines the flexibility and thermal properties of the myosin motor. These characteristics of the SH1 helix may contribute to the pathogenesis of the human diseases caused by mutations within this structural element.

  14. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD

    Directory of Open Access Journals (Sweden)

    Yu Wei-Hsuan

    2012-05-01

    Full Text Available Abstract Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C. The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6

  15. A smallest 6 kda metalloprotease, mini-matrilysin, in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD).

    Science.gov (United States)

    Yu, Wei-Hsuan; Huang, Po-Tsang; Lou, Kuo-Long; Yu, Shuan-Su C; Lin, Chen

    2012-05-29

    The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?" We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3 day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are

  16. The swimming of a perfect deforming helix

    Science.gov (United States)

    Koens, Lyndon; Zhang, Hang; Mourran, Ahmed; Lauga, Eric

    2017-11-01

    Many bacteria rotate helical flagellar filaments in order to swim. When at rest or rotated counter-clockwise these flagella are left handed helices but they undergo polymorphic transformations to right-handed helices when the motor is reversed. These helical deformations themselves can generate motion, with for example Rhodobacter sphaeroides using the polymorphic transformation of the flagellum to generate rotation, or Spiroplasma propagating a change of helix handedness across its body's length to generate forward motion. Recent experiments reported on an artificial helical microswimmer generating motion without a propagating change in handedness. Made of a temperature sensitive gel, these swimmers moved by changing the dimensions of the helix in a non-reciprocal way. Inspired by these results and helix's ubiquitous presence in the bacterial world, we investigate how a deforming helix moves within a viscous fluid. Maintaining a single handedness along its entire length, we discuss how a perfect deforming helix can create a non-reciprocal swimming stroke, identify its principle directions of motion, and calculate the swimming kinematics asymptotically.

  17. An extended CCR5 ECL2 peptide forms a helix that binds HIV-1 gp120 through non-specific hydrophobic interactions.

    Science.gov (United States)

    Abayev, Meital; Moseri, Adi; Tchaicheeyan, Oren; Kessler, Naama; Arshava, Boris; Naider, Fred; Scherf, Tali; Anglister, Jacob

    2015-05-01

    C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing extracellular loops 1 and 3 (ECL1 and ECL3) were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study, we found that, in aqueous buffer, the segment Q188-Q194 in an elongated ECL2 peptide (R168-K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two-dimensional saturation transfer difference NMR spectroscopy and dynamic filtering studies revealed involvement of Y187, F189, W190 and F193 of the helical segment in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule, and therefore could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions that are not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides, as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. The structures and NMR data of ECL2S (Q186-T195) were deposited under Protein Data Bank ID 2mzx and BioMagResBank ID 25505. © 2015 FEBS.

  18. Gold helix photonic metamaterial as broadband circular polarizer.

    Science.gov (United States)

    Gansel, Justyna K; Thiel, Michael; Rill, Michael S; Decker, Manuel; Bade, Klaus; Saile, Volker; von Freymann, Georg; Linden, Stefan; Wegener, Martin

    2009-09-18

    We investigated propagation of light through a uniaxial photonic metamaterial composed of three-dimensional gold helices arranged on a two-dimensional square lattice. These nanostructures are fabricated via an approach based on direct laser writing into a positive-tone photoresist followed by electrochemical deposition of gold. For propagation of light along the helix axis, the structure blocks the circular polarization with the same handedness as the helices, whereas it transmits the other, for a frequency range exceeding one octave. The structure is scalable to other frequency ranges and can be used as a compact broadband circular polarizer.

  19. Structure of a prokaryotic fumarate transporter reveals the architecture of the SLC26 family.

    Science.gov (United States)

    Geertsma, Eric R; Chang, Yung-Ning; Shaik, Farooque R; Neldner, Yvonne; Pardon, Els; Steyaert, Jan; Dutzler, Raimund

    2015-10-01

    The SLC26 family of membrane proteins combines a variety of functions within a conserved molecular scaffold. Its members, besides coupled anion transporters and channels, include the motor protein Prestin, which confers electromotility to cochlear outer hair cells. To gain insight into the architecture of this protein family, we characterized the structure and function of SLC26Dg, a facilitator of proton-coupled fumarate symport, from the bacterium Deinococcus geothermalis. Its modular structure combines a transmembrane unit and a cytoplasmic STAS domain. The membrane-inserted domain consists of two intertwined inverted repeats of seven transmembrane segments each and resembles the fold of the unrelated transporter UraA. It shows an inward-facing, ligand-free conformation with a potential substrate-binding site at the interface between two helix termini at the center of the membrane. This structure defines the common framework for the diverse functional behavior of the SLC26 family.

  20. Structure and function of the C-terminal domain of MrpA in the Bacillus subtilis Mrp-antiporter complex--the evolutionary progenitor of the long horizontal helix in complex I.

    Science.gov (United States)

    Virzintiene, Egle; Moparthi, Vamsi K; Al-Eryani, Yusra; Shumbe, Leonard; Górecki, Kamil; Hägerhäll, Cecilia

    2013-10-11

    MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. Structural basis for TatA oligomerization: an NMR study of Escherichia coli TatA dimeric structure.

    Science.gov (United States)

    Zhang, Yi; Hu, Yunfei; Li, Hongwei; Jin, Changwen

    2014-01-01

    Many proteins are transported across lipid membranes by protein translocation systems in living cells. The twin-arginine transport (Tat) system identified in bacteria and plant chloroplasts is a unique system that transports proteins across membranes in their fully-folded states. Up to date, the detailed molecular mechanism of this process remains largely unclear. The Escherichia coli Tat system consists of three essential transmembrane proteins: TatA, TatB and TatC. Among them, TatB and TatC form a tight complex and function in substrate recognition. The major component TatA contains a single transmembrane helix followed by an amphipathic helix, and is suggested to form the translocation pore via self-oligomerization. Since the TatA oligomer has to accommodate substrate proteins of various sizes and shapes, the process of its assembly stands essential for understanding the translocation mechanism. A structure model of TatA oligomer was recently proposed based on NMR and EPR observations, revealing contacts between the transmembrane helices from adjacent subunits. Herein we report the construction and stabilization of a dimeric TatA, as well as the structure determination by solution NMR spectroscopy. In addition to more extensive inter-subunit contacts between the transmembrane helices, we were also able to observe interactions between neighbouring amphipathic helices. The side-by-side packing of the amphipathic helices extends the solvent-exposed hydrophilic surface of the protein, which might be favourable for interactions with substrate proteins. The dimeric TatA structure offers more detailed information of TatA oligomeric interface and provides new insights on Tat translocation mechanism.

  2. Rationally designed transmembrane peptide mimics of the multidrug transporter protein Cdr1 act as antagonists to selectively block drug efflux and chemosensitize azole-resistant clinical isolates of Candida albicans.

    Science.gov (United States)

    Maurya, Indresh Kumar; Thota, Chaitanya Kumar; Verma, Sachin Dev; Sharma, Jyotsna; Rawal, Manpreet Kaur; Ravikumar, Balaguru; Sen, Sobhan; Chauhan, Neeraj; Lynn, Andrew M; Chauhan, Virander Singh; Prasad, Rajendra

    2013-06-07

    Drug-resistant pathogenic fungi use several families of membrane-embedded transporters to efflux antifungal drugs from the cells. The efflux pump Cdr1 (Candida drug resistance 1) belongs to the ATP-binding cassette (ABC) superfamily of transporters. Cdr1 is one of the most predominant mechanisms of multidrug resistance in azole-resistant (AR) clinical isolates of Candida albicans. Blocking drug efflux represents an attractive approach to combat the multidrug resistance of this opportunistic human pathogen. In this study, we rationally designed and synthesized transmembrane peptide mimics (TMPMs) of Cdr1 protein (Cdr1p) that correspond to each of the 12 transmembrane helices (TMHs) of the two transmembrane domains of the protein to target the primary structure of the Cdr1p. Several FITC-tagged TMPMs specifically bound to Cdr1p and blocked the efflux of entrapped fluorescent dyes from the AR (Gu5) isolate. These TMPMs did not affect the efflux of entrapped fluorescent dye from cells expressing the Cdr1p homologue Cdr2p or from cells expressing a non-ABC transporter Mdr1p. Notably, the time correlation of single photon counting fluorescence measurements confirmed the specific interaction of FITC-tagged TMPMs with their respective TMH. By using mutant variants of Cdr1p, we show that these TMPM antagonists contain the structural information necessary to target their respective TMHs of Cdr1p and specific binding sites that mediate the interactions between the mimics and its respective helix. Additionally, TMPMs that were devoid of any demonstrable hemolytic, cytotoxic, and antifungal activities chemosensitize AR clinical isolates and demonstrate synergy with drugs that further improved the therapeutic potential of fluconazole in vivo.

  3. Solvent-Directed Switch of a Left-Handed 10/12-Helix into a Right-Handed 12/10-Helix in Mixed β-Peptides.

    Science.gov (United States)

    Thodupunuri, Prashanth; Katukuri, Sirisha; Ramakrishna, Kallaganti V S; Sharma, Gangavaram V M; Kunwar, Ajit C; Sarma, Akella V S; Hofmann, Hans-Jörg

    2017-02-17

    Present study describes the synthesis and conformational analysis of β-peptides from C-linked carbo-β-amino acids [β-Caa (l) ] with a d-lyxo furanoside side chain and β-hGly in 1:1 alternation. NMR and CD investigations on peptides with an (S)-β-Caa (l) monomer at the N-terminus revealed a right-handed 10/12-mixed helix. An unprecedented solvent-directed "switch" both in helical pattern and handedness was observed when the sequence begins with a β-hGly residue instead of a (S)-β-Caa (l) constituent. NMR studies on these peptides in chloroform indicated a left-handed 10/12-helix, while the CD spectrum in methanol inferred a right-handed secondary structure. The NMR data for these peptides in CD 3 OH showed the presence of a right-handed 12/10-helix. NMR investigations in acetonitrile indicated the coexistence of both helix types. Quantum chemical studies predicted a small energy difference of 0.3 kcal/mol between the two helix types, which may explain the possibility of solvent influence. Examples for a solvent-directed switch of both the H-bonding pattern and the handedness of foldamer helices are rare so far. A comparable solvent effect was not found in the corresponding peptides with (R)-β-Caa (l) residues, where right-handed 12/10-helices are predominating.

  4. Structural dynamics of soluble chloride intracellular channel protein CLIC1 examined by amide hydrogen-deuterium exchange mass spectrometry.

    Science.gov (United States)

    Stoychev, Stoyan H; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L; Dirr, Heini W

    2009-09-08

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1, but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2, and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilizing domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix alpha1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand beta2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer.

  5. Spontaneous formation of structurally diverse membrane channel architectures from a single antimicrobial peptide

    Science.gov (United States)

    Wang, Yukun; Chen, Charles H.; Hu, Dan; Ulmschneider, Martin B.; Ulmschneider, Jakob P.

    2016-11-01

    Many antimicrobial peptides (AMPs) selectively target and form pores in microbial membranes. However, the mechanisms of membrane targeting, pore formation and function remain elusive. Here we report an experimentally guided unbiased simulation methodology that yields the mechanism of spontaneous pore assembly for the AMP maculatin at atomic resolution. Rather than a single pore, maculatin forms an ensemble of structurally diverse temporarily functional low-oligomeric pores, which mimic integral membrane protein channels in structure. These pores continuously form and dissociate in the membrane. Membrane permeabilization is dominated by hexa-, hepta- and octamers, which conduct water, ions and small dyes. Pores form by consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models. The diversity of the pore architectures--formed by a single sequence--may be a key feature in preventing bacterial resistance and could explain why sequence-function relationships in AMPs remain elusive.

  6. The C-Terminal RpoN Domain of sigma54 Forms an unpredictedHelix-Turn-Helix Motif Similar to domains of sigma70

    Energy Technology Data Exchange (ETDEWEB)

    Doucleff, Michaeleen; Malak, Lawrence T.; Pelton, Jeffrey G.; Wemmer, David E.

    2005-11-01

    The ''{delta}'' subunit of prokaryotic RNA-polymerase allows gene-specific transcription initiation. Two {sigma} families have been identified, {sigma}{sup 70} and {sigma}{sup 54}, which use distinct mechanisms to initiate transcription and share no detectable sequence homology. Although the {sigma}{sup 70}-type factors have been well characterized structurally by x-ray crystallography, no high-resolution structural information is available for the {sigma}{sup 54}-type factors. Here we present the NMR derived structure of the C-terminal domain of {sigma}{sup 54} from Aquifex aeolicus. This domain (Thr323 to Gly389), which contains the highly conserved RpoN box sequence, consists of a poorly structured N-terminal tail followed by a three-helix bundle, which is surprisingly similar to domains of the {sigma}{sup 70}-type proteins. Residues of the RpoN box, which have previously been shown to be critical for DNA binding, form the second helix of an unpredicted helix-turn-helix motif. This structure's homology with other DNA binding proteins, combined with previous biochemical data, suggest how the C-terminal domain of {sigma}{sup 54} binds to DNA.

  7. The Discovery of the Double Helix

    CERN Multimedia

    CERN. Geneva

    2011-01-01

    Professor James D. Watson has kindly agreed to make a presentation on the 1953 finding of the Double Helix at the Cavendish Laboratory by Francis Crick and himself. Being one of the greatest scientific discoveries in human history, little else needs to be added.

  8. The Modular Construction of DNA Double Helix

    Indian Academy of Sciences (India)

    DNA or the left-handed double helix,. Z- DNA. Construction of the Module .... 12. DNA, namely, replication and transcription. In the former case, Figure 3. 8 would represent a DNA single strand-generated by splitting of the mother duplex ...

  9. Helix reactor: great potential for flow chemistry

    NARCIS (Netherlands)

    Geerdink, P.; Runstraat, A. van den; Roelands, C.P.M.; Goetheer, E.L.V.

    2009-01-01

    The Helix reactor is highly suited for precise reaction control based on good hydrodynamics. The hydrodynamics are controlled by the Dean vortices, which create excellent heat transfer properties, approach plug flow and avoid turbulence. The flexibility of this reactor has been demonstrated using a

  10. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

    Science.gov (United States)

    Zhang, Xianjun; Zhao, Fei; Wu, Yiran; Yang, Jun; Han, Gye Won; Zhao, Suwen; Ishchenko, Andrii; Ye, Lintao; Lin, Xi; Ding, Kang; Dharmarajan, Venkatasubramanian; Griffin, Patrick R.; Gati, Cornelius; Nelson, Garrett; Hunter, Mark S.; Hanson, Michael A.; Cherezov, Vadim; Stevens, Raymond C.; Tan, Wenfu; Tao, Houchao; Xu, Fei

    2017-05-01

    The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

  11. Homologue Structure of the SLAC1 Anion Channel for Closing Stomata in Leaves

    Energy Technology Data Exchange (ETDEWEB)

    Y Chen; L Hu; M Punta; R Bruni; B Hillerich; B Kloss; B Rost; J Love; S Siegelbaum; W Hendrickson

    2011-12-31

    The plant SLAC1 anion channel controls turgor pressure in the aperture-defining guard cells of plant stomata, thereby regulating the exchange of water vapour and photosynthetic gases in response to environmental signals such as drought or high levels of carbon dioxide. Here we determine the crystal structure of a bacterial homologue (Haemophilus influenzae) of SLAC1 at 1.20 {angstrom} resolution, and use structure-inspired mutagenesis to analyse the conductance properties of SLAC1 channels. SLAC1 is a symmetrical trimer composed from quasi-symmetrical subunits, each having ten transmembrane helices arranged from helical hairpin pairs to form a central five-helix transmembrane pore that is gated by an extremely conserved phenylalanine residue. Conformational features indicate a mechanism for control of gating by kinase activation, and electrostatic features of the pore coupled with electrophysiological characteristics indicate that selectivity among different anions is largely a function of the energetic cost of ion dehydration.

  12. Structured and disordered facets of the GPCR fold.

    Science.gov (United States)

    Venkatakrishnan, A J; Flock, Tilman; Prado, Daniel Estévez; Oates, Matt E; Gough, Julian; Madan Babu, M

    2014-08-01

    The seven-transmembrane (7TM) helix fold of G-protein coupled receptors (GPCRs) has been adapted for a wide variety of physiologically important signaling functions. Here, we discuss the diversity in the structured and disordered regions of GPCRs based on the recently published crystal structures and sequence analysis of all human GPCRs. A comparison of the structures of rhodopsin-like receptors (class A), secretin-like receptors (class B), metabotropic receptors (class C) and frizzled receptors (class F) shows that the relative arrangement of the transmembrane helices is conserved across all four GPCR classes although individual receptors can be activated by ligand binding at varying positions within and around the transmembrane helical bundle. A systematic analysis of GPCR sequences reveals the presence of disordered segments in the cytoplasmic side, abundant post-translational modification sites, evidence for alternative splicing and several putative linear peptide motifs that have the potential to mediate interactions with cytosolic proteins. While the structured regions permit the receptor to bind diverse ligands, the disordered regions appear to have an underappreciated role in modulating downstream signaling in response to the cellular state. An integrated paradigm combining the knowledge of structured and disordered regions is imperative for gaining a holistic understanding of the GPCR (un)structure-function relationship. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. High constitutive activity of a virus-encoded seven transmembrane receptor in the absence of the conserved DRY motif (Asp-Arg-Tyr) in transmembrane helix 3

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Kledal, Thomas N; Schwartz, Thue W

    2005-01-01

    -driven transcriptional activity through a pertussis toxin-sensitive manner. Gs and Gq were not activated constitutively as determined by the lack of inositol phosphate turnover and activities of the three transcription factors: cAMP response element-binding protein (CREB), nuclear factor-kappaB, and nuclear factor...

  14. Reversible Dimerization of Acid-Denatured ACBP Controlled by Helix A4

    DEFF Research Database (Denmark)

    Fieber, Wolfgang; Kragelund, Birthe Brandt; Meldal, Morten Peter

    2005-01-01

    denaturing conditions at pH 2.3, helix conformations are still populated in 24% of the ensemble of molecules. The structure of HA4 at atomic resolution was assessed using nuclear magnetic resonance (NMR) spectroscopy. Long-range NOEs between remote residues at opposite peptide ends suggested the formation...... of dimers and revealed a cooperative stabilization of helix A4 in this process. This emphasizes its special role in the structure formation in the denatured state of ACBP. No dimers are formed in the presence of guanidine hydrochloride, which underlines the fundamental difference between the nature...

  15. Molecular Insights into the Transmembrane Domain of the Thyrotropin Receptor.

    Directory of Open Access Journals (Sweden)

    Vanessa Chantreau

    Full Text Available The thyrotropin receptor (TSHR is a G protein-coupled receptor (GPCR that is member of the leucine-rich repeat subfamily (LGR. In the absence of crystal structure, the success of rational design of ligands targeting the receptor internal cavity depends on the quality of the TSHR models built. In this subfamily, transmembrane helices (TM 2 and 5 are characterized by the absence of proline compared to most receptors, raising the question of the structural conformation of these helices. To gain insight into the structural properties of these helices, we carried out bioinformatics and experimental studies. Evolutionary analysis of the LGR family revealed a deletion in TM5 but provided no information on TM2. Wild type residues at positions 2.58, 2.59 or 2.60 in TM2 and/or at position 5.50 in TM5 were substituted to proline. Depending on the position of the proline substitution, different effects were observed on membrane expression, glycosylation, constitutive cAMP activity and responses to thyrotropin. Only proline substitution at position 2.59 maintained complex glycosylation and high membrane expression, supporting occurrence of a bulged TM2. The TSHR transmembrane domain was modeled by homology with the orexin 2 receptor, using a protocol that forced the deletion of one residue in the TM5 bulge of the template. The stability of the model was assessed by molecular dynamics simulations. TM5 straightened during the equilibration phase and was stable for the remainder of the simulations. Our data support a structural model of the TSHR transmembrane domain with a bulged TM2 and a straight TM5 that is specific of glycoprotein hormone receptors.

  16. Tunable single photonic defect-mode in cholesteric liquid crystals with laser-induced local modifications of helix

    International Nuclear Information System (INIS)

    Yoshida, Hiroyuki; Lee, Chee Heng; Fujii, Akihiko; Ozaki, Masanori

    2006-01-01

    The authors demonstrate a tunable single photonic defect-mode in a single cholesteric liquid crystal material based on a structural defect introduced by local modification of the helix. An unpolymerized region of cholesteric liquid crystal acting as the defect was left between two polymerized regions via a two-photon excitation laser-lithography process. Upon polymerization, the cholesteric liquid crystal helix elongated and became thermally stable, and a single photonic defect mode was exhibited due to the contrast in the helix pitch at the defect. The defect mode showed tunability upon heating, and a 36 nm redshift was seen over a temperature range of 30 deg. C

  17. pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides.

    Science.gov (United States)

    Takei, Toshiaki; Tsumoto, Kouhei; Okonogi, Atsuhito; Kimura, Akiko; Kojima, Shuichi; Yazaki, Kazumori; Takei, Tsunetomo; Ueda, Takuya; Miura, Kin-ichiro

    2015-05-01

    We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)3, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)3, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)3, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)3; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation. © 2015 The Protein Society.

  18. Triple helix interactions for eco-innovation

    DEFF Research Database (Denmark)

    Hermann, Roberto Rivas; Riisgaard, Henrik; Remmen, Arne

    the role of science parks in promoting eco-innovation. This study uses qualitative data gathered in two units of analysis: Panama Canal Authority and City of Knowledge Science Park. The study examines how Triple Helix interactions have built the regional system of eco-innovation at the Panama Canal....... Overall, the research found that the Panamanian national innovation system facilitates eco-innovation by: providing research and development, building competence and financing of innovation processes. The “green maritime route” is an example of institutional eco-innovation promoted by the Panama Canal...... Authority with insights from consultants, universities and donnor agencies. The proximity of the science park to the canal, has hitherto not yielded with the creation of a “green cluster”, which could be a precedent to promote eco-innovations. These findings suggest that, Triple Helix interactions...

  19. FPGA helix tracking algorithm for PANDA

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Yutie; Galuska, Martin; Gessler, Thomas; Kuehn, Wolfgang; Lange, Jens Soeren; Muenchow, David; Spruck, Bjoern [II. Physikalisches Institut, Giessen University (Germany); Ye, Hua [Institute of High Energy Physics, Beijing (China); Collaboration: PANDA-Collaboration

    2015-07-01

    The PANDA detector is a general-purpose detector for physics with high luminosity cooled antiproton beams, planed to operate at the FAIR facility in Darmstadt, Germany. The central detector includes a silicon Micro Vertex Detector (MVD) and a Straw Tube Tracker (STT). Without any hardware trigger, large amounts of raw data are streaming into the data acquisition system. The data reduction task is performed in the online system by reconstruction algorithms programmed on FPGAs (Field Programmable Gate Arrays) as first level and on a farm of GPUs or PCs as a second level. One important part in the system is the online track reconstruction. In this presentation, an online tracking algorithm for helix tracking reconstruction in the solenoidal field is shown. The tracking algorithm is composed by two parts, a road finding module followed by an iterative helix parameter calculation module. A performance study using C++ and the status of the VHDL implementation are presented.

  20. Nucleic acid helices: I. Structure of M1 RNA from E. coli as determined bypsoralen crosslinking. II. Thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing 3.0 M tetramethylammonium chloride

    International Nuclear Information System (INIS)

    Lipson, S.E.

    1987-01-01

    This work includes two different investigations examining nucleic acid helices. The first study discusses secondary and tertiary interactions in the RNA moiety of ribonuclease P from Escherichia coli. The second study discusses the thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing tetramethylammonium chloride. The RNA moiety of ribonuclease P from Escherichia coli (M1 RNA) has been photoreacted with 4'-hydroxymethyl-4,5'8-trimethylpsoralen and long wave UV light (320-380 nm) in a buffer in which the M1 RNA alone acts as a true catalyst of tRNA processing. Limited specific digestion followed by two dimensional gel electrophoresis yields fragments crosslinked by HMT. The positions of the crosslinks have been determined to within ±15 nucleotides by photoreversal of the isolated crosslinked fragments and enzymatic sequencing of the resulting RNA. Further assignments of the exact locations of the crosslinks have been made on the known photoreactivity of the psoralen with different bases

  1. PDBTM: Protein Data Bank of transmembrane proteins after 8 years

    OpenAIRE

    Kozma, D?niel; Simon, Istv?n; Tusn?dy, G?bor E.

    2012-01-01

    The PDBTM database (available at http://pdbtm.enzim.hu), the first comprehensive and up-to-date transmembrane protein selection of the Protein Data Bank, was launched in 2004. The database was created and has been continuously updated by the TMDET algorithm that is able to distinguish between transmembrane and non-transmembrane proteins using their 3D atomic coordinates only. The TMDET algorithm can locate the spatial positions of transmembrane proteins in lipid bilayer as well. During the la...

  2. Evidence for genetic control of adult weight plasticity in the snail Helix aspersa

    DEFF Research Database (Denmark)

    Ros, Mathieu; Sorensen, Daniel; Waagepetersen, Rasmus Plenge

    2004-01-01

    of adult weight in the snail Helix aspersa. Several models of heterogeneous variance are fitted using a Bayesin, MCMC approach. Exploratory analyses using posterior predictive model checking and model comparisons based on the deviance information criterion favor a model postulating a genetically structured...... is illustrated numerically using estimates of parameters derived from the snail data set....

  3. Extension of Helix 12 in Munc18-1 Induces Vesicle Priming

    DEFF Research Database (Denmark)

    Munch, Anders S; Kedar, Girish H; van Weering, Jan R T

    2016-01-01

    Munc18-1 is essential for vesicle fusion and participates in the docking of large dense-core vesicles to the plasma membrane. Recent structural data suggest that conformational changes in the 12th helix of the Munc18-1 domain 3a within the Munc18-1:syntaxin complex result in an additional interac...

  4. Microsecond and nanosecond polyproline II helix formation in aqueous nanodrops measured by mass spectrometry.

    Science.gov (United States)

    Mortensen, Daniel N; Williams, Evan R

    2016-10-06

    The 1.5 μs and <400 ns time constants for the formation of polyproline II helix structures in 21 and 16 residue peptides, respectively, are measured using rapid mixing from theta-glass emitters coupled with mass spectrometry. Results from these studies should serve as useful benchmarks for comparison with computational simulation results.

  5. A basic helix-loop-helix transcription factor DvIVS determines flower color intensity in cyanic dahlia cultivars.

    Science.gov (United States)

    Ohno, Sho; Deguchi, Ayumi; Hosokawa, Munetaka; Tatsuzawa, Fumi; Doi, Motoaki

    2013-08-01

    The study was aimed to identify the factors that regulate the intensity of flower color in cyanic dahlia (Dahlia variabilis), using fifteen cultivars with different color intensities in their petals. The cultivars were classified into three groups based on their flavonoid composition: ivory white cultivars with flavones; purple and pink cultivars with flavones and anthocyanins; and red cultivars with flavones, anthocyanins, and chalcones. Among the purple, pink, and ivory white cultivars, an inverse relationship was detected between lightness, which was used as an indicator for color intensity and anthocyanin content. A positive correlation was detected between anthocyanin contents and the expression of some structural genes in the anthocyanin synthesis pathway that are regulated by DvIVS, a basic helix-loop-helix transcription factor. A positive correlation between anthocyanin content and expression of DvIVS was also found. The promoter region of DvIVS was classified into three types, with cultivars carrying Type 1 promoter exhibited deep coloring, those carrying Type 2 and/or Type 3 exhibited pale coloring, and those carrying Type 1 and Type 2 and/or Type 3 exhibited medium coloring. The transcripts of the genes from these promoters encoded full-length predicted proteins. These results suggested that the genotype of the promoter region in DvIVS is one of the key factors determining the flower color intensity.

  6. Nucleic acid helices: I. Structure of M1 RNA from E. coli as determined bypsoralen crosslinking. II. Thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing 3. 0 M tetramethylammonium chloride

    Energy Technology Data Exchange (ETDEWEB)

    Lipson, S.E.

    1987-01-01

    This work includes two different investigations examining nucleic acid helices. The first study discusses secondary and tertiary interactions in the RNA moiety of ribonuclease P from Escherichia coli. The second study discusses the thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing tetramethylammonium chloride. The RNA moiety of ribonuclease P from Escherichia coli (M1 RNA) has been photoreacted with 4{prime}-hydroxymethyl-4,5{prime}8-trimethylpsoralen and long wave UV light (320-380 nm) in a buffer in which the M1 RNA alone acts as a true catalyst of tRNA processing. Limited specific digestion followed by two dimensional gel electrophoresis yields fragments crosslinked by HMT. The positions of the crosslinks have been determined to within {plus minus}15 nucleotides by photoreversal of the isolated crosslinked fragments and enzymatic sequencing of the resulting RNA. Further assignments of the exact locations of the crosslinks have been made on the known photoreactivity of the psoralen with different bases.

  7. Effects of ligands on unfolding of the amyloid β-peptide central helix: mechanistic insights from molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Mika Ito

    Full Text Available Polymerization of the amyloid β-peptide (Aβ, a process which requires that the helical structure of Aβ unfolds beforehand, is suspected to cause neurodegeneration in Alzheimer's disease. According to recent experimental studies, stabilization of the Aβ central helix counteracts Aβ polymerization into toxic assemblies. The effects of two ligands (Dec-DETA and Pep1b, which were designed to bind to and stabilize the Aβ central helix, on unfolding of the Aβ central helix were investigated by molecular dynamics simulations. It was quantitatively demonstrated that the stability of the Aβ central helix is increased by both ligands, and more effectively by Pep1b than by Dec-DETA. In addition, it was shown that Dec-DETA forms parallel conformations with β-strand-like Aβ, whereas Pep1b does not and instead tends to bend unwound Aβ. The molecular dynamics results correlate well with previous experiments for these ligands, which suggest that the simulation method should be useful in predicting the effectiveness of novel ligands in stabilizing the Aβ central helix. Detailed Aβ structural changes upon loss of helicity in the presence of the ligands are also revealed, which gives further insight into which ligand may lead to which path subsequent to unwinding of the Aβ central helix.

  8. Left-handed polyproline-II helix revisited: proteins causing proteopathies.

    Science.gov (United States)

    Adzhubei, Alexei A; Anashkina, Anastasia A; Makarov, Alexander A

    2017-09-01

    Left-handed polyproline-II type helix is a regular conformation of polypeptide chain not only of fibrous, but also of folded and natively unfolded proteins and peptides. It is the only class of regular secondary structure substantially represented in non-fibrous proteins and peptides on a par with right-handed alpha-helix and beta-structure. In this study, we have shown that polyproline-II helix is abundant in several peptides and proteins involved in proteopathies, the amyloid-beta peptides, protein tau and prion protein. Polyproline-II helices form two interaction sites in the amyloid-beta peptides, which are pivotal for pathogenesis of Alzheimer's disease (AD). It also with high probability is the structure of the majority of tau phosphorylation sites, important for tau hyperphosphorylation and formation of neurofibrillary tangles, a hallmark of AD. Polyproline-II helices form large parts of the structure of the folded domain of prion protein. They can undergo conversion to beta-structure as a result of relatively small change of one torsional angle of polypeptide chain. We hypothesize that in prions and amyloids, in general polyproline-II helices can serve as structural elements of the normal structure as well as dormant nuclei of structure conversion, and thus play important role in structure changes leading to the formation of fibrils.

  9. Transmembrane topology of FRO2, a ferric chelate reductase from Arabidopsis thaliana.

    Science.gov (United States)

    Schagerlöf, Ulrika; Wilson, Greer; Hebert, Hans; Al-Karadaghi, Salam; Hägerhäll, Cecilia

    2006-09-01

    Iron uptake in Arabidopsis thaliana is mediated by ferric chelate reductase FRO2, a transmembrane protein belonging to the flavocytochrome b family. There is no high resolution structural information available for any member of this family. We have determined the transmembrane topology of FRO2 experimentally using the alkaline phosphatase fusion method. The resulting topology is different from that obtained by theoretical predictions and contains 8 transmembrane helices, 4 of which build up the highly conserved core of the protein. This core is present in the entire flavocytochrome b family. The large water soluble domain of FRO2, which contains NADPH, FAD and oxidoreductase sequence motifs, was located on the inside of the membrane.

  10. Government and Governance of Regional Triple Helix Interactions

    Science.gov (United States)

    Danson, Mike; Todeva, Emanuela

    2016-01-01

    This conceptual paper contributes to the discussion of the role of regional government and regional Triple Helix constellations driving economic development and growth within regional boundaries. The impact of regionalism and subsidiarity on regional Triple Helix constellations, and the questions of governmentality, governance and institutional…

  11. FPGA helix tracking algorithm for PANDA

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Yutie; Galuska, Martin; Gessler, Thomas; Kuehn, Wolfgang; Lange, Jens Soeren; Muenchow, David [II. Physikalisches Institut, University of Giessen (Germany); Ye, Hua [Institute of High Energy Physics, CAS (China); Collaboration: PANDA-Collaboration

    2016-07-01

    The PANDA detector is a general-purpose detector for physics with high luminosity cooled antiproton beams, planed to operate at the FAIR facility in Darmstadt, Germany. The central detector includes a silicon Micro Vertex Detector (MVD) and a Straw Tube Tracker (STT). Without any hardware trigger, large amounts of raw data are streaming into the data acquisition system. The data reduction task is performed in the online system by reconstruction algorithms programmed on FPGAs (Field Programmable Gate Arrays) as first level and on a farm of GPUs or PCs as a second level. One important part in the system is the online track reconstruction. In this presentation, an online tracking algorithm for helix tracking reconstruction in the solenoidal field is shown. The VHDL-based algorithm is tested with different types of events, at different event rate. Furthermore, a study of T0 extraction from the tracking algorithm is performed. A concept of simultaneous tracking and T0 determination is presented.

  12. FPGA helix tracking algorithm for PANDA

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Yutie; Galuska, Martin; Gessler, Thomas; Hu, Jifeng; Kuehn, Wolfgang; Lange, Jens Soeren; Muenchow, David; Spruck, Bjoern [II. Physikalisches, Giessen University (Germany); Ye, Hua [II. Physikalisches, Giessen University (Germany); Institute of High Energy Physics, Beijing (China); Collaboration: PANDA-Collaboration

    2014-07-01

    The PANDA detector is a general-purpose detector for physics with high luminosity cooled antiproton beams, planed to operate at the FAIR facility in Darmstadt, Germany. The central detector includes a silicon Micro Vertex Detector (MVD) and a Straw Tube Tracker (STT). Without any hardware trigger, large amounts of raw data are streaming into the data acquisition system. The data reduction task is performed in the online system by reconstruction algorithms programmed in VHDL (Very High Speed Integrated Circuit Hardware Description Language) on FPGAs (Field Programmable Gate Arrays) as first level and on a farm of GPUs or PCs as a second level. One important part in the system is the online track reconstruction. In this presentation, an online tracking finding algorithm for helix track reconstruction in the solenoidal field is shown. A performance study using C++ and the status of the VHDL implementation are presented.

  13. Evolution of vertebrate interferon inducible transmembrane proteins

    Directory of Open Access Journals (Sweden)

    Hickford Danielle

    2012-04-01

    Full Text Available Abstract Background Interferon inducible transmembrane proteins (IFITMs have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates. Results Five IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein. Conclusions Like eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution.

  14. Basic helix-loop-helix transcription factors and epidermal cell fate determination in Arabidopsis.

    Science.gov (United States)

    Zhao, Hongtao; Li, Xia; Ma, Ligeng

    2012-12-01

    Cell fate determination is an important process in multicellular organisms. Plant epidermis is a readily-accessible, well-used model for the study of cell fate determination. Our knowledge of cell fate determination is growing steadily due to genetic and molecular analyses of root hairs, trichomes, and stomata, which are derived from the epidermal cells of roots and aerial tissues. Studies have shown that a large number of factors are involved in the establishment of these cell types, especially members of the basic helix-loop-helix (bHLH) superfamily, which is an important family of transcription factors. In this mini-review, we focus on the role of bHLH transcription factors in cell fate determination in Arabidopsis.

  15. Volumetric Physics of Polypeptide Coil-Helix Transitions.

    Science.gov (United States)

    Krobath, Heinrich; Chen, Tao; Chan, Hue Sun

    2016-11-15

    Volumetric properties of proteins bear directly on their biological functions in hyperbaric environments and are useful in general as a biophysical probe. To gain insight into conformation-dependent protein volume, we developed an implicit-solvent atomic chain model that transparently embodies two physical origins of volume: (1) a fundamental geometric term capturing the van der Waals volume of the protein and the particulate, finite-size nature of the water molecules, modeled together by the volume encased by the protein's molecular surface, and (2) a physicochemical term for other solvation effects, accounted for by empirical proportionality relationships between experimental partial molar volumes and solvent-accessible surface areas of model compounds. We tested this construct by Langevin dynamics simulations of a 16-residue polyalanine. The simulated trajectories indicate an average volume decrease of 1.73 ± 0.1 Å 3 /residue for coil-helix transition, ∼80% of which is caused by a decrease in geometric void/cavity volume, and a robust positive activation volume for helical hydrogen bond formation originating from the transient void created by an approaching donor-acceptor pair and nearby atoms. These findings are consistent with prior experiments with alanine-rich peptides and offer an atomistic analysis of the observed overall volume changes. The results suggest, in general, that hydrostatic pressure likely stabilizes helical conformations of short peptides but slows the process of helix formation. In contrast, hydrostatic pressure is more likely to destabilize natural globular proteins because of the void volume entrapped in their folded structures. The conceptual framework of our model thus affords a coherent physical rationalization for experiments.

  16. Large deformation of helix F during the photoreaction cycle of Pharaonis halorhodopsin in complex with azide.

    Science.gov (United States)

    Nakanishi, Taichi; Kanada, Soun; Murakami, Midori; Ihara, Kunio; Kouyama, Tsutomu

    2013-01-22

    Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl(-) and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (∼4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. Efficient Fatigue Analysis of Helix Elements in Umbilicals and Flexible Risers: Theory and Applications

    Directory of Open Access Journals (Sweden)

    Geir Skeie

    2012-01-01

    Full Text Available Fatigue analysis of structural components such as helix tensile armors and steel tubes is a critical design issue for dynamic umbilicals and flexible pipes. The basis for assessment of fatigue damage of such elements is the long-term stress cycle distribution at critical locations on the helix elements caused by long-term environmental loading on the system. The long-term stress cycle distribution will hence require global dynamic time domain analysis followed by a detailed cross-sectional analysis in a large number of irregular sea states. An overall computational consistent and efficient fatigue analysis scheme is outlined with due regard of the cross-sectional analysis technique required for fatigue stress calculation with particular attention to the helix elements. The global cross-section is exposed to pure bending, tensile, torsion, and pressure loading. The state of the different cross-section elements is based on the global response. Special emphasis is placed on assessment of friction stresses caused by the stick-slip behavior of helix elements in bending that are of special importance for fatigue life assessments. The described cross-sectional analysis techniques are based on an extensive literature survey and are hence considered to represent industry consensus. The performance of the described calculation scheme is illustrated by case studies.

  18. MutHTP: Mutations in Human Transmembrane Proteins.

    Science.gov (United States)

    A, Kulandaisamy; S, Binny Priya; R, Sakthivel; Tarnovskaya, Svetlana; Bizin, Ilya; Hönigschmid, Peter; Frishman, Dmitrij; Gromiha, M Michael

    2018-02-01

    We have developed a novel database, MutHTP, which contains information on 183395 disease-associated and 17827 neutral mutations in human transmembrane proteins. For each mutation site MutHTP provides a description of its location with respect to the membrane protein topology, structural environment (if available) and functional features. Comprehensive visualization, search, display and download options are available. The database is publicly available at http://www.iitm.ac.in/bioinfo/MutHTP/. The website is implemented using HTML, PHP and javascript and supports recent versions of all major browsers, such as Firefox, Chrome and Opera. gromiha@iitm.ac.in. Supplementary data are available at Bioinformatics online. © The Author (2018). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  19. Effect of ionizing radiation on transmembrane potential of Streptococcus

    International Nuclear Information System (INIS)

    Fomenko, B.S.; Akoev, I.G.

    1979-01-01

    Treatment of Streptococcus faecalis with ionizing radiation at doses of 5 to 100 krad is shown to reduce the energy-dependent accumulation of dibenzyldimethylammonium (DDA + ) by the cell. Since transmembrane potential is the moving force of DDA + transport across the membrane, the decrease in DDA + accumulation is suggested to be due to potential reduction. This radiation effect was not due to inactivation of the potential-generating mechanism; thus, the ATPase activity and glycolytic activity of the irradiated cells were higher than in the control. At the same time, the membranes exhibited an increased permeability for K + and protons, which is probably due to structural rearrangements in the membranes after irradiation. It is suggested that the potential reduction results from the increase in proton permeability of membranes

  20. The Effect of a Helix-Coil Transition on the Extension Elasticity

    Science.gov (United States)

    Buhot, Arnaud; Halperin, Avi

    2000-03-01

    The secondary structure of a polymer affects its deformation behavior in accordance with the Le Chatelier principle. An important example of such secondary structure is the alpha helix encountered in polypeptides. Similar structure was recently proposed for PEO in aqueous media. Our discussion concerns the coupling of the cooperative helix-coil transition and the extension elasticity. In particular, we analyze the extension of a long single chain by use of optical tweezers or AFM. We consider chains that exist in the coil-state when unperturbed. The transition nevertheless occurs because the extension favors the low entropy helical state. As a result, the corresponding force law exhibits a plateau. The analysis of this situation involves two ingredients: (I) the stretching free energy penalty for a rod-coil mutiblock copolymer (II) the entropy associated with the possible placements of the rod and coil blocks.

  1. GPCR-I-TASSER: A hybrid approach to G protein-coupled receptor structure modeling and the application to the human genome

    Science.gov (United States)

    Zhang, Jian; Yang, Jianyi; Jang, Richard; Zhang, Yang

    2015-01-01

    SUMMARY Experimental structure determination remains very difficult for G protein-coupled receptors (GPCRs). We propose a new hybrid protocol to construct GPCR structure models that integrates experimental mutagenesis data with ab initio transmembrane (TM) helix assembly simulations. The method was tested on 24 known GPCRs where the ab initio TM-helix assembly procedure constructed the correct fold for 20 cases. When combined with weak-homology and sparse mutagenesis restraints, the method generated correct folds for all the tested cases with an average C-alpha RMSD 2.4 Å in the TM-regions. The new hybrid protocol was applied to model all 1026 GPCRs in the human genome, where 923 have a high confidence score that are expected to have correct folds; these contain many pharmaceutically important families with no previously solved structures, including Trace amine, Prostanoids, Releasing hormones, Melanocortins, Vasopressin and Neuropeptide Y receptors. The results demonstrate new progress on genome-wide structure modeling of transmembrane proteins. PMID:26190572

  2. DNA-Tile Structures Induce Ionic Currents through Lipid Membranes.

    Science.gov (United States)

    Göpfrich, Kerstin; Zettl, Thomas; Meijering, Anna E C; Hernández-Ainsa, Silvia; Kocabey, Samet; Liedl, Tim; Keyser, Ulrich F

    2015-05-13

    Self-assembled DNA nanostructures have been used to create man-made transmembrane channels in lipid bilayers. Here, we present a DNA-tile structure with a nominal subnanometer channel and cholesterol-tags for membrane anchoring. With an outer diameter of 5 nm and a molecular weight of 45 kDa, the dimensions of our synthetic nanostructure are comparable to biological ion channels. Because of its simple design, the structure self-assembles within a minute, making its creation scalable for applications in biology. Ionic current recordings demonstrate that the tile structures enable ion conduction through lipid bilayers and show gating and voltage-switching behavior. By demonstrating the design of DNA-based membrane channels with openings much smaller than that of the archetypical six-helix bundle, our work showcases their versatility inspired by the rich diversity of natural membrane components.

  3. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  4. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  5. Cooperative formation of native-like tertiary contacts in the ensemble of unfolded states of a four-helix protein

    DEFF Research Database (Denmark)

    Bruun, Susanne W; Iesmantavicius, Vytautas; Danielsson, Jens

    2010-01-01

    In studies of the ensembles of unfolded structures of a four-helix bundle protein, we have detected the presence of potential precursors of native tertiary structures. These observations were based on the perturbation of NMR chemical shifts of the protein backbone atoms by single site mutations. ...

  6. Virus-Encoded 7 Transmembrane Receptors

    DEFF Research Database (Denmark)

    Mølleskov-Jensen, Ann-Sofie; Oliveira, MarthaTrindade; Farrell, Helen Elizabeth

    2015-01-01

    have acquired a range of distinctive characteristics. This chapter reviews key features of the v7TMRs which are likely to impact upon their functional roles: trafficking properties, ligand specificity, and signaling capacity. Rapid, constitutive endocytosis, reminiscent of cellular “scavenger......Herpesviruses are an ancient group which have exploited gene capture of multiple cellular modulators of the immune response. Viral homologues of 7 transmembrane receptors (v7TMRs) are a consistent feature of beta- and gammaherpesviruses; the majority of the v7TMRs are homologous to cellular...... chemokine receptors (CKRs). Conserved families of v7TMRs distinguish between beta- versus gammaherpesviruses; furthermore, significant divisions within these subfamilies, such as between genera of the gammaherpesviruses or between the primate and rodent cytomegaloviruses, coincide with specific v7TMR gene...

  7. Specificity of transmembrane protein palmitoylation in yeast.

    Directory of Open Access Journals (Sweden)

    Ayelén González Montoro

    Full Text Available Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs, characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD. There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as

  8. Molecular mechanisms for generating transmembrane proton gradients

    Science.gov (United States)

    Gunner, M.R.; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side. PMID:23507617

  9. Effect of Hedera helix on lung histopathology in chronic asthma.

    Science.gov (United States)

    Hocaoglu, Arzu Babayigit; Karaman, Ozkan; Erge, Duygu Olmez; Erbil, Guven; Yilmaz, Osman; Kivcak, Bijen; Bagriyanik, H Alper; Uzuner, Nevin

    2012-12-01

    Hedera helix is widely used to treat bronchial asthma for many years. However, effects of this herb on lung histopathology is still far from clear. We aimed to determine the effect of oral administration of Hedera helix on lung histopathology in a murine model of chronic asthma.BALB/c mice were divided into four groups; I (Placebo), II (Hedera helix), III (Dexamethasone) and IV (Control). All mice except controls were sensitized and challenged with ovalbumin. Then, mice in group I received saline, group II 100 mg/kg Hedera helix and group III 1 mg/kg dexamethasone via orogastic gavage once daily for one week. Airway histopathology was evaluated by using light and electron microscopy in all groups.Goblet cell numbers and thicknesses of basement membrane were found significantly lower in group II, but there was no statistically significant difference in terms of number of mast cells, thicknesses of epithelium and subepithelial smooth muscle layers between group I and II. When Hedera helix and dexamethasone groups were compared with each other, thickness of epithelium, subepithelial muscle layers, number of mast cells and goblet cells of group III were significantly ameliorated when compared with the group II. Although Hedera helix administration reduced only goblet cell counts and the thicknesses of basement membrane in the asthmatic airways, dexamethasone ameliorated all histopathologic parameters except thickness of basement membrane better than Hedera helix.

  10. A rare polyglycine type II-like helix motif in naturally occurring proteins.

    Science.gov (United States)

    Warkentin, Eberhard; Weidenweber, Sina; Schühle, Karola; Demmer, Ulrike; Heider, Johann; Ermler, Ulrich

    2017-11-01

    Common structural elements in proteins such as α-helices or β-sheets are characterized by uniformly repeating, energetically favorable main chain conformations which additionally exhibit a completely saturated hydrogen-bonding network of the main chain NH and CO groups. Although polyproline or polyglycine type II helices (PP II or PG II ) are frequently found in proteins, they are not considered as equivalent secondary structure elements because they do not form a similar self-contained hydrogen-bonding network of the main chain atoms. In this context our finding of an unusual motif of glycine-rich PG II -like helices in the structure of the acetophenone carboxylase core complex is of relevance. These PG II -like helices form hexagonal bundles which appear to fulfill the criterion of a (largely) saturated hydrogen-bonding network of the main-chain groups and therefore may be regarded in this sense as a new secondary structure element. It consists of a central PG II -like helix surrounded by six nearly parallel PG II -like helices in a hexagonal array, plus an additional PG II -like helix extending the array outwards. Very related structural elements have previously been found in synthetic polyglycine fibers. In both cases, all main chain NH and CO groups of the central PG II -helix are saturated by either intra- or intermolecular hydrogen-bonds, resulting in a self-contained hydrogen-bonding network. Similar, but incomplete PG II -helix patterns were also previously identified in a GTP-binding protein and an antifreeze protein. © 2017 Wiley Periodicals, Inc.

  11. Approaches to ab initio molecular replacement of α-helical transmembrane proteins.

    Science.gov (United States)

    Thomas, Jens M H; Simkovic, Felix; Keegan, Ronan; Mayans, Olga; Zhang, Chengxin; Zhang, Yang; Rigden, Daniel J

    2017-12-01

    α-Helical transmembrane proteins are a ubiquitous and important class of proteins, but present difficulties for crystallographic structure solution. Here, the effectiveness of the AMPLE molecular replacement pipeline in solving α-helical transmembrane-protein structures is assessed using a small library of eight ideal helices, as well as search models derived from ab initio models generated both with and without evolutionary contact information. The ideal helices prove to be surprisingly effective at solving higher resolution structures, but ab initio-derived search models are able to solve structures that could not be solved with the ideal helices. The addition of evolutionary contact information results in a marked improvement in the modelling and makes additional solutions possible.

  12. The effect of C-terminal helix on the stability of FF domain studied by molecular dynamics simulation.

    Science.gov (United States)

    Zhao, Liling; Cao, Zanxia; Wang, Jihua

    2012-01-01

    To investigate the effect of C-terminal helix on the stability of the FF domain, we studied the native domain FF3-71 from human HYPA/FBP11 and the truncated version FF3-60 with C-terminal helix being deleted by molecular dynamics simulations with GROMACS package and GROMOS 43A1 force field. The results indicated that the structures of truncated version FF3-60 were evident different from those of native partner FF3-71. Compared with FF3-71, the FF3-60 lost some native contacts and exhibited some similar structural characters to those of intermediate state. The C-terminal helix played a major role in stabilizing the FF3-71 domain. To a certain degree, the FF domain had a tendency to form an intermediate state without the C-terminal helix. In our knowledge, this was the first study to examine the role of C-terminal helix of FF domain in detail by molecular dynamics simulations, which was useful to understand the three-state folding mechanism of the small FF domain.

  13. Effects of radiation on DNA's double helix

    Science.gov (United States)

    2003-01-01

    The blueprint of life, DNA's double helix is found in the cells of everything from bacteria to astronauts. Exposure to radiation(depicted at right) such as X-rays (upper) or heavy ion particles (lower), can damage DNA and cause dire consequences both to the organism itself and to future generations. One of NASA's main goals is to develop better radiation shielding materials to protect astronauts from destructive radiation in space. This is particularly important for long space missions. NASA has selected researchers to study materials that provide better shielding. This research is managed by NASA's Office of Biological and Physical Research and is supported by the Microgravity Science and Applications Department at NASA's Marshall Center. During International Space Station Expedition Six, the Extravehicular Activity Radiation Monitoring (EVARM) will continue to measure radiation dosage encountered by the eyes, internal organs and skin during specific spacewalks, and relate it to the type of activity, location and other factors. An analysis of this information may be useful in mitigating potential exposure to space walkers in the future. (Illustration by Dr. Frank Cucinotta, NASA/Johnson Space Center, and Prem Saganti, Lockheed Martin)

  14. Visualizing water molecules in transmembrane proteins using radiolytic labeling methods.

    Science.gov (United States)

    Orban, Tivadar; Gupta, Sayan; Palczewski, Krzysztof; Chance, Mark R

    2010-02-09

    Essential to cells and their organelles, water is both shuttled to where it is needed and trapped within cellular compartments and structures. Moreover, ordered waters within protein structures often colocalize with strategically placed polar or charged groups critical for protein function, yet it is unclear if these ordered water molecules provide structural stabilization, mediate conformational changes in signaling, neutralize charged residues, or carry out a combination of all these functions. Structures of many integral membrane proteins, including G protein-coupled receptors (GPCRs), reveal the presence of ordered water molecules that may act like prosthetic groups in a manner quite unlike bulk water. Identification of "ordered" waters within a crystalline protein structure requires sufficient occupancy of water to enable its detection in the protein's X-ray diffraction pattern, and thus, the observed waters likely represent a subset of tightly bound functional waters. In this review, we highlight recent studies that suggest the structures of ordered waters within GPCRs are as conserved (and thus as important) as conserved side chains. In addition, methods of radiolysis, coupled to structural mass spectrometry (protein footprinting), reveal dynamic changes in water structure that mediate transmembrane signaling. The idea of water as a prosthetic group mediating chemical reaction dynamics is not new in fields such as catalysis. However, the concept of water as a mediator of conformational dynamics in signaling is just emerging, because of advances in both crystallographic structure determination and new methods of protein footprinting. Although oil and water do not mix, understanding the roles of water is essential to understanding the function of membrane proteins.

  15. Helix Electrohydrodynamic Printing of Highly Aligned Serpentine Micro/Nanofibers

    Directory of Open Access Journals (Sweden)

    Yongqing Duan

    2017-09-01

    Full Text Available Micro/nano serpentine structures have widespread applications in flexible/stretchable electronics; however, challenges still exist for low-cost, high-efficiency and controllable manufacturing. Helix electrohydrodynamic printing (HE-printing has been proposed here to realize controllable direct-writing of large area, highly aligned serpentine micro/nanofibers by introducing the rope coiling effect into printing process. By manipulating the flying trajectory and solidification degree of the micro/nano jet, the solidified micro/nanofiber flying in a stabilized helical manner and versatile serpentine structures deposited on a moving collector have been achieved. Systematic experiments and theoretical analysis were conducted to study the transformation behavior and the size changing rules for various deposited microstructures, and highly aligned serpentine microfibers were directly written by controlling the applied voltage, nozzle-to-collector distance and collector velocity. Furthermore, a hyper-stretchable piezoelectric device that can detect stretching, bending and pressure has been successfully fabricated using the printed serpentine micro/nanofibers, demonstrating the potential of HE-printing in stretchable electronics manufacturing.

  16. The X-Ray Crystal Structure of the Keratin 1-Keratin 10 Helix 2B Heterodimer Reveals Molecular Surface Properties and Biochemical Insights into Human Skin Disease

    Energy Technology Data Exchange (ETDEWEB)

    Bunick, Christopher G.; Milstone, Leonard M.

    2017-01-01

    Keratins 1 (K1) and 10 (K10) are the primary keratins expressed in differentiated epidermis. Mutations in K1/K10 are associated with human skin diseases. We determined the crystal structure of the complex between the distal (2B) helices of K1 and K10 to better understand how human keratin structure correlates with function. The 3.3 Å resolution structure confirms many features inferred by previous biochemical analyses, but adds unexpected insights. It demonstrates a parallel, coiled-coil heterodimer with a predominantly hydrophobic intermolecular interface; this heterodimer formed a higher order complex with a second K1-K10-2B heterodimer via a Cys401K10 disulfide link, although the bond angle is unanticipated. The molecular surface analysis of K1-K10-2B identified several pockets, one adjacent to the disulfide linkage and conserved in K5-K14. The solvent accessible surface area of the K1-K10 structure is 20–25% hydrophobic. The 2B region contains mixed acidic and basic patches proximally (N-terminal), whereas it is largely acidic distally (C-terminal). Mapping of conserved and nonconserved residues between K1-K10 and K5-K14 onto the structure demonstrated the majority of unique residues align along the outer helical ridge. Finally, the structure permitted a fresh analysis of the deleterious effects caused by K1/K10 missense mutations found in patients with phenotypic skin disease.

  17. Hedera helix L. and damages in Tlos Ancient City

    Directory of Open Access Journals (Sweden)

    Elinç, Z.K.

    2013-03-01

    Full Text Available There are various plant types in Tlos Ancient City of Fethiye district in the Province of Mugla, a city where different residential ruins of Lycia Civilization starting from Classical Age until Byzantine Period. Tlos is an important city in West-Lycia and is situated right on the control point of Lycia Way. Hedera helix L. is one of the plants living in this area, which attracts the attention as it mostly harms the ancient ruins. One of the most important reasons why Hedera helix L. is growing commonly in this region is the perfect ecological circumstances for the growth of this plant of the location where this ancient city is situated in. Additionally the fact that the ruins of the city are left on their fate, is another perfect circumstance for the Hedera helix L. to grow. Climbing or creeping stems of Hedera helix L. stick easily to the objects it touches and encircle them. Due to this characteristic, the walls of the ancient city are covered by this plant. Nevertheless, Hedera helix L. does not only harm the ancient constructions and natural rocks but also woody plants. The harm caused by dried out or cut Hedera helix L. are more than the harm caused by them when they were untouched. The subject of this study is to prove the shape and level of the harm caused by Hedera helix L. on ancient ruins of Tlos. At the same time, this study will underline the fighting methods against Hedera helix L. by comparing similar studies in other countries. Knowledge collected after this study will offer an insight into the excavation and restoration studies undertaken in all Mediterranean countries.

  18. Conformational change from antiparallel beta-sheet to alpha-helix in a series of depsipeptide, -(Leu-Leu-Lac)(n)-: syntheses, spectroscopic studies, and crystal structures of Boc-Leu-Lac-OEt and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2).

    Science.gov (United States)

    Oku, Hiroyuki; Yamada, Keiichi; Katakai, Ryoichi

    2008-04-01

    The depsipeptides Boc-Leu-Lac-OEt (1) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 1, 2) (2 and 3, respectively) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized and studied by crystallographic, CD spectroscopic, and ESI-MS analyses. In the packing cells, those three compounds adopt beta-strand conformations. Each molecule is linked into a dimer (1) or an infinite assembly (2 and 3) by tight hydrogen bonds of the type NH...O==C. Interestingly, the hexamer, 3 shows the first example of antiparallel pleated beta-sheet crystal structure for a depsipeptide molecule. In the packing cells, especially for 3, the ester groups O--C==O are perpendicularly oriented to the amide groups NH--C==O and beta-sheet planes to avoid the interaction between --O--(ester) and O==C. Therefore, when the chain length become longer, the O...O==C repulsion interaction works as a beta-sheet breaker and hence promotes an alpha-helical structure as observed for Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (4) (Oku et al. Biopolymers 2004, 75, 242-254) and Boc-(Leu-Leu-Lac)(n)-OEt (n = 4-6) (5-7) (Katakai et al., Biopolymers 1996, 38, 285-290), in which the O...O==C repulsion does not cause significant structural changes in alpha-helical main chains. Therefore from the structural and spectroscopic analyses, we have found governing factors for the specificity in the beta-sheet and alpha-helix decision in this series of depsipeptides, -(Leu-Leu-Lac)(n)-.

  19. Nanoparticle biofabrication using English ivy (Hedera helix

    Directory of Open Access Journals (Sweden)

    Burris Jason N

    2012-10-01

    Full Text Available Abstract Background English ivy (Hedera helix is well known for its adhesive properties and climbing ability. Essential to its ability to adhere to vertical surfaces is the secretion of a nanocomposite adhesive containing spherical nanoparticles, 60–85 nm in diameter, produced exclusively by root hairs present on adventitious roots. These organic nanoparticles have shown promise in biomedical and cosmetic applications, and represent a safer alternative to metal oxide nanoparticles currently available. Results It was discovered that the maximum adventitious root production was achieved by a 4 h application of 1 mg/ml indole-3 butyric acid (IBA to juvenile English ivy shoot segments cultured in custom vessels. After incubation of the shoots under continuous light at 83 μmol/m2 s at 20°C for 2 weeks, the adventitious roots were harvested from the culture system and it was possible to isolate 90 mg of dry weight nanoparticles per 12 g of roots. The nanoparticle morphology was characterized by atomic force microscopy, and found to be similar to previous studies. Conclusions An enhanced system for the production of English ivy adventitious roots and their nanoparticles by modifying GA7 Magenta boxes and identifying the optimal concentration of IBA for adventitious root growth was developed. This system is the first such platform for growing and harvesting organic nanoparticles from plants, and represents an important step in the development of plant-based nanomanufacturing. It is a significant improvement on the exploitation of plant systems for the formation of metallic nanoparticles, and represents a pathway for the generation of bulk ivy nanoparticles for translation into biomedical applications.

  20. An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.

    Directory of Open Access Journals (Sweden)

    Ling Liu

    2009-03-01

    Full Text Available Brome mosaic virus (BMV protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a(Pol and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a(Pol accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a(Pol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

  1. Guided Transport of a Transmembrane Nanochannel

    Science.gov (United States)

    Dutt, Meenakshi; Kuksenok, Olga; Balazs, Anna

    2011-03-01

    Via the Dissipative Particle Dynamics approach, we design a system that allows transport of a nanochannel to a desired location by applying an external force. Each nanochannel encompasses an ABA architecture, with a hydrophobic shaft (B) with two hydrophilic ends (A). One of the hydrophilic ends of the nanochannel is functionalized with hydrophilic functional groups, or hairs. The hydrophilic hairs serve a dual role: (1) control transport across the membrane barrier when the channel diffuses freely in the membrane, and (2) enable the channel relocation to a specific membrane site. Our system comprises a transmembrane hairy nanochannel with the hairs extending into solution. In our earlier work, we demonstrated the spontaneous insertion of such a hairy nanochannel into a lipid bilayer (Nanoscale DOI: 10.1039/C0NR00578A). First, we hold a suitably functionalized pipette stationary above the membrane while the nanochannel freely diffuses within the membrane. For an optimal range of parameters, we demonstrate that the hairs find the pipette and spontaneously anchor onto it. We then show that by moving the pipette for a range of velocities, we can effectively transport the channel to any location within the membrane. This prototype system can provide guidelines for designing a number of biomimetic applications.

  2. Performance of Process Damping in Machining Titanium Alloys at Low Cutting Speed with Different Helix Tools

    International Nuclear Information System (INIS)

    Shaharun, M A; Yusoff, A R; Reza, M S; Jalal, K A

    2012-01-01

    Titanium is a strong, lustrous, corrosion-resistant and transition metal with a silver color to produce strong lightweight alloys for industrial process, automotive, medical instruments and other applications. However, it is very difficult to machine the titanium due to its poor machinability. When machining titanium alloys with the conventional tools, the wear rate of the tool is rapidly accelerate and it is generally difficult to achieve at high cutting speed. In order to get better understanding of machining titanium alloy, the interaction between machining structural system and the cutting process which result in machining instability will be studied. Process damping is a useful phenomenon that can be exploited to improve the limited productivity of low speed machining. In this study, experiments are performed to evaluate the performance of process damping of milling under different tool helix geometries. The results showed that the helix of 42° angle is significantly increase process damping performance in machining titanium alloy.

  3. Structure modeling of all identified G protein-coupled receptors in the human genome.

    Directory of Open Access Journals (Sweden)

    Yang Zhang

    2006-02-01

    Full Text Available G protein-coupled receptors (GPCRs, encoded by about 5% of human genes, comprise the largest family of integral membrane proteins and act as cell surface receptors responsible for the transduction of endogenous signal into a cellular response. Although tertiary structural information is crucial for function annotation and drug design, there are few experimentally determined GPCR structures. To address this issue, we employ the recently developed threading assembly refinement (TASSER method to generate structure predictions for all 907 putative GPCRs in the human genome. Unlike traditional homology modeling approaches, TASSER modeling does not require solved homologous template structures; moreover, it often refines the structures closer to native. These features are essential for the comprehensive modeling of all human GPCRs when close homologous templates are absent. Based on a benchmarked confidence score, approximately 820 predicted models should have the correct folds. The majority of GPCR models share the characteristic seven-transmembrane helix topology, but 45 ORFs are predicted to have different structures. This is due to GPCR fragments that are predominantly from extracellular or intracellular domains as well as database annotation errors. Our preliminary validation includes the automated modeling of bovine rhodopsin, the only solved GPCR in the Protein Data Bank. With homologous templates excluded, the final model built by TASSER has a global C(alpha root-mean-squared deviation from native of 4.6 angstroms, with a root-mean-squared deviation in the transmembrane helix region of 2.1 angstroms. Models of several representative GPCRs are compared with mutagenesis and affinity labeling data, and consistent agreement is demonstrated. Structure clustering of the predicted models shows that GPCRs with similar structures tend to belong to a similar functional class even when their sequences are diverse. These results demonstrate the usefulness

  4. Changes in the reproductive system of the snail Helix aspersa caused by mucus from the love dart

    NARCIS (Netherlands)

    Koene, J M; Chase, R.

    The function of the love dart in certain species of terrestrial snails is unknown. In Helix aspersa, the dart is a sharp calcareous structure that is used to pierce the partner's skin during courtship. When expelled, the dart is covered with a thick mucus. The hypothesis tested here is that the

  5. Portrait of a discovery. Watson, Crick, and the double helix.

    Science.gov (United States)

    de Chadarevian, Soraya

    2003-03-01

    This essay examines an iconic image of twentieth-century science: Antony Barrington Brown's photograph of James Watson, Francis Crick, and the double-helical model of DNA. The detailed reconstruction of the production, reception, and uses of the photograph reveals the central role of the image in making the discovery it portrays. Taken in May 1953, two full months after the scientists built the model, to accompany a report on the structure in Time magazine, the photograph (like the report) was never published. It came into circulation only fifteen years later, as an illustration in Watson's best-selling book The Double Helix. While the image served as a historical document and advertisement for the book, only the book provided the description that made the image as well as the people and the model it represented famous. The history of the image provides insights into the retrospective construction of the discovery, which has since been celebrated as the origin of a new science of life.

  6. MemBrain: improving the accuracy of predicting transmembrane helices.

    Directory of Open Access Journals (Sweden)

    Hongbin Shen

    Full Text Available Prediction of transmembrane helices (TMH in alpha helical membrane proteins provides valuable information about the protein topology when the high resolution structures are not available. Many predictors have been developed based on either amino acid hydrophobicity scale or pure statistical approaches. While these predictors perform reasonably well in identifying the number of TMHs in a protein, they are generally inaccurate in predicting the ends of TMHs, or TMHs of unusual length. To improve the accuracy of TMH detection, we developed a machine-learning based predictor, MemBrain, which integrates a number of modern bioinformatics approaches including sequence representation by multiple sequence alignment matrix, the optimized evidence-theoretic K-nearest neighbor prediction algorithm, fusion of multiple prediction window sizes, and classification by dynamic threshold. MemBrain demonstrates an overall improvement of about 20% in prediction accuracy, particularly, in predicting the ends of TMHs and TMHs that are shorter than 15 residues. It also has the capability to detect N-terminal signal peptides. The MemBrain predictor is a useful sequence-based analysis tool for functional and structural characterization of helical membrane proteins; it is freely available at http://chou.med.harvard.edu/bioinf/MemBrain/.

  7. System and methods for predicting transmembrane domains in membrane proteins and mining the genome for recognizing G-protein coupled receptors

    Science.gov (United States)

    Trabanino, Rene J; Vaidehi, Nagarajan; Hall, Spencer E; Goddard, William A; Floriano, Wely

    2013-02-05

    The invention provides computer-implemented methods and apparatus implementing a hierarchical protocol using multiscale molecular dynamics and molecular modeling methods to predict the presence of transmembrane regions in proteins, such as G-Protein Coupled Receptors (GPCR), and protein structural models generated according to the protocol. The protocol features a coarse grain sampling method, such as hydrophobicity analysis, to provide a fast and accurate procedure for predicting transmembrane regions. Methods and apparatus of the invention are useful to screen protein or polynucleotide databases for encoded proteins with transmembrane regions, such as GPCRs.

  8. Crystal structure of a claudin provides insight into the architecture of tight junctions.

    Science.gov (United States)

    Suzuki, Hiroshi; Nishizawa, Tomohiro; Tani, Kazutoshi; Yamazaki, Yuji; Tamura, Atsushi; Ishitani, Ryuichiro; Dohmae, Naoshi; Tsukita, Sachiko; Nureki, Osamu; Fujiyoshi, Yoshinori

    2014-04-18

    Tight junctions are cell-cell adhesion structures in epithelial cell sheets that surround organ compartments in multicellular organisms and regulate the permeation of ions through the intercellular space. Claudins are the major constituents of tight junctions and form strands that mediate cell adhesion and function as paracellular barriers. We report the structure of mammalian claudin-15 at a resolution of 2.4 angstroms. The structure reveals a characteristic β-sheet fold comprising two extracellular segments, which is anchored to a transmembrane four-helix bundle by a consensus motif. Our analyses suggest potential paracellular pathways with distinctive charges on the extracellular surface, providing insight into the molecular basis of ion homeostasis across tight junctions.

  9. An unusual helix-turn-helix protease inhibitory motif in a novel trypsin inhibitor from seeds of Veronica (Veronica hederifolia L.).

    Science.gov (United States)

    Conners, Rebecca; Konarev, Alexander V; Forsyth, Jane; Lovegrove, Alison; Marsh, Justin; Joseph-Horne, Timothy; Shewry, Peter; Brady, R Leo

    2007-09-21

    The storage tissues of many plants contain protease inhibitors that are believed to play an important role in defending the plant from invasion by pests and pathogens. These proteinaceous inhibitor molecules belong to a number of structurally distinct families. We describe here the isolation, purification, initial inhibitory properties, and three-dimensional structure of a novel trypsin inhibitor from seeds of Veronica hederifolia (VhTI). The VhTI peptide inhibits trypsin with a submicromolar apparent K(i) and is expected to be specific for trypsin-like serine proteases. VhTI differs dramatically in structure from all previously described families of trypsin inhibitors, consisting of a helix-turn-helix motif, with the two alpha helices tightly associated by two disulfide bonds. Unusually, the crystallized complex is in the form of a stabilized acyl-enzyme intermediate with the scissile bond of the VhTI inhibitor cleaved and the resulting N-terminal portion of the inhibitor remaining attached to the trypsin catalytic serine 195 by an ester bond. A synthetic, truncated version of the VhTI peptide has also been produced and co-crystallized with trypsin but, surprisingly, is seen to be uncleaved and consequently forms a noncovalent complex with trypsin. The VhTI peptide shows that effective enzyme inhibitors can be constructed from simple helical motifs and provides a new scaffold on which to base the design of novel serine protease inhibitors.

  10. Salt- and pH-Triggered Helix-Coil Transition of Ionic Polypeptides under Physiology Conditions.

    Science.gov (United States)

    Yuan, Jingsong; Zhang, Yi; Sun, Yue; Cai, Zhicheng; Yang, Lijiang; Lu, Hua

    2018-03-26

    Controlling the helix-coil transition of polypeptides under physiological conditions is an attractive way toward smart functional materials. Here we report the synthesis of a series of tertiary amine-functionalized ethylene glycol (EGx)-linked polypeptide electrolytes with their secondary structures tunable under physiological conditions. The resultant polymers, denoted as P(EGxDMA-Glu) (x =1, 2, and 3), show excellent aqueous solubility (> 10 mg/mL) regardless of their charge states. Unlike poly-L-lysine that can form helix only at pH above 10, P(EGxDMA-Glu) undergo a pH-dependent helix-coil switch with their transition points within physiological range (~ pH 5.3-6.5). Meanwhile, P(EGxDMA-Glu) exhibit unusual salt-induced helical conformation presumably owing to the unique properties of EGx linkers. Together, the current work highlights the importance of fine-tuning the linker chemistry in achieving conformation-switchable polypeptides, and represents a facile approach towards stimuli-responsive biopolymers for advanced biological applications.

  11. Sample Extraction Bsaed on Helix Scattering for Polarimetric SAR Calibratio

    Science.gov (United States)

    Chang, Y.; Yang, J.; Li, P.; Zhao, L.; Shi, L.

    2017-09-01

    Polarimetric calibration (PolCAL) of Synthetic Aperture Radar (SAR) images is a significant preprocessing for further applications. Since the reflection symmetry property of distributed objects can provide stable constraints for PolCAL. It is reasonable to extract these reference samples before calibration. The helix scattering generally appears in complex urban area and disappears for a natural scatterer, making it a good measure to extract distributed objects. In this paper, a novel technique that extracts reflecting symmetry samples is proposed by using helix scattering. The helix scattering information is calculated by Yamaguchi four-component decomposition algorithm. An adaptive threshold selection algorithm based on generalized Gaussian distribution is also utilized to scale the helix scattering components automatically, getting rid of the problem of various numerical range. The extracting results will be taken as PolCAL reference samples and the Quegan method are utilized to calibrate these PolSAR images. A C-band airborne PolSAR data was taken as examples to evaluate its ability in improving calibration precision. Traditional method i.e. extracting samples with span power was also evaluated as contrast experiment. The results showed that the samples extracting method based on helix scattering can improve the Polcal precision preferably.

  12. SAMPLE EXTRACTION BSAED ON HELIX SCATTERING FOR POLARIMETRIC SAR CALIBRATIO

    Directory of Open Access Journals (Sweden)

    Y. Chang

    2017-09-01

    Full Text Available Polarimetric calibration (PolCAL of Synthetic Aperture Radar (SAR images is a significant preprocessing for further applications. Since the reflection symmetry property of distributed objects can provide stable constraints for PolCAL. It is reasonable to extract these reference samples before calibration. The helix scattering generally appears in complex urban area and disappears for a natural scatterer, making it a good measure to extract distributed objects. In this paper, a novel technique that extracts reflecting symmetry samples is proposed by using helix scattering. The helix scattering information is calculated by Yamaguchi four-component decomposition algorithm. An adaptive threshold selection algorithm based on generalized Gaussian distribution is also utilized to scale the helix scattering components automatically, getting rid of the problem of various numerical range. The extracting results will be taken as PolCAL reference samples and the Quegan method are utilized to calibrate these PolSAR images. A C-band airborne PolSAR data was taken as examples to evaluate its ability in improving calibration precision. Traditional method i.e. extracting samples with span power was also evaluated as contrast experiment. The results showed that the samples extracting method based on helix scattering can improve the Polcal precision preferably.

  13. Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.

    Science.gov (United States)

    Hamouda, Ayman K; Stewart, Deirdre S; Husain, S Shaukat; Cohen, Jonathan B

    2011-06-10

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive

  14. Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor*

    Science.gov (United States)

    Hamouda, Ayman K.; Stewart, Deirdre S.; Husain, S. Shaukat; Cohen, Jonathan B.

    2011-01-01

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC50 = 4 μm, whereas it inhibited the binding of [3H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC50 values of 2.5 and 0.7 mm, respectively. Similar to [3H]TDBzl-etomidate, [3H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [3H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive modulators (TDBzl

  15. Receptor tyrosine kinase structure and function in health and disease

    Directory of Open Access Journals (Sweden)

    Oleg A. Karpov

    2015-09-01

    Full Text Available Receptor tyrosine kinases (RTKs are membrane proteins that control the flow of information through signal transduction pathways, impacting on different aspects of cell function. RTKs are characterized by a ligand-binding ectodomain, a single transmembrane α-helix, a cytosolic region comprising juxtamembrane and kinase domains followed by a flexible C-terminal tail. Somatic and germline RTK mutations can induce aberrant signal transduction to give rise to cardiovascular, developmental and oncogenic abnormalities. RTK overexpression occurs in certain cancers, correlating signal strength and disease incidence. Diverse RTK activation and signal transduction mechanisms are employed by cells during commitment to health or disease. Small molecule inhibitors are one means to target RTK function in disease initiation and progression. This review considers RTK structure, activation, and signal transduction and evaluates biological relevance to therapeutics and clinical outcomes.

  16. Surfactant in the gas mantle of the snail Helix aspersa.

    Science.gov (United States)

    Daniels, C B; Wood, P G; Loptako, O V; Codd, J R; Johnston, S D; Orgeig, S

    1999-01-01

    Surfactant occurs in cyclically inflating and deflating, gas-holding structures of vertebrates to reduce the surface tension of the inner fluid lining, thereby preventing collapse and decreasing the work of inflation. Here we determined the presence of surfactant in material lavaged from the airspace in the gas mantle of the pulmonate snail Helix aspersa. Surfactant is characterized by the presence of disaturated phospholipid (DSP), especially disaturated phosphatidylcholine (PC), lavaged from the airspace, by the presence of lamellated osmiophilic bodies (LBs) in the airspaces and epithelial tissue, and by the ability of the lavage to reduce surface tension of fluid in a surface balance. Lavage had a DSP/phospholipid (PL) ratio of 0.085, compared to 0.011 in membranes, with the major PL being PC (45.3%). Cholesterol, the primary fluidizer for pulmonary surfactant, was similar in lavage and in lipids extracted from cell homogenates (cholesterol/PL: 0.04 and 0. 03, respectively). LBs were found in the tissues and airspaces. The surface activity of the lavage material is defined as the ability to reduce surface tension under compression to values much lower than that of water. In addition, surface-active lipids will vary surface tension, increasing it upon inspiration as the surface area expands. By these criteria, the surface activity of lavaged material was poor and most similar to that shown by pulmonary lavage of fish and toads. Snail surfactant displays structures, a biochemical PL profile, and biophysical properties similar to surfactant obtained from primitive fish, teleost swim bladders, the lung of the Dipnoan Neoceratodus forsteri, and the amphibian Bufo marinus. However, the cholesterol/PL and cholesterol/DSP ratios are more similar to the amphibian B. marinus than to the fish, and this similarity may indicate a crucial physicochemical relationship for these lipids.

  17. Surfactant protein C peptides with salt-bridges (“ion-locks” promote high surfactant activities by mimicking the α-helix and membrane topography of the native protein

    Directory of Open Access Journals (Sweden)

    Frans J. Walther

    2014-07-01

    Full Text Available Background. Surfactant protein C (SP-C; 35 residues in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as ‘helical adjuvants’ to maintain activity by overriding the β-sheet propensities of the native sequences.Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges (“ion-locks” promote surface activity by mimicking the α-helix and membrane topography of native SP-C.Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines. Ion-lock SP-C molecules were prepared by incorporating single or double Glu−–Lys+ into the parent SP-C’s. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D exchange FTIR, and also Membrane Protein Explorer (MPEx hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency.Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with ‘amyloid-like’ properties. The enhanced

  18. Metal ion site engineering indicates a global toggle switch model for seven-transmembrane receptor activation

    DEFF Research Database (Denmark)

    Elling, Christian E; Frimurer, Thomas M; Gerlach, Lars-Ole

    2006-01-01

    Much evidence indicates that, during activation of seven-transmembrane (7TM) receptors, the intracellular segments of the transmembrane helices (TMs) move apart with large amplitude, rigid body movements of especially TM-VI and TM-VII. In this study, AspIII:08 (Asp113), the anchor point...... in sites constructed between positions III:08 (Asp or His), VI:16 (preferentially Cys), and/or VII:06 (preferentially Cys). In molecular models built over the backbone conformation of the inactive rhodopsin structure, the heavy atoms that coordinate the metal ion were located too far away from each other...... ion sites, we propose a global toggle switch mechanism for 7TM receptor activation in which inward movement of the extracellular segments of especially TM-VI and, to some extent, TM-VII is coupled to the well established outward movement of the intracellular segments of these helices. We suggest...

  19. Definition of the G protein-coupled receptor transmembrane bundle binding pocket and calculation of receptor similarities for drug design

    DEFF Research Database (Denmark)

    Gloriam, David Erik Immanuel; Foord, Steven M; Blaney, Frank E

    2009-01-01

    Recent advances in structural biology for G-protein-coupled receptors (GPCRs) have provided new opportunities to improve the definition of the transmembrane binding pocket. Here a reference set of 44 residue positions accessible for ligand binding was defined through detailed analysis of all...... to endogenous ligand types, although it revealed subdivision of certain classes, notably peptide and lipid receptors. The transmembrane binding site reference set, particularly when coupled with a means of identifying the subset of ligand binding residues, provides a general paradigm for understanding...

  20. Agrobacterium tumefaciens VirC2 enhances T-DNA transfer and virulence through its C-terminal ribbon-helix-helix DNA-binding fold.

    Science.gov (United States)

    Lu, Jun; den Dulk-Ras, Amke; Hooykaas, Paul J J; Glover, J N Mark

    2009-06-16

    Agrobacterium tumefaciens VirC2 stimulates processing of single-stranded T-DNA that is translocated into plants to induce tumor formation, but how VirC2 functions is unclear. Here, we report the 1.7-A X-ray crystal structure of its trypsin-resistant C-terminal domain, VirC2(82-202), which reveals a form of the ribbon-helix-helix (RHH) DNA-binding fold contained within a single polypeptide chain. DNA-binding assays and mutagenesis indicate that VirC2 uses this RHH fold to bind double-stranded DNA but not single-stranded DNA. Mutations that severely affect VirC2 DNA binding are highly deleterious for both T-DNA transfer into yeast and the virulence of A. tumefaciens in different plants including Nicotiana glauca and Kalanchoe daigremontiana. These data suggest that VirC2 enhances T-DNA transfer and virulence through DNA binding with its RHH fold. The RHH fold of VirC2 is the first crystal structure representing a group of predicted RHH proteins that facilitate endonucleolytic processing of DNA for horizontal gene transfer.

  1. Structure of Concatenated HAMP Domains Provides a Mechanism for Signal Transduction

    Energy Technology Data Exchange (ETDEWEB)

    Airola, Michael V.; Watts, Kylie J.; Bilwes, Alexandrine M.; Crane, Brian R. (Cornell); (Lorma Linda U)

    2010-08-23

    HAMP domains are widespread prokaryotic signaling modules found as single domains or poly-HAMP chains in both transmembrane and soluble proteins. The crystal structure of a three-unit poly-HAMP chain from the Pseudomonas aeruginosa soluble receptor Aer2 defines a universal parallel four-helix bundle architecture for diverse HAMP domains. Two contiguous domains integrate to form a concatenated di-HAMP structure. The three HAMP domains display two distinct conformations that differ by changes in helical register, crossing angle, and rotation. These conformations are stabilized by different subsets of conserved residues. Known signals delivered to HAMP would be expected to switch the relative stability of the two conformations and the position of a coiled-coil phase stutter at the junction with downstream helices. We propose that the two conformations represent opposing HAMP signaling states and suggest a signaling mechanism whereby HAMP domains interconvert between the two states, which alternate down a poly-HAMP chain.

  2. Identification of helix capping and β-turn motifs from NMR chemical shifts

    International Nuclear Information System (INIS)

    Shen Yang; Bax, Ad

    2012-01-01

    We present an empirical method for identification of distinct structural motifs in proteins on the basis of experimentally determined backbone and 13 C β chemical shifts. Elements identified include the N-terminal and C-terminal helix capping motifs and five types of β-turns: I, II, I′, II′ and VIII. Using a database of proteins of known structure, the NMR chemical shifts, together with the PDB-extracted amino acid preference of the helix capping and β-turn motifs are used as input data for training an artificial neural network algorithm, which outputs the statistical probability of finding each motif at any given position in the protein. The trained neural networks, contained in the MICS (motif identification from chemical shifts) program, also provide a confidence level for each of their predictions, and values ranging from ca 0.7–0.9 for the Matthews correlation coefficient of its predictions far exceed those attainable by sequence analysis. MICS is anticipated to be useful both in the conventional NMR structure determination process and for enhancing on-going efforts to determine protein structures solely on the basis of chemical shift information, where it can aid in identifying protein database fragments suitable for use in building such structures.

  3. Identification of helix capping and {beta}-turn motifs from NMR chemical shifts

    Energy Technology Data Exchange (ETDEWEB)

    Shen Yang; Bax, Ad, E-mail: bax@nih.gov [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2012-03-15

    We present an empirical method for identification of distinct structural motifs in proteins on the basis of experimentally determined backbone and {sup 13}C{sup {beta}} chemical shifts. Elements identified include the N-terminal and C-terminal helix capping motifs and five types of {beta}-turns: I, II, I Prime , II Prime and VIII. Using a database of proteins of known structure, the NMR chemical shifts, together with the PDB-extracted amino acid preference of the helix capping and {beta}-turn motifs are used as input data for training an artificial neural network algorithm, which outputs the statistical probability of finding each motif at any given position in the protein. The trained neural networks, contained in the MICS (motif identification from chemical shifts) program, also provide a confidence level for each of their predictions, and values ranging from ca 0.7-0.9 for the Matthews correlation coefficient of its predictions far exceed those attainable by sequence analysis. MICS is anticipated to be useful both in the conventional NMR structure determination process and for enhancing on-going efforts to determine protein structures solely on the basis of chemical shift information, where it can aid in identifying protein database fragments suitable for use in building such structures.

  4. Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia

    DEFF Research Database (Denmark)

    Shamloo, Mehrdad; Soriano, Liza; von Schack, David

    2006-01-01

    Basic helix-loop-helix PAS domain proteins form a growing family of transcription factors. These proteins are involved in the process of adaptation to cellular stresses and environmental factors such as a change in oxygen concentration. We describe the identification and characterization of a rec...... lead to a decrease in the 200 kDa form and a simultaneous increase in the 100 kDa immunoreactivity. This could indicate a novel regulatory mechanism for activation and/or deactivation of this protein in response to ischemic brain injury....

  5. Cystic fibrosis transmembrane conductance regulator protein expression in the male excretory duct system during development.

    Science.gov (United States)

    Marcorelles, Pascale; Gillet, Danièle; Friocourt, Gaëlle; Ledé, Françoise; Samaison, Laura; Huguen, Geneviève; Ferec, Claude

    2012-03-01

    Sterility due to bilateral destruction in utero or in early infancy resulting in congenital absence of the vas deferens is the rule in male patients with cystic fibrosis. To understand the developmental pattern of this anomaly, the microscopic morphology of the male excretory system was analyzed during development and the expression of the cystic fibrosis transmembrane conductance regulator protein was explored by immunohistochemistry. We observed that cystic fibrosis fetuses had no excretory ducts agenesis or obstruction until 22 weeks of gestation. However, a focal inflammatory pattern and mucinous plugs in the oldest cystic fibrosis case suggested a disruptive mechanism. Immunolabeling of cytoplasmic epithelial cystic fibrosis transmembrane conductance regulator protein was demonstrated in all cystic fibrosis and control cases with a similar pattern of expression of the protein between age-matched controls and cystic fibrosis cases. At midgestation, an apical intensification appeared in both cystic fibrosis and control cases and was stable during the remainder of fetal life. No gradient of intensity could be detected between the different segments of the excretory tract. These findings are different from those reported in adults. The absence of any morphologic anomaly until 22 weeks of gestation, the focal destruction of the epithelial structures during the second trimester, and the chronological pattern of expression of cystic fibrosis transmembrane conductance regulator are of interest for a better understanding of the pathophysiology of this disease. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. The Triple Helix Model and the Knowledge-Based Economy

    NARCIS (Netherlands)

    Leydesdorff, L.; Meyer, M.

    2010-01-01

    The Triple Helix model of university-industry-government relations can be generalized from a neo-institutional model of networks of relations to a neo-evolutionary model of how three selection environments operate upon one another. Two selection mechanisms operating upon each other can mutually

  7. Living Labs as boundary-spanners between Triple Helix actors

    NARCIS (Netherlands)

    van Geenhuizen, M.S.

    2016-01-01

    Living labs are an increasingly popular methodology to enhance innovation. Living labs aim to span boundaries between different organizations, among others Triple helix actors, by acting as a network organization typically in a real-life environment to foster co-creation by user-groups. This paper

  8. Uncovering molecular structural mechanisms of signaling mediated by the prion protein

    Energy Technology Data Exchange (ETDEWEB)

    Romano, Sebastian A.; Linden, Rafael [Universidade Federal do Rio de Janeiro (IBCCF/UFRl), RJ (Brazil). Inst. de Biofisica Carlos Chagas Filho; Cordeiro, Yraima; Rocha e Lima, Luis M.T. da [Universidade Federal do Rio de Janeiro (FF/UFRl), RJ (Brazil). Fac. de Farmacia; Lopes, Marilene H. [Instituto Ludwig de Pesquisa de Cancer, Sao Paulo, SP (Brazil); Silva, Jerson L.; Foguel, Debora [Universidade Federal do Rio de Janeiro (IBqM/UFRl), RJ (Brazil). Inst. de Bioquimica Medica

    2009-07-01

    The glycosyl phosphatidylinositol (GPI) - anchored prion protein (PrP{sup c}), usually associated with neurodegenerative diseases, modulates various cellular responses and may scaffold multiprotein cell surface signaling complexes. Engagement of PrP{sup c} with the secretable cochaperone hop/STI 1 induces neurotrophic transmembrane signals through unknown molecular mechanisms. We addressed whether interaction of Pr P{sup c} and hop STI 1 entails structural rearrangements relevant for signaling. Circular dichroism and fluorescence spectroscopy showed that PrP{sup c}:hop/STI 1 interaction triggers loss of PrP helical structures, involving at least a perturbation of the Pr P{sup c}{sub 143-153} beta-helix. Novel SAXS models revealed a significant C-terminal compaction of hop/STI 1 when bound to PrP{sup c}. Differing from a recent dimeric model of human hop/STI 1, both size exclusion chromatography and SAXS data support a monomeric form of free murine hop/STI 1. Changes in the Pr P{sup c}{sub 143-153} beta-helix may engage the transmembrane signaling protein laminin receptor precursor and neural cell adhesion molecule, both of which bind that domain of Pr P{sup c}, and further ligands may be engaged by the tertiary structural changes of hop/STI 1. These reciprocal structural modifications indicate a versatile mechanism for signaling mediated by Pr P{sup c}:hop/STI 1 interaction, consistent with the hypothesis that Pr P{sup c} scaffolds multiprotein signaling complexes at the cell surface. (author)

  9. Structural Dynamics of Soluble Chloride Intracellular Channel Protein CLIC1 Examined by Amide Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS)†

    Science.gov (United States)

    Stoychev, Stoyan H.; Nathaniel, Christos; Fanucchi, Sylvia; Brock, Melissa; Li, Sheng; Asmus, Kyle; Woods, Virgil L.; Dirr, Heini W.

    2009-01-01

    Chloride intracellular channel protein 1 (CLIC1) functions as an anion channel in plasma and nuclear membranes when its soluble monomeric form converts to an integral-membrane form. The transmembrane region of CLIC1 is located in its thioredoxin-like domain 1 but the mechanism whereby the protein converts to its membrane conformation has yet to be determined. Since channel formation in membranes is enhanced at low pH (5 to 5.5), a condition that is found at the surface of membranes, the structural dynamics of soluble CLIC1 was studied at pH 7 and at pH 5.5 in the absence of membranes by amide hydrogen-deuterium exchange mass spectrometry (DXMS). Rapid hydrogen exchange data indicate that CLIC1 displays a similar core structure at these pH values. Domain 1 is less stable than the all-helical domain 2 and, while the structure of domain 1 remains intact, its conformational flexibility is further increased in an acidic environment (pH 5.5). In the absence of membrane, an acidic environment appears to prime the solution structure of CLIC1 by destabilising domain 1 in order to lower the activation energy barrier for its conversion to the membrane-insertion conformation. The significantly enhanced H/D-exchange rates at pH 5.5 displayed by two segments (peptides 11-31 and 68-82) could be due to the protonation of acidic residues in salt bridges. One of these segments (peptide 11-31) includes part of the transmembrane region which, in the solution structure, consists of helix α1. This helix is intrinsically stable and is most likely retained in the membrane conformation. Strand β2, another element of the transmembrane region, displays a propensity to form a helical structure and has putative N- and C-capping motifs, suggesting that it too most likely forms a helix in a lipid bilayer. PMID:19650640

  10. Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix

    Science.gov (United States)

    Inoguchi, Noriko; Oshlo, Jake R.; Natarajan, Chandrasekhar; Weber, Roy E.; Fago, Angela; Storz, Jay F.; Moriyama, Hideaki

    2013-01-01

    The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128. PMID:23545644

  11. The Modular Construction of DNA Double Helix

    Indian Academy of Sciences (India)

    In the annals of science, rarely if ever, has any molecule captured the imagination of mankind as DNA. Within five decades of the discovery, DNA structure has been able to disseminate knowl- edge of key aspects related to life. From grade levels to research studies, DNA is described, examined and analyzed from diverse.

  12. Structural model of the open–closed–inactivated cycle of prokaryotic voltage-gated sodium channels

    Science.gov (United States)

    Bagnéris, Claire; Naylor, Claire E.; McCusker, Emily C.

    2015-01-01

    In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels. PMID:25512599

  13. A hidden Markov model for prediction transmembrane helices in proteinsequences

    DEFF Research Database (Denmark)

    Sonnhammer, Erik L.L.; von Heijne, Gunnar; Krogh, Anders Stærmose

    1998-01-01

    , helix caps on either side, loop on the cytoplasmic side, two loops for the non-cytoplasmic side, and a globular domain state in the middle of each loop. The two loop paths on the non-cytoplasmic side are used to model short and long loops separately, which corresponds biologically to the two known...

  14. Interaction of batrachotoxin with the local anesthetic receptor site in transmembrane segment IVS6 of the voltage-gated sodium channel.

    Science.gov (United States)

    Linford, N J; Cantrell, A R; Qu, Y; Scheuer, T; Catterall, W A

    1998-11-10

    The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics. We have investigated the interaction of BTX with amino acid residues I1760, F1764, and Y1771, which form part of local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1764 (mutant F1764A) reduces tritiated BTX-A-20-alpha-benzoate binding affinity, causing a 60-fold increase in Kd. Alanine substitution for I1760, which is adjacent to F1764 in the predicted IVS6 transmembrane alpha helix, causes only a 4-fold increase in Kd. In contrast, mutant Y1771A shows no change in BTX binding affinity. For wild-type and mutant Y1771A, BTX shifted the voltage for half-maximal activation approximately 40 mV in the hyperpolarizing direction and increased the percentage of noninactivating sodium current to approximately 60%. In contrast, these BTX effects were eliminated completely for the F1764A mutant and were reduced substantially for mutant I1760A. Our data suggest that the BTX receptor site shares overlapping but nonidentical molecular determinants with the local anesthetic receptor site in transmembrane segment IVS6 as well as having unique molecular determinants in transmembrane segment IS6, as demonstrated in previous work. Evidently, BTX conforms to a domain-interface allosteric model of ligand binding and action, as previously proposed for calcium agonist and antagonist drugs acting on L-type calcium channels.

  15. Transmembrane voltage: Potential to induce lateral microdomains.

    Czech Academy of Sciences Publication Activity Database

    Malínský, Jan; Tanner, W.; Opekarová, Miroslava

    2016-01-01

    Roč. 1861, č. 8 (2016), s. 806-811 ISSN 1388-1981 R&D Projects: GA ČR(CZ) GA15-10641S Institutional support: RVO:68378041 Keywords : membrane microdomain * membrane potential * fluorescence spectroscopy * membrane structure * fluorescence microscopy Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.547, year: 2016

  16. Mutational analysis of photosystem I of Synechocystis sp. PCC 6803: the role of four conserved aromatic residues in the j-helix of PsaB.

    Directory of Open Access Journals (Sweden)

    Wu Xu

    Full Text Available Photosystem I is the light-driven plastocyanin-ferredoxin oxidoreductase in the photosynthetic electron transfer of cyanobacteria and plants. Two histidyl residues in the symmetric transmembrane helices A-j and B-j provide ligands for the P700 chlorophyll molecules of the reaction center of photosystem I. To determine the role of conserved aromatic residues adjacent to the histidyl molecule in the helix of B-j, we generated six site-directed mutants of the psaB gene in Synechocystis sp. PCC 6803. Three mutant strains with W645C, W643C/A644I and S641C/V642I substitutions could grow photoautotrophically and showed no obvious reduction in the photosystem I activity. Kinetics of P700 re-reduction by plastocyanin remained unaltered in these mutants. In contrast, the strains with H651C/L652M, F649C/G650I and F647C substitutions could not grow under photoautotrophic conditions because those mutants had low photosystem I activity, possibly due to low levels of proteins. A procedure to select spontaneous revertants from the mutants that are incapable to photoautotrophic growth resulted in three revertants that were used in this study. The molecular analysis of the spontaneous revertants suggested that an aromatic residue at F647 and a small residue at G650 may be necessary for maintaining the structural integrity of photosystem I. The (P700⁺-P700 steady-state absorption difference spectrum of the revertant F647Y has a ∼5 nm narrower peak than the recovered wild-type, suggesting that additional hydroxyl group of this revertant may participate in the interaction with the special pair while the photosystem I complexes of the F649C/G650T and H651Q mutants closely resemble the wild-type spectrum. The results presented here demonstrate that the highly conserved residues W645, W643 and F649 are not critical for maintaining the integrity and in mediating electron transport from plastocyanin to photosystem I. Our data suggest that an aromatic residue is

  17. Contributions of H G Khorana to Understanding Transmembrane ...

    Indian Academy of Sciences (India)

    IAS Admin

    GENERAL | ARTICLE. Contributions of H G Khorana to Understanding. Transmembrane Signal Transduction. David L Farrens and Thomas P Sakmar. Heptahelical G protein-coupled receptors (GPCRs) are lo- cated in the cell's plasma membrane and are responsible for transmitting chemical signals across the lipid bilayer.

  18. Modelling of a transmembrane evaporation module for desalination of seawater

    NARCIS (Netherlands)

    Guijt, C.M.; Racz, I.G.; van Heuven, Jan Willem; Reith, T.; de Haan, A.B.

    1999-01-01

    Transmembrane evaporation (often called membrane distillation) carried out in a countercurrent flow module, in which incoming cold seawater is heated by the condensing product water flow, is a promising technology for low-cost seawater desalination. This paper presents a model for preliminary design

  19. Dissecting the functions of conserved prolines within transmembrane helices of the D2 dopamine receptor.

    Science.gov (United States)

    Van Arnam, Ethan B; Lester, Henry A; Dougherty, Dennis A

    2011-10-21

    G protein-coupled receptors (GPCRs) contain a number of conserved proline residues in their transmembrane helices, and it is generally assumed these play important functional and/or structural roles. Here we use unnatural amino acid mutagenesis, employing α-hydroxy acids and proline analogues, to examine the functional roles of five proline residues in the transmembrane helices of the D2 dopamine receptor. The well-known tendency of proline to disrupt helical structure is important at all sites, while we find no evidence for a functional role for backbone amide cis-trans isomerization, another feature associated with proline. At most proline sites, the loss of the backbone NH is sufficient to explain the role of the proline. However, at one site, P210(5.50), a substituent on the backbone N appears to be essential for proper function. Interestingly, the pattern in functional consequences that we see is mirrored in the pattern of structural distortions seen in recent GPCR crystal structures.

  20. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

    Directory of Open Access Journals (Sweden)

    Michael Mühle

    Full Text Available The transmembrane envelope (TM protein gp41 of the human immunodeficiency virus-1 (HIV-1 plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS. Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb directed against the membrane proximal external region (MPER suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in

  1. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins: roles in protein folding, stability, and function.

    Science.gov (United States)

    Smith, Everett Clinton; Smith, Stacy E; Carter, James R; Webb, Stacy R; Gibson, Kathleen M; Hellman, Lance M; Fried, Michael G; Dutch, Rebecca Ellis

    2013-12-13

    Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion.

  2. Nanomechanics of single keratin fibres: A Raman study of the alpha helix -> beta sheet transition and water effect

    OpenAIRE

    Colomban, Philippe; Paquin, Raphaël

    2007-01-01

    International audience; The use of micro-Raman spectroscopy, through chemical bond nano-scale probes, allows the changes in conformations (α helix -> β sheet), chain orientation, disconnection of disulfide bonds (-20%) and the increase of intra and inter-chain distances during the strain to be distinguished. The combination of micro-Raman spectroscopy and a allows a quantitative measure of the extension of chemical bonds in the peptidic chain during loading. The nano-structural transformation...

  3. A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation.

    Science.gov (United States)

    Reshat, Reshat; Priestley, Catherine C; Gooderham, Nigel J

    2012-11-01

    Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.

  4. Helix unfolding/refolding characterizes the functional dynamics of Staphylococcus aureus Clp protease.

    Science.gov (United States)

    Ye, Fei; Zhang, Jie; Liu, Hongchuan; Hilgenfeld, Rolf; Zhang, Ruihan; Kong, Xiangqian; Li, Lianchun; Lu, Junyan; Zhang, Xinlei; Li, Donghai; Jiang, Hualiang; Yang, Cai-Guang; Luo, Cheng

    2013-06-14

    The ATP-dependent Clp protease (ClpP) plays an essential role not only in the control of protein quality but also in the regulation of bacterial pathogen virulence, making it an attractive target for antibacterial treatment. We have previously determined the crystal structures of Staphylococcus aureus ClpP (SaClpP) in two different states, extended and compressed. To investigate the dynamic switching of ClpP between these states, we performed a series of molecular dynamics simulations. During the structural transition, the long and straight helix E in the extended SaClpP monomer underwent an unfolding/refolding process, resulting in a kinked helix very similar to that in the compressed monomer. As a stable intermediate in the molecular dynamics simulation, the compact state was suggested and subsequently identified in x-ray crystallographic experiment. Our combined studies also determined that Ala(140) acted as a "hinge" during the transition between the extended and compressed states, and Glu(137) was essential for stabilizing the compressed state. Overall, this study provides molecular insights into the dynamics and mechanism of the functional conformation changes of SaClpP. Given the highly conserved sequences of ClpP proteins among different species, these findings potentially reflect a switching mechanism for the dynamic process shared in the whole ClpP family in general and thus aid in better understand the principles of Clp protease assembly and function.

  5. Opposite Displacement of Helix F in Attractant and Repellent Signaling by Sensory Rhodopsin-Htr Complexes*

    Science.gov (United States)

    Sasaki, Jun; Tsai, Ah-lim; Spudich, John L.

    2011-01-01

    Two forms of the phototaxis receptor sensory rhodopsin I distinguished by differences in its photoactive site have been shown to be directly correlated with attractant and repellent signaling by the dual-signaling protein. In prior studies, differences in the photoactive site defined the two forms, namely the direction of light-induced proton transfer from the chromophore and the pKa of an Asp counterion to the protonated chromophore. Here, we show by both in vivo and in vitro measurements that the two forms are distinct protein conformers with structural similarities to two conformers seen in the light-driven proton transport cycle of the related protein bacteriorhodopsin. Measurements of spontaneous cell motility reversal frequencies, an in vivo measure of histidine kinase activity in the phototaxis system, indicate that the two forms are a photointerconvertible pair, with one conformer activating and the other inhibiting the kinase. Protein conformational changes in these photoconversions monitored by site-directed spin labeling show that opposite structural changes in helix F, distant from the photoactive site, correspond to the opposite phototaxis signals. The results provide the first direct evidence that displacements of helix F are directly correlated with signaling and impact our understanding of the sensory rhodopsin I signaling mechanism and the evolution of diverse functionality in this protein family. PMID:21454480

  6. Opposite displacement of helix F in attractant and repellent signaling by sensory rhodopsin-Htr complexes.

    Science.gov (United States)

    Sasaki, Jun; Tsai, Ah-lim; Spudich, John L

    2011-05-27

    Two forms of the phototaxis receptor sensory rhodopsin I distinguished by differences in its photoactive site have been shown to be directly correlated with attractant and repellent signaling by the dual-signaling protein. In prior studies, differences in the photoactive site defined the two forms, namely the direction of light-induced proton transfer from the chromophore and the pK(a) of an Asp counterion to the protonated chromophore. Here, we show by both in vivo and in vitro measurements that the two forms are distinct protein conformers with structural similarities to two conformers seen in the light-driven proton transport cycle of the related protein bacteriorhodopsin. Measurements of spontaneous cell motility reversal frequencies, an in vivo measure of histidine kinase activity in the phototaxis system, indicate that the two forms are a photointerconvertible pair, with one conformer activating and the other inhibiting the kinase. Protein conformational changes in these photoconversions monitored by site-directed spin labeling show that opposite structural changes in helix F, distant from the photoactive site, correspond to the opposite phototaxis signals. The results provide the first direct evidence that displacements of helix F are directly correlated with signaling and impact our understanding of the sensory rhodopsin I signaling mechanism and the evolution of diverse functionality in this protein family.

  7. Hierarchical cascades of instability govern the mechanics of coiled coils: helix unfolding precedes coil unzipping.

    Science.gov (United States)

    Hamed, Elham; Keten, Sinan

    2014-07-15

    Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Poly(A) Tail Recognition by a Viral RNA Element Through Assembly of a Triple Helix

    Energy Technology Data Exchange (ETDEWEB)

    M Mitton-Fry; S DeGregorio; J Wang; T Steitz; J Steitz

    2011-12-31

    Kaposi's sarcoma-associated herpesvirus produces a highly abundant, nuclear noncoding RNA, polyadenylated nuclear (PAN) RNA, which contains an element that prevents its decay. The 79-nucleotide expression and nuclear retention element (ENE) was proposed to adopt a secondary structure like that of a box H/ACA small nucleolar RNA (snoRNA), with a U-rich internal loop that hybridizes to and protects the PAN RNA poly(A) tail. The crystal structure of a complex between the 40-nucleotide ENE core and oligo(A){sub 9} RNA at 2.5 angstrom resolution reveals that unlike snoRNAs, the U-rich loop of the ENE engages its target through formation of a major-groove triple helix. A-minor interactions extend the binding interface. Deadenylation assays confirm the functional importance of the triple helix. Thus, the ENE acts as an intramolecular RNA clamp, sequestering the PAN poly(A) tail and preventing the initiation of RNA decay.

  9. Crowding effect on helix-coil transition: Beyond entropic stabilization

    International Nuclear Information System (INIS)

    Koutsioubas, A.; Lairez, D.; Combet, S.; Longeville, S.; Fadda, G. C.; Zalczer, G.

    2012-01-01

    We report circular dichroism measurements on the helix-coil transition of poly(L-glutamic acid) in solution with polyethylene glycol (PEG) as a crowding agent. The PEG solutions have been characterized by small angle neutron scattering and are well described by the picture of a network of mesh size ξ, usual for semi-dilute chains in good solvent. We show that the increase of PEG concentration stabilizes the helices and increases the transition temperature. But more unexpectedly, we also notice that the increase of concentration of crowding agent reduces the mean helix extent at the transition, or in other words reduces its cooperativity. This result cannot be taken into account for by an entropic stabilization mechanism. Comparing the mean length of helices at the transition and the mesh size of the PEG network, our results strongly suggest two regimes: helices shorter or longer than the mesh size.

  10. Impact of signal peptide and transmembrane segments on expression and biochemical properties of a lipase from Bacillus sphaericus 205y.

    Science.gov (United States)

    Masomian, Malihe; Jasni, Azmiza Syawani; Rahman, Raja Noor Zaliha Raja Abd; Salleh, Abu Bakar; Basri, Mahiran

    2017-12-20

    A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an α/β hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Single-Molecule Spectroscopic Investigations of Amphipathic Helix Formation

    Science.gov (United States)

    Cunningham, Joy Ann; Okamoto, Kenji; English, Douglas

    2004-03-01

    We are using single molecule spectroscopy to examine surface-induced conformational states occurring through interaction of a polypeptide with an interface. Specifically, we investigate the folding of amphipathic helices by using single-molecule fluorescence resonance energy transfer to construct peptide conformational distributions in solution and at interfaces. Analysis of the conformational distributions and kinetics of peptides in different environments reveals properties of the free energy surface for helix formation at an interface relative to formation in solution.

  12. Chiral transformation: From single nanowire to double helix

    KAUST Repository

    Wang, Yong

    2011-12-21

    We report a new type of water-soluble ultrathin Au-Ag alloy nanowire (NW), which exhibits unprecedented behavior in a colloidal solution. Upon growth of a thin metal (Pd, Pt, or Au) layer, the NW winds around itself to give a metallic double helix. We propose that the winding originates from the chirality within the as-synthesized Au-Ag NWs, which were induced to untwist upon metal deposition. © 2011 American Chemical Society.

  13. Facilitating Quintuple helix innovation with urban living labs

    OpenAIRE

    Baccarne, Bastiaan; Schuurman, Dimitri; De Marez, Lieven

    2015-01-01

    This paper discusses the Urban Living Lab approach as a way to put the Quintuple Helix model for innovation into practice. In this analysis we focus on the concepts innovation democracy, ‘mode 3’ knowledge production, the innovation ecosystem as a system of societal subsystems and socioecological transition. The empirical analysis is performed by means of a multidimensional case study design, applied on a project-based ad hoc collaborative innovation development process in an ecological doma...

  14. Snail mucus - glandular origin and composition in Helix pomatia.

    Science.gov (United States)

    Greistorfer, Sophie; Klepal, Waltraud; Cyran, Norbert; Gugumuck, Andreas; Rudoll, Livia; Suppan, Johannes; von Byern, Janek

    2017-06-01

    Apart from their well-known culinary use, gastropod species such as Helix, which have a hydrogel-like mucus, are increasingly being exploited for cosmetic, bioengineering and medical applications. However, not only are the origin and composition of these "sticky" secretions far from being fully characterized, the number and morphology of the mucus glands involved is also uncertain. This study aims to characterize in detail the cutaneous glands of the Helix pomatia foot on morphological, histochemical and immunohistochemical levels. Hereby the focus is on the gland position and appearance on the foot sole as well as on the chemical nature of the different gland secretions. At least five different gland types can be distinguished by their microanatomy; three are located on the dorsal side and two on the ventral side of the foot sole. Most glands are reactive for acidic proteins and sugars such as mannose and fucose, indicating the presence of acidic glycosaminoglycans. One dorsal gland type shows high reactivity for acidic proteins only. The isolated mucus includes a certain amount of the elements chlorine, potassium and calcium; evidence for lipids was also confirmed in the isolated mucus. The present results for Helix pomatia show a clear difference in the number of glands compared to the related species Helix aspersa (only four mucus glands); histochemically, the glands of both species similarly produce acidic proteins as well as acidic glycosaminoglycans. While calcium ions are known to play a role in mucus formation, the presence and function of other ions such as potassium still need to be clarified. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Cellular uptake of Aib-containing amphipathic helix peptide.

    Science.gov (United States)

    Wada, Shun-ichi; Tsuda, Hirokazu; Okada, Terumi; Urata, Hidehito

    2011-10-01

    Cell-penetrating peptides (CPPs) are useful tools for the delivery of hydrophilic bioactive molecules, such as peptides, proteins, and oligonucleotides, across the cell membrane. To realize the delivery of therapeutic macromolecules by CPPs, the CPPs are required to show resistance to protease and no cytotoxicity. In order to produce potent non-toxic and protease-resistant CPPs with high cellular uptake, we designed an amphipathic helix peptide using α-aminoisobutyric acid (Aib, U) and named it MAP(Aib). In the MAP(Aib) molecule, five Aib residues are aligned on the hydrophobic face of the helix and five lysine (K) residues are aligned on the hydrophilic face. MAP(Aib) showed potent resistance to trypsin and pronase compared with MAP, an amphipathic helix peptide formed by usual amino acids. Fluorescein-labeled MAP(Aib) efficiently traversed the A549 cell membrane, diffusing into the cytoplasm and slightly into the nucleus without exerting any cytotoxicity. In contrast, MAP was poorly taken up by the cell. These results indicate that the incorporation of Aib residues into CPPs markedly improves cellular uptake and MAP(Aib) may be a useful tool for the delivery of hydrophilic macromolecules. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Class-B GPCR activation: is ligand helix-capping the key?

    Science.gov (United States)

    Neumann, Jean-Michel; Couvineau, Alain; Murail, Samuel; Lacapère, Jean-Jacques; Jamin, Nadège; Laburthe, Marc

    2008-07-01

    The class B family of G-protein-coupled receptors (GPCRs) regulates essential physiological functions such as exocrine and endocrine secretions, feeding behaviour, metabolism, growth, and neuro- and immuno-modulations. These receptors are activated by endogenous peptide hormones including secretin, glucagon, vasoactive intestinal peptide, corticotropin-releasing factor and parathyroid hormone. We have identified a common structural motif that is encoded in all class B GPCR-ligand N-terminal sequences. We propose that this local structure, a helix N-capping motif, is formed upon receptor binding and constitutes a key element underlying class B GPCR activation. The folded backbone conformation imposed by the capping structure could serve as a template for a rational design of drugs targeting class B GPCRs in several diseases.

  17. TMFoldWeb: a web server for predicting transmembrane protein fold class.

    Science.gov (United States)

    Kozma, Dániel; Tusnády, Gábor E

    2015-09-17

    Here we present TMFoldWeb, the web server implementation of TMFoldRec, a transmembrane protein fold recognition algorithm. TMFoldRec uses statistical potentials and utilizes topology filtering and a gapless threading algorithm. It ranks template structures and selects the most likely candidates and estimates the reliability of the obtained lowest energy model. The statistical potential was developed in a maximum likelihood framework on a representative set of the PDBTM database. According to the benchmark test the performance of TMFoldRec is about 77 % in correctly predicting fold class for a given transmembrane protein sequence. An intuitive web interface has been developed for the recently published TMFoldRec algorithm. The query sequence goes through a pipeline of topology prediction and a systematic sequence to structure alignment (threading). Resulting templates are ordered by energy and reliability values and are colored according to their significance level. Besides the graphical interface, a programmatic access is available as well, via a direct interface for developers or for submitting genome-wide data sets. The TMFoldWeb web server is unique and currently the only web server that is able to predict the fold class of transmembrane proteins while assigning reliability scores for the prediction. This method is prepared for genome-wide analysis with its easy-to-use interface, informative result page and programmatic access. Considering the info-communication evolution in the last few years, the developed web server, as well as the molecule viewer, is responsive and fully compatible with the prevalent tablets and mobile devices.

  18. Modeling of arylamide helix mimetics in the p53 peptide binding site of hDM2 suggests parallel and anti-parallel conformations are both stable.

    Directory of Open Access Journals (Sweden)

    Jonathan C Fuller

    Full Text Available The design of novel α-helix mimetic inhibitors of protein-protein interactions is of interest to pharmaceuticals and chemical genetics researchers as these inhibitors provide a chemical scaffold presenting side chains in the same geometry as an α-helix. This conformational arrangement allows the design of high affinity inhibitors mimicking known peptide sequences binding specific protein substrates. We show that GAFF and AutoDock potentials do not properly capture the conformational preferences of α-helix mimetics based on arylamide oligomers and identify alternate parameters matching solution NMR data and suitable for molecular dynamics simulation of arylamide compounds. Results from both docking and molecular dynamics simulations are consistent with the arylamides binding in the p53 peptide binding pocket. Simulations of arylamides in the p53 binding pocket of hDM2 are consistent with binding, exhibiting similar structural dynamics in the pocket as simulations of known hDM2 binders Nutlin-2 and a benzodiazepinedione compound. Arylamide conformations converge towards the same region of the binding pocket on the 20 ns time scale, and most, though not all dihedrals in the binding pocket are well sampled on this timescale. We show that there are two putative classes of binding modes for arylamide compounds supported equally by the modeling evidence. In the first, the arylamide compound lies parallel to the observed p53 helix. In the second class, not previously identified or proposed, the arylamide compound lies anti-parallel to the p53 helix.

  19. In Vitro and In Vivo Studies on the Structural Organization of Chs3 from Saccharomyces cerevisiae.

    Science.gov (United States)

    Gohlke, Simon; Muthukrishnan, Subbaratnam; Merzendorfer, Hans

    2017-03-25

    Chitin biosynthesis in yeast is accomplished by three chitin synthases (Chs) termed Chs1, Chs2 and Chs3, of which the latter accounts for most of the chitin deposited within the cell wall. While the overall structures of Chs1 and Chs2 are similar to those of other chitin synthases from fungi and arthropods, Chs3 lacks some of the C-terminal transmembrane helices raising questions regarding its structure and topology. To fill this gap of knowledge, we performed bioinformatic analyses and protease protection assays that revealed significant information about the catalytic domain, the chitin-translocating channel and the interfacial helices in between. In particular, we identified an amphipathic, crescent-shaped α-helix attached to the inner side of the membrane that presumably controls the channel entrance and a finger helix pushing the polymer into the channel. Evidence has accumulated in the past years that chitin synthases form oligomeric complexes, which may be necessary for the formation of chitin nanofibrils. However, the functional significance for living yeast cells has remained elusive. To test Chs3 oligomerization in vivo, we used bimolecular fluorescence complementation. We detected oligomeric complexes at the bud neck, the lateral plasma membrane, and in membranes of Golgi vesicles, and analyzed their transport route using various trafficking mutants.

  20. A bifunctional spin label reports the structural topology of phospholamban in magnetically-aligned bicelles

    Science.gov (United States)

    McCaffrey, Jesse E.; James, Zachary M.; Svensson, Bengt; Binder, Benjamin P.; Thomas, David D.

    2016-01-01

    We have applied a bifunctional spin label and EPR spectroscopy to determine membrane protein structural topology in magnetically-aligned bicelles, using monomeric phospholamban (PLB) as a model system. Bicelles are a powerful tool for studying membrane proteins by NMR and EPR spectroscopies, where magnetic alignment yields topological constraints by resolving the anisotropic spectral properties of nuclear and electron spins. However, EPR bicelle studies are often hindered by the rotational mobility of monofunctional Cys-linked spin labels, which obscures their orientation relative to the protein backbone. The rigid and stereospecific TOAC label provides high orientational sensitivity but must be introduced via solid-phase peptide synthesis, precluding its use in large proteins. Here we show that a bifunctional methanethiosulfonate spin label attaches rigidly and stereospecifically to Cys residues at i and i + 4 positions along PLB's transmembrane helix, thus providing orientational resolution similar to that of TOAC, while being applicable to larger membrane proteins for which synthesis is impractical. Computational modeling and comparison with NMR data shows that these EPR experiments provide accurate information about helix tilt relative to the membrane normal, thus establishing a robust method for determining structural topology in large membrane proteins with a substantial advantage in sensitivity over NMR.

  1. Evaluation of a high-precision gear measuring machine for helix measurement using helix and wedge artifacts

    Science.gov (United States)

    Taguchi, Tetsuya; Kondo, Yohan

    2016-08-01

    High-precision gears are required for advanced motion and power transmission. The reliability of the measured value becomes important as the gear accuracy increases, and the establishment of a traceability system is needed. Therefore, a high-precision gear measuring machine (GMM) with a smaller uncertainty is expected to improve the gear calibration uncertainty. For this purpose, we developed a prototype of a high-precision GMM that adopts a direct drive mechanism and other features. Then, the high measurement capability of the developed GMM was verified using gear artifacts. Recently, some new measurement methods using simple shapes such as spheres and planes have been proposed as standards. We have verified the tooth profile measurement using a sphere artifact and reported the results that the developed GMM had a high capability in tooth profile measurement. Therefore, we attempted to devise a new evaluation method for helix measurement using a wedge artifact (WA) whose plane was treated as the tooth flank, and the high measurement capability of the developed GMM was verified. The results will provide a part of information to fully assess measurement uncertainty as our future work. This paper describes the evaluation results of the developed GMM for helix measurement using both a helix artifact and the WA, and discusses the effectiveness of the WA as a new artifact to evaluate the GMMs.

  2. The effect of k-cubic Dresselhaus spin—orbit coupling on the decay time of persistent spin helix states in semiconductor two-dimensional electron gases

    International Nuclear Information System (INIS)

    Chai Zheng; Hu Mao-Jin; Wang Rui-Qiang; Hu Liang-Bin

    2014-01-01

    We study the theoretical effect of k-cubic (i.e. cubic-in-momentum) Dresselhaus spin—orbit coupling on the decay time of persistent spin helix states in semiconductor two-dimensional electron gases. We show that the decay time of persistent spin helix states may be suppressed substantially by k-cubic Dresselhaus spin—orbit coupling, and after taking the effect of k-cubic Dresselhaus spin—orbit interaction into account, the theoretical results obtained accord both qualitatively and quantitatively with other recent experimental results. (condensed matter: electronic structure, electrical, magnetic, and optical properties)

  3. Exposed hydrophobic residues in human immunodeficiency virus type 1 Vpr helix-1 are important for cell cycle arrest and cell death.

    Directory of Open Access Journals (Sweden)

    R Anthony Barnitz

    Full Text Available The human immunodeficiency virus type 1 (HIV-1 accessory protein viral protein R (Vpr is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M blockade and cytopathicity. Mutations of the exposed hydrophobic residues along helix-1 were found to reduce Vpr-induced cell cycle arrest and cell death as well. The levels of toxicity during virion delivery of Vpr correlated with G2/M arrest. Thus, the exposed hydrophobic amino acids in the amino-terminal helix-1 are important for the cell cycle arrest and cytopathicity functions of Vpr.

  4. Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

    Directory of Open Access Journals (Sweden)

    Balda Maria S

    2009-12-01

    Full Text Available Abstract Background Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction. Results Here we identify a new transmembrane component of tight junctions: MarvelD3, a four-span transmembrane protein. Its predicted transmembrane helices form a Marvel (MAL and related proteins for vesicle traffic and membrane link domain, a structural motif originally discovered in proteins involved in membrane apposition and fusion events, such as the tight junction proteins occludin and tricellulin. In mammals, MarvelD3 is expressed as two alternatively spliced isoforms. Both isoforms exhibit a broad tissue distribution and are expressed by different types of epithelial as well as endothelial cells. MarvelD3 co-localises with occludin at tight junctions in intestinal and corneal epithelial cells. RNA interference experiments in Caco-2 cells indicate that normal MarvelD3 expression is not required for the formation of functional tight junctions but depletion results in monolayers with increased transepithelial electrical resistance. Conclusions Our data indicate that MarvelD3 is a third member of the tight junction-associated occludin family of transmembrane proteins. Similar to occludin, normal expression of MarvelD3 is not essential for the formation of functional tight junctions. However, MarvelD3 functions as a determinant of epithelial paracellular permeability properties.

  5. Stakeholder engagement in quattro helix model for mobile phone reverse logistics in Indonesia: a conceptual framework

    Science.gov (United States)

    Maheswari, H.; Yudoko, G.; Adhiutama, A.

    2017-12-01

    The number of e-waste from mobile phone industry is still dominating until now. This is happened because there is no mutual commitment from all of parties i.e. businesses, government, and societies to reduce the use of mobile phone that has the shortest product life cycle. There are many researches study about firms’ motivation and government’s role, other discuss about actions of communities in supporting reverse logistics implementation. Unfortunately, research about engagement mechanism that involving all parties is still rare. Therefore, it is important to find the engagement model through this conceptual paper and it is expected useful to build the novel model. Through literature review, the results of this research are establishing the Quattro helix model as the appropriate structure to build the robust team by exploring stakeholder theories; mapping the engagement model either in form of collaboration or participation that consider stakeholders’ role and motivation and finding six types of engagement that consider their interest; and determining the novel model of engagement through Quattro helix model for implementing reverse logistics in handling e-waste by describing the linkage and the gaps among existing model.

  6. Nuclear receptor ligand-binding domains: reduction of helix H12 dynamics to favour crystallization

    Energy Technology Data Exchange (ETDEWEB)

    Nahoum, Virginie; Lipski, Alexandra; Quillard, Fabien; Guichou, Jean-François [INSERM, U554, 34090 Montpellier (France); Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale (CBS), 34090 Montpellier (France); Boublik, Yvan [CNRS, UMR5237, Centre de Recherche de Biochimie Macromoléculaire (CRBM), 34293 Montpellier (France); Pérez, Efrèn [Universidade de Vigo, Departamento de Quimica Organica, Facultad de Química, 36310 Vigo (Spain); Germain, Pierre [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), BP 10142, 67404 Illkirch CEDEX (France); Lera, Angel R. de [Universidade de Vigo, Departamento de Quimica Organica, Facultad de Química, 36310 Vigo (Spain); Bourguet, William, E-mail: bourguet@cbs.cnrs.fr [INSERM, U554, 34090 Montpellier (France); Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale (CBS), 34090 Montpellier (France)

    2008-07-01

    Attempts have been made to crystallize the ligand-binding domain of the human retinoid X receptor in complex with a variety of newly synthesized ligands. An inverse correlation was observed between the ‘crystallizability’ and the structural dynamics of the various receptor–ligand complexes. Crystallization trials of the human retinoid X receptor α ligand-binding domain (RXRα LBD) in complex with various ligands have been carried out. Using fluorescence anisotropy, it has been found that when compared with agonists these small-molecule effectors enhance the dynamics of the RXRα LBD C-terminal helix H12. In some cases, the mobility of this helix could be dramatically reduced by the addition of a 13-residue co-activator fragment (CoA). In keeping with these observations, crystals have been obtained of the corresponding ternary RXRα LBD–ligand–CoA complexes. In contrast, attempts to crystallize complexes with a highly mobile H12 remained unsuccessful. These experimental observations substantiate the previously recognized role of co-regulator fragments in facilitating the crystallization of nuclear receptor LBDs.

  7. Role of the future creative universities in the triple helix of science and technology corridors

    Directory of Open Access Journals (Sweden)

    Iraj nabipour

    2015-01-01

    Full Text Available The science and technology corridor is a complex cluster containing universities, science parks, research centers, high-tech companies, venture capital, institutional and physical infrastructures, and human capital in a defined geography with its unique management and legal structure in association with the business space and knowledge-based products. In fact, the science and technology corridor reflects the concept of development based on the knowledge region (the especial region for science and technology. The knowledge region is clearly a triple helix phenomenon par excellence: universities, governments and businesses combine their efforts to construct a common advantage which they would not be able to offer on their own. The future creative universities in connection with the knowledge city-regions not only will deal with innovation and entrepreneurial training but also produce a competitive, vibrant environment with high indices for quality of life and full of green technologies. In this article, we will present functional interactions of the creative universities in the triple helix, particularly the missions for the Iranian universities of medical sciences. As a theoretical model, the complex interactions of Bushehr University of Medical Sciences and Health Services with Bushehr Science and Technology Corridor will be discussed.

  8. Tubular Unimolecular Transmembrane Channels: Construction Strategy and Transport Activities.

    Science.gov (United States)

    Si, Wen; Xin, Pengyang; Li, Zhan-Ting; Hou, Jun-Li

    2015-06-16

    Lipid bilayer membranes separate living cells from their environment. Membrane proteins are responsible for the processing of ion and molecular inputs and exports, sensing stimuli and signals across the bilayers, which may operate in a channel or carrier mechanism. Inspired by these wide-ranging functions of membrane proteins, chemists have made great efforts in constructing synthetic mimics in order to understand the transport mechanisms, create materials for separation, and develop therapeutic agents. Since the report of an alkylated cyclodextrin for transporting Cu(2+) and Co(2+) by Tabushi and co-workers in 1982, chemists have constructed a variety of artificial transmembrane channels by making use of either the multimolecular self-assembly or unimolecular strategy. In the context of the design of unimolecular channels, important advances have been made, including, among others, the tethering of natural gramicidin A or alamethicin and the modification of various macrocycles such as crown ethers, cyclodextrins, calixarenes, and cucurbiturils. Many of these unimolecular channels exhibit high transport ability for metal ions, particularly K(+) and Na(+). Concerning the development of artificial channels based on macrocyclic frameworks, one straightforward and efficient approach is to introduce discrete chains to reinforce their capability to insert into bilayers. Currently, this approach has found the widest applications in the systems of crown ethers and calixarenes. We envisioned that for macrocycle-based unimolecular channels, control of the arrangement of the appended chains in the upward and/or downward direction would favor the insertion of the molecular systems into bilayers, while the introduction of additional interactions among the chains would further stabilize a tubular conformation. Both factors should be helpful for the formation of new efficient channels. In this Account, we discuss our efforts in designing new unimolecular artificial channels from

  9. A genome-wide identification and analysis of the basic helix-loop-helix transcription factors in the ponerine ant, Harpegnathos saltator

    Directory of Open Access Journals (Sweden)

    Liu Ake

    2012-08-01

    Full Text Available Abstract Background The basic helix-loop-helix (bHLH transcription factors and their homologs form a superfamily that plays essential roles in transcriptional networks of multiple developmental processes. bHLH family members have been identified in over 20 organisms, including fruit fly, zebrafish, human and mouse. Result In this study, we conducted a genome-wide survey for bHLH sequences, and identified 57 bHLH sequences encoded in complete genome sequence of the ponerine ant, Harpegnathos saltator. Phylogenetic analysis of the bHLH domain sequences classified these genes into 38 bHLH families with 23, 14, 10, 1, 8 and 1 members in group A, B, C, D, E and F, respectively. The number of PabHLHs (ponerine ant bHLHs with introns is higher than many other insect species, and they are found to have introns with average lengths only inferior to those of pea aphid. In addition, two H. saltator bHLHs named PaCrp1 and PaSide locate on two separate contigs in the genome. Conclusions A putative full set of PabHLH genes is comparable with other insect species and genes encoding Oligo, MyoRb and Figα were not found in genomes of all insect species of which bHLH family members have been identified. Moreover, in-family phylogenetic analyses indicate that the PabHLH genes are more closely related with Apis mellifera than others. The present study will serve as a solid foundation for further investigations into the structure and function of bHLH proteins in the regulation of H. saltator development.

  10. The ER membrane protein complex is a transmembrane domain insertase

    Science.gov (United States)

    Guna, Alina; Volkmar, Norbert; Christianson, John C.; Hegde, Ramanujan S.

    2018-01-01

    Insertion of proteins into membranes is an essential cellular process. The extensive biophysical and topological diversity of membrane proteins necessitates multiple insertion pathways that remain incompletely defined. Here, we found that known membrane insertion pathways fail to effectively engage tail-anchored membrane proteins with moderately hydrophobic transmembrane domains. These proteins are instead shielded in the cytosol by calmodulin. Dynamic release from calmodulin allowed sampling of the endoplasmic reticulum (ER), where the conserved ER membrane protein complex (EMC) was shown to be essential for efficient insertion in vitro and in cells. Purified EMC in synthetic liposomes catalyzed insertion of its substrates in a reconstituted system. Thus, EMC is a transmembrane domain insertase, a function that may explain its widely pleiotropic membrane-associated phenotypes across organisms. PMID:29242231

  11. Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jun; Byrne, Noel; Wang, John; Bricogne, Gerard; Brown, Frank K.; Chobanian, Harry R.; Colletti, Steven L.; Di Salvo, Jerry; Thomas-Fowlkes, Brande; Guo, Yan; Hall, Dawn L.; Hadix, Jennifer; Hastings, Nicholas B.; Hermes, Jeffrey D.; Ho, Thu; Howard, Andrew D.; Josien, Hubert; Kornienko, Maria; Lumb, Kevin J.; Miller, Michael W.; Patel, Sangita B.; Pio, Barbara; Plummer, Christopher W.; Sherborne, Bradley S.; Sheth, Payal; Souza, Sarah; Tummala, Srivanya; Vonrhein, Clemens; Webb, Maria; Allen, Samantha J.; Johnston, Jennifer M.; Weinglass, Adam B.; Sharma, Sujata; Soisson, Stephen M. (Merck); (Globel Phasing)

    2017-06-05

    Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

  12. The HIV-1 Integrase α4-Helix Involved in LTR-DNA Recognition Is also a Highly Antigenic Peptide Element

    Science.gov (United States)

    Azzi, Sandy; Parissi, Vincent; Maroun, Richard G.; Eid, Pierre; Mauffret, Olivier; Fermandjian, Serge

    2010-01-01

    Monoclonal antibodies (MAbas) constitute remarkable tools to analyze the relationship between the structure and the function of a protein. By immunizing a mouse with a 29mer peptide (K159) formed by residues 147 to 175 of the HIV-1 integrase (IN), we obtained a monoclonal antibody (MAba4) recognizing an epitope lying in the N-terminal portion of K159 (residues 147–166 of IN). The boundaries of the epitope were determined in ELISA assays using peptide truncation and amino acid substitutions. The epitope in K159 or as a free peptide (pep-a4) was mostly a random coil in solution, while in the CCD (catalytic core domain) crystal, the homologous segment displayed an amphipathic helix structure (α4-helix) at the protein surface. Despite this conformational difference, a strong antigenic crossreactivity was observed between pep-a4 and the protein segment, as well as K156, a stabilized analogue of pep-a4 constrained into helix by seven helicogenic mutations, most of them involving hydrophobic residues. We concluded that the epitope is freely accessible to the antibody inside the protein and that its recognition by the antibody is not influenced by the conformation of its backbone and the chemistry of amino acids submitted to helicogenic mutations. In contrast, the AA →Glu mutations of the hydrophilic residues Gln148, Lys156 and Lys159, known for their interactions with LTRs (long terminal repeats) and inhibitors (5 CITEP, for instance), significantly impaired the binding of K156 to the antibody. Moreover, we found that in competition ELISAs, the processed and unprocessed LTR oligonucleotides interfered with the binding of MAba4 to IN and K156, confirming that the IN α4-helix uses common residues to interact with the DNA target and the MAba4 antibody. This also explains why, in our standard in vitro concerted integration assays, MAba4 strongly impaired the IN enzymatic activity. PMID:21209864

  13. Transmembrane electron transport and the neutral theory of evolution.

    Science.gov (United States)

    Scherer, S

    1984-01-01

    Based on the concept of "pairs of basic functional states" the evolution of the first chemiosmotic mechanism of energy conversion is discussed in terms of point mutations, gene duplications and of the neutral theory of evolution. A model for estimating the overall probability of the evolutionary step in question is presented, both for the "selectionist" and "neutralist" position. It is concluded that, concerning the present stage of knowledge, the evolution of transmembrane electron transport is an unsolved problem in evolutionary biology.

  14. The Mutation P.T613a in the Pore Helix of the Kv 11.1 Potassium Channel is Associated with Long Qt Syndrome

    DEFF Research Database (Denmark)

    Poulsen, Kristian L; Hotait, Mostafa; Calloe, Kirstine

    2015-01-01

    BACKGROUND: Loss-of-function mutations in the voltage gated potassium channel Kv 11.1 have been associated with the Long QT Syndrome (LQTS) type 2. We identified the p.T613A mutation in Kv 11.1 in a family with LQTS. T613A is located in the outer part of the pore helix, a structure that is involved...

  15. Metal bridges between the PhoQ sensor domain and the membrane regulate transmembrane signaling.

    Science.gov (United States)

    Cho, Uhn Soo; Bader, Martin W; Amaya, Maria F; Daley, Margaret E; Klevit, Rachel E; Miller, Samuel I; Xu, Wenqing

    2006-03-10

    Bacterial histidine kinases respond to environmental stimuli by transducing a signal from an extracytosolic sensor domain to a cytosolic catalytic domain. Among them, PhoQ promotes bacterial virulence and is tightly repressed by the divalent cations such as calcium and magnesium. We have determined the crystal structure of the PhoQ sensor domain from Salmonella typhimurium in the Ca2+-bound state, which reveals a highly negatively charged surface that is in close proximity to the inner membrane. This acidic surface binds at least three Ca2+, which mediate the PhoQ-membrane interaction. Mutagenesis analysis indicates that structural integrity at the membrane proximal region of the PhoQ sensor domain promotes metal-mediated repression. We propose that depletion or displacement of divalent cations leads to charge repulsion between PhoQ and the membrane, which initiates transmembrane signaling through a change in orientation between the PhoQ sensor domain and membrane. Therefore, both PhoQ and the membrane are required for extracytosolic sensing and transmembrane signaling.

  16. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors.

    Science.gov (United States)

    Culhane, Kelly J; Liu, Yuting; Cai, Yingying; Yan, Elsa C Y

    2015-01-01

    Although family B G protein-coupled receptors (GPCRs) contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs.

  17. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

    Directory of Open Access Journals (Sweden)

    Kelly J Culhane

    2015-11-01

    Full Text Available Although family B G protein-coupled receptors (GPCRs contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs.

  18. Nanoporous microbead supported bilayers: stability, physical characterization, and incorporation of functional transmembrane proteins.

    Energy Technology Data Exchange (ETDEWEB)

    Davis, Ryan W. (University of New Mexico, Albuquerque, NM); Brozik, James A. (University of New Mexico, Albuquerque, NM); Brozik, Susan Marie; Cox, Jason M. (University of New Mexico, Albuquerque, NM); Lopez, Gabriel P. (University of New Mexico, Albuquerque, NM); Barrick, Todd A. (University of New Mexico, Albuquerque, NM); Flores, Adrean (University of New Mexico, Albuquerque, NM)

    2007-03-01

    The introduction of functional transmembrane proteins into supported bilayer-based biomimetic systems presents a significant challenge for biophysics. Among the various methods for producing supported bilayers, liposomal fusion offers a versatile method for the introduction of membrane proteins into supported bilayers on a variety of substrates. In this study, the properties of protein containing unilamellar phosphocholine lipid bilayers on nanoporous silica microspheres are investigated. The effects of the silica substrate, pore structure, and the substrate curvature on the stability of the membrane and the functionality of the membrane protein are determined. Supported bilayers on porous silica microspheres show a significant increase in surface area on surfaces with structures in excess of 10 nm as well as an overall decrease in stability resulting from increasing pore size and curvature. Comparison of the liposomal and detergent-mediated introduction of purified bacteriorhodopsin (bR) and the human type 3 serotonin receptor (5HT3R) are investigated focusing on the resulting protein function, diffusion, orientation, and incorporation efficiency. In both cases, functional proteins are observed; however, the reconstitution efficiency and orientation selectivity are significantly enhanced through detergent-mediated protein reconstitution. The results of these experiments provide a basis for bulk ionic and fluorescent dye-based compartmentalization assays as well as single-molecule optical and single-channel electrochemical interrogation of transmembrane proteins in a biomimetic platform.

  19. Transient Non-Native Helix Formation during the Folding of b-Lactoglobulin

    Directory of Open Access Journals (Sweden)

    Masamichi Ikeguchi

    2014-02-01

    Full Text Available In ideal proteins, only native interactions are stabilized step-by-step in a smooth funnel-like energy landscape. In real proteins, however, the transient formation of non-native structures is frequently observed. In this review, the transient formation of non-native structures is described using the non-native helix formation during the folding of b-lactoglobulin as a prominent example. Although b-lactoglobulin is a predominantly b-sheet protein, it has been shown to form non-native helices during the early stage of folding. The location of non-native helices, their stabilization mechanism, and their role in the folding reaction are discussed.

  20. Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

    Science.gov (United States)

    Xi, Zhiqun; Gao, Ying; Sirinakis, George; Guo, Honglian; Zhang, Yongli

    2012-01-01

    The biological functions of coiled coils generally depend on efficient folding and perfect pairing of their α-helices. Dynamic changes in the helical registry that lead to staggered helices have only been proposed for a few special systems and not found in generic coiled coils. Here, we report our observations of multiple staggered helical structures of two canonical coiled coils. The partially folded structures are formed predominantly by coiled coil misfolding and occasionally by helix sliding. Using high-resolution optical tweezers, we characterized their energies and transition kinetics at a single-molecule level. The staggered states occur less than 2% of the time and about 0.1% of the time at zero force. We conclude that dynamic changes in helical registry may be a general property of coiled coils. Our findings should have broad and unique implications in functions and dysfunctions of proteins containing coiled coils. PMID:22451899

  1. Stretched versus compressed exponential kinetics in α-helix folding

    International Nuclear Information System (INIS)

    Hamm, Peter; Helbing, Jan; Bredenbeck, Jens

    2006-01-01

    In a recent paper (J. Bredenbeck, J. Helbing, J.R. Kumita, G.A. Woolley, P. Hamm, α-helix formation in a photoswitchable peptide tracked from picoseconds to microseconds by time resolved IR spectroscopy, Proc. Natl. Acad. Sci USA 102 (2005) 2379), we have investigated the folding of a photo-switchable α-helix with a kinetics that could be fit by a stretched exponential function exp(-(t/τ) β ). The stretching factor β became smaller as the temperature was lowered, a result which has been interpreted in terms of activated diffusion on a rugged energy surface. In the present paper, we discuss under which conditions diffusion problems occur with stretched exponential kinetics (β 1). We show that diffusion problems do have a strong tendency to yield stretched exponential kinetics, yet, that there are conditions (strong perturbation from equilibrium, performing the experiment in the folding direction) under which compressed exponential kinetics would be expected instead. We discuss the kinetics on free energy surfaces predicted by simple initiation-propagation models (zipper models) of α-helix folding, as well as by folding funnel models. We show that our recent experiment has been performed under condition for which models with strong downhill driving force, such as the zipper model, would predict compressed, rather than stretched exponential kinetics, in disagreement with the experimental observation. We therefore propose that the free energy surface along a reaction coordinate that governs the folding kinetics must be relatively flat and has a shape similar to a 1D golf course. We discuss how this conclusion can be unified with the thermodynamically well established zipper model by introducing an additional kinetic reaction coordinate

  2. The self-propulsion of a helix in granular matter

    Science.gov (United States)

    Valdes, Rogelio; Angeles, Veronica; de La Calleja, Elsa; Zenit, Roberto

    2017-11-01

    The effect of the shape of helicoidal on the displacement of magnetic robots in granular media is studied experimentally. We quantify the influences of three main parameters of the shape of the helicoidal swimmers: body diameter, step, and the angle. We compare the experimental measurements with an empirically modified resistive force theory prediction that accounts for the static friction coefficient of the particles of the granular material, leading to good agreement. Comparisons are also made with the granular resistive force theory proposed by Goldman and collaborators. We found an optimal helix angle to produce movement and determined a relationship between the swimmer size and speed.

  3. Some quality parameters of land snail meat - Helix pomatia

    Directory of Open Access Journals (Sweden)

    Tojagić Slobodan N.

    2004-01-01

    Full Text Available Considering the tradition in our regions to collect land snails (Helix pomatia for export, which is "disrupted" by social control resulting in limited possibilities to develop this attractive activity, there is a great interest lately for land snail breeding and fattening at farms. For this reason it is necessary to investigate systematically the possibilities to develop this activity in a longer period and in larger areas. The first investigations, although covering only nutritive and health safety aspects of the edible parts yielded the results presented in this work. Chemical composition, the content of some elements and organochlorine insecticides were followed as unavoidable in human living and environment.

  4. Toxic effects of Cadmium on the garden snail (Helix aspersa)

    Energy Technology Data Exchange (ETDEWEB)

    Russell, L.K. (Northrop Services Inc., Corvallis, OR); DeHaven, J.I.; Botts, R.P.

    1981-05-01

    Spreading treated municipal wastes on agricultural and forest lands is becoming an established method of disposal. However, there is concern about the deleterious effects of toxicants, particularly cadmium, in the sludges. Cadmium concentrations in sewage sludge have been reported as high as 1500 ppM. The work reported here is a part of a larger project to investigate the ecological effects of municipal wastes on forest lands. Snails, Helix aspersa, were chosen to examine the entrance of cadmium into terrestrial food chains. This experiment was designed to determine cadmium accumulation, acute toxicity, and behavioral, reproductive and growth responses with increasing levels of cadmium.

  5. Ab initio theory of helix <-> coil phase transition

    DEFF Research Database (Denmark)

    Yakubovich, Alexander V.; Solov'yov, Ilia; Solov'yov, Andrey V.

    2008-01-01

    In this paper, we suggest a theoretical method based on the statistical mechanics for treating the alpha-helix random coil transition in alanine polypeptides. We consider this process as a first-order phase transition and develop a theory which is free of model parameters and is based solely...... on fundamental physical principles. It describes essential thermodynamical properties of the system such as heat capacity, the phase transition temperature and others from the analysis of the polypeptide potential energy surface calculated as a function of two dihedral angles, responsible for the polypeptide...

  6. Identification of the roles of individual amino acid residues of the helix E of the major antenna of photosystem II (LHCII) by alanine scanning mutagenesis.

    Science.gov (United States)

    Liu, Cheng; Rao, Yan; Zhang, Lei; Yang, Chunhong

    2014-10-01

    The functions of the helix E (W97-F105), an amphiphilic lumenal 310 helix of the major antenna of photosystem II (LHCII), are still unidentified. To elucidate the roles of individual amino acid residue of the helix E, alanine scanning mutagenesis has been performed to mutate every residue of this domain to alanine. The influence of every alanine substitution on the structure and function of LHCII has been investigated biochemically and spectroscopically. The results show that all mutations have little impact on the pigment binding and configuration. However, many mutants presented decreased thermo- or photo-stability compared with the wild type, highlighting the significance of this helix to the stability of LHCII. The most critical residue for stability is W97. The mutant W97A yielded very fragile trimeric pigment protein complexes. The structural analysis revealed that the hydrogen bonding and aromatic interactions between W97, F195, F194 and a water molecule contributed greatly to the stability of LHCII. Moreover, Q103A and F105A have been identified to be able to reinforce the tendency of aggregation in vitro. The structural analysis suggested that the enhancement in aggregation formation for Q103A and F105A might be attributed to the changing hydrophobicity of the region. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  7. Spontaneously amplified homochiral organic-inorganic nano-helix complexes via self-proliferation

    Science.gov (United States)

    Zhai, Halei; Quan, Yan; Li, Li; Liu, Xiang-Yang; Xu, Xurong; Tang, Ruikang

    2013-03-01

    Most spiral coiled biomaterials in nature, such as gastropod shells, are homochiral, and the favoured chiral feature can be precisely inherited. This inspired us that selected material structures, including chirality, could be specifically replicated into the self-similar populations; however, a physicochemical understanding of the material-based heritage is unknown. We study the homochirality by using calcium phosphate mineralization in the presence of racemic amphiphilic molecules and biological protein. The organic-inorganic hybrid materials with spiral coiling characteristics are produced at the nanoscale. The resulted helixes are chiral with the left- and right-handed characteristics, which are agglomerated hierarchically to from clusters and networks. It is interesting that each cluster or network is homochiral so that the enantiomorphs can be separated readily. Actually, each homochiral architecture is evolved from an original chiral helix, demonstrating the heritage of the matrix chirality during the material proliferation under a racemic condition. By using the Ginzburg-Landaue expression we find that the chiral recognition in the organic-inorganic hybrid formation may be determined by a spontaneous chiral separation and immobilization of asymmetric amphiphilic molecules on the mineral surface, which transferred the structural information from the mother matrix to the descendants by an energetic control. This study shows how biomolecules guide the selective amplification of chiral materials via spontaneous self-replication. Such a strategy can be applied generally in the design and production of artificial materials with self-similar structure characteristics.Most spiral coiled biomaterials in nature, such as gastropod shells, are homochiral, and the favoured chiral feature can be precisely inherited. This inspired us that selected material structures, including chirality, could be specifically replicated into the self-similar populations; however, a

  8. Assessing GPCR homology models constructed from templates of various transmembrane sequence identities: Binding mode prediction and docking enrichment.

    Science.gov (United States)

    Loo, Jason S E; Emtage, Abigail L; Ng, Kar Weng; Yong, Alene S J; Doughty, Stephen W

    2018-03-01

    GPCR crystal structures have become more readily accessible in recent years. However, homology models of GPCRs continue to play an important role as many GPCR structures remain unsolved. The new crystal structures now available provide not only additional templates for homology modelling but also the opportunity to assess the performance of homology models against their respective crystal structures and gain insight into the performance of such models. In this study we have constructed homology models from templates of various transmembrane sequence identities for eight GPCR targets to better understand the relationship between transmembrane sequence identity and model quality. Model quality was assessed relative to the crystal structure in terms of structural accuracy as well as performance in two typical structure-based drug design applications: ligand binding pose prediction and docking enrichment in virtual screening. Crystal structures significantly outperformed homology models in both assessments. Accurate ligand binding pose prediction was possible but difficult to achieve using homology models, even with the use of induced fit docking. In virtual screening using homology models still conferred significant enrichment compared to random selection, with a clear benefit also observed in using models optimized through induced fit docking. Our results indicate that while homology models that are reasonably accurate structurally can be constructed, without significant refinement homology models will be outperformed by crystal structures in ligand binding pose prediction and docking enrichment regardless of the template used, primarily due to the extremely high level of structural accuracy needed for such applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Improving membrane binding as a design strategy for amphipathic peptide hormones: 2-helix variants of PYY3-36.

    Science.gov (United States)

    Pedersen, Søren L; Bhatia, Vikram K; Jurt, Simon; Paulsson, Johan F; Pedersen, Maria H; Jorgensen, Rasmus; Holst, Birgitte; Stamou, Dimitrios; Vrang, Niels; Zerbe, Oliver; Jensen, Knud J

    2012-09-01

    It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3-36 is believed to perform its appetite-suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3-36 via its amphipathic α-helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3-36. We first studied membrane binding of PYY3-36. Next, we designed a series of PYY3-36 analogs to increase membrane-binding affinity by substituting the N-terminal segment with a de novo designed α-helical, amphipathic sequence. These 2-helix variants of PYY3-36 were assembled by solid-phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4 nM for membranes, was structurally characterized by NMR in the membrane-bound state, which clearly showed that it formed the expected 2-helix. The topology of the peptide-micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3-36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2-helix variant of PYY3-36 will be useful as a tool compound for studying peptide-membrane interactions. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

  10. Structure and Inhibition of the neu-erbB-2 Receptor

    National Research Council Canada - National Science Library

    Stern, David

    1998-01-01

    ... its hydrophobic transmembrane domain. The goal of the research project has been to obtain the high resolution structure of the receptor transmembrane domain and to establish the mechanism by which the Glu664 mutation leads to receptor activation...

  11. Clear cell hidradenocarcinoma of the ear helix: report of primary ear helix adnexal carcinoma with regional lymph node metastasis.

    Science.gov (United States)

    Bae, Tae Hui; Kang, Shin Hyuk; Kim, Han Koo; Kim, Woo Seob; Kim, Mi Kyung

    2014-07-01

    Clear cell hidradenocarcinoma is a rare tumor of eccrine sweat gland origin that has a predilection for the head and neck. It has an indolent growth pattern and a higher incidence of regional and distant metastases. Metastasizing adnexal carcinomas are rare; thus, currently there is no uniform treatment guideline. We report a case of an 89-year-old female patient with clear cell hidradenocarcinoma manifesting in the right ear helix that metastasized to the right parotid gland who was treated by wide local excision and radiation therapy.

  12. Construction and genetic selection of small transmembrane proteins that activate the human erythropoietin receptor.

    Science.gov (United States)

    Cammett, Tobin J; Jun, Susan J; Cohen, Emily B; Barrera, Francisco N; Engelman, Donald M; Dimaio, Daniel

    2010-02-23

    This work describes a genetic approach to isolate small, artificial transmembrane (TM) proteins with biological activity. The bovine papillomavirus E5 protein is a dimeric, 44-amino acid TM protein that transforms cells by specifically binding and activating the platelet-derived growth factor beta receptor (PDGFbetaR). We used the E5 protein as a scaffold to construct a retrovirus library expressing approximately 500,000 unique 44-amino acid proteins with randomized TM domains. We screened this library to select small, dimeric TM proteins that were structurally unrelated to erythropoietin (EPO), but specifically activated the human EPO receptor (hEPOR). These proteins did not activate the murine EPOR or the PDGFbetaR. Genetic studies with one of these activators suggested that it interacted with the TM domain of the hEPOR. Furthermore, this TM activator supported erythroid differentiation of primary human hematopoietic progenitor cells in vitro in the absence of EPO. Thus, we have changed the specificity of a protein so that it no longer recognizes its natural target but, instead, modulates an entirely different protein. This represents a novel strategy to isolate small artificial proteins that affect diverse membrane proteins. We suggest the word "traptamer" for these transmembrane aptamers.

  13. ER-mediated control for abundance, quality, and signaling of transmembrane immune receptors in plants

    Directory of Open Access Journals (Sweden)

    Nico eTintor

    2014-02-01

    Full Text Available Plants recognize a wide range of microbes with cell-surface and intracellular immune receptors. Transmembrane pattern recognition receptors (PRRs initiate immune responses upon recognition of cognate ligands characteristic of microbes or aberrant cellular states, designated microbe-associated molecular patterns (MAMPs or danger-associated molecular patterns (DAMPs, respectively. Pattern-triggered immunity (PTI provides a first line of defense that restricts the invasion and propagation of both adapted and non-adapted pathogens. Receptor kinases (RKs and receptor-like proteins (RLPs with an extracellular leucine-rich repeat (LRR or lysine-motif (LysM domain are extensively used as PRRs. The correct folding of the extracellular domain of these receptors is under quality control (QC in the endoplasmic reticulum (ER, which thus provides a critical step in plant immunity. Genetic and structural insight suggests that ERQC regulates not only the abundance and quality of transmembrane receptors but also affects signal sorting between multi-branched pathways downstream of the receptor. However, ERQC dysfunction can also positively stimulate plant immunity, possibly through cell death and DAMP signaling pathways.

  14. Phenoloxidase activity of Helix aspersa maxima (garden snail, gastropod) hemocyanin.

    Science.gov (United States)

    Raynova, Yuliana; Doumanova, Lyuba; Idakieva, Krassimira Nikolova

    2013-12-01

    The oxygen-transporting protein, hemocyanin (Hc), of the garden snail Helix aspersa maxima (HaH) was isolated and kinetically characterized. Kinetic parameters of the reaction of catalytic oxidation of catechol to quinone, catalyzed by native HaH were determined: the V max value amounted to 22 nmol min(-1) mg(-1), k cat to 1.1 min(-1). Data were compared to those reported for other molluscan Hcs and phenoloxidases (POs). The o-diphenoloxidase activity of the native HaH is about five times higher than the activity determined for the Hcs of the terrestrial snail Helix pomatia and of the marine snail Rapana thomasiana (k cat values of 0.22 and 0.25 min(-1), respectively). The K m values obtained for molluscan Hcs from different species are comparable to those for true POs, but the low catalytic efficiency of Hcs is probably related to inaccessibility of the active sites to potential substrates. Upon treatment of HaH with subtilisin DY, the enzyme activity against substrate catechol was considerably increased. The relatively high proteolytically induced o-diPO activity of HaH allowed using it for preparation of a biosensor for detection of catechol.

  15. α-helix formation rate of oligopeptides at subzero temperatures

    Science.gov (United States)

    Qin, Zhi-jie; Shimizu, Akio; Li, Jinsong; Ikeguchi, Masamichi; Shinjo, Masaji; Kihara, Hiroshi

    2014-01-01

    In 1999, Clarke et al. ((1999) Proc. Natl. Acad. Sci. USA 96, 7232–7237) reported that the nucleation rate of α-helix of oligopeptide AK16 is as slow as 60 ms. In the present study, we measured the nucleation rate of oligopeptide, C17 (DLTDDIMCVKKILDKVG, corresponding to α-helical region of 84th to 100th amino acids of bovine α-lactalbumin) using the same method as Clarke et al. We found only initial bursts of the increase of α-helices at temperatures higher than −50°C in the presence of 70% methanol. The result with AK16 was the same as Clarke et al. reported. We also found that the folding rate of polyglutamic acid is too fast to be detected by the stopped-flow apparatus at 4°C. These results demonstrate that the α-helix formation rates in C17, AK16 and polyglutamic acid are shorter than the dead time of the stopped-flow apparatus (6 ms). PMID:27493493

  16. Molecular pharmacology of promiscuous seven transmembrane receptors sensing organic nutrients

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Johansen, Lars Dan; Bräuner-Osborne, Hans

    2009-01-01

    A number of highly promiscuous seven transmembrane (7TM) receptors have been cloned and characterized within the last few years. It is noteworthy that many of these receptors are activated broadly by amino acids, proteolytic degradation products, carbohydrates, or free fatty acids and are expressed...... receptors FFA1, FFA2, FFA3, GPR84, and GPR120. The involvement of the individual receptors in sensing of food intake has been validated to different degrees because of limited availability of specific pharmacological tools and/or receptor knockout mice. However, as a group, the receptors represent potential...

  17. Role of protein dynamics in transmembrane receptor signalling

    DEFF Research Database (Denmark)

    Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

    2018-01-01

    Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... and computation have been used to study receptor dynamics. Recent studies on three distinct classes of receptors (G-protein coupled receptors, ligand-gated ion-channels and single-pass receptors) are highlighted to show that conformational changes across a range of time-scales and length-scales are central...

  18. Promiscuous Seven Transmembrane Receptors Sensing L-α-amino Acids

    DEFF Research Database (Denmark)

    Smajilovic, Sanela; Wellendorph, Petrine; Bräuner-Osborne, Hans

    2014-01-01

    A number of nutrient sensing seven trans-membrane (7TM) receptors have been identified and characterized over the past few years. While the sensing mechanisms to carbohydrates and free fatty acids are well understood, the molecular basis of amino acid sensing has recently come to the limelight. T....... The present review describes the current status of promiscuous L-α-amino acid sensors, the calcium sensing receptor (CaSR), the GPRC6A receptor, the T1R1/T1R3 receptor and also their molecular pharmacology, expression pattern and physiological significance....

  19. Phylogeny, Functional Annotation, and Protein Interaction Network Analyses of the Xenopus tropicalis Basic Helix-Loop-Helix Transcription Factors

    Directory of Open Access Journals (Sweden)

    Wuyi Liu

    2013-01-01

    Full Text Available The previous survey identified 70 basic helix-loop-helix (bHLH proteins, but it was proved to be incomplete, and the functional information and regulatory networks of frog bHLH transcription factors were not fully known. Therefore, we conducted an updated genome-wide survey in the Xenopus tropicalis genome project databases and identified 105 bHLH sequences. Among the retrieved 105 sequences, phylogenetic analyses revealed that 103 bHLH proteins belonged to 43 families or subfamilies with 46, 26, 11, 3, 15, and 4 members in the corresponding supergroups. Next, gene ontology (GO enrichment analyses showed 65 significant GO annotations of biological processes and molecular functions and KEGG pathways counted in frequency. To explore the functional pathways, regulatory gene networks, and/or related gene groups coding for Xenopus tropicalis bHLH proteins, the identified bHLH genes were put into the databases KOBAS and STRING to get the signaling information of pathways and protein interaction networks according to available public databases and known protein interactions. From the genome annotation and pathway analysis using KOBAS, we identified 16 pathways in the Xenopus tropicalis genome. From the STRING interaction analysis, 68 hub proteins were identified, and many hub proteins created a tight network or a functional module within the protein families.

  20. Basic helix-loop-helix transcription factor TCF21 is a downstream target of the male sex determining gene SRY.

    Directory of Open Access Journals (Sweden)

    Ramji K Bhandari

    Full Text Available The cascade of molecular events involved in mammalian sex determination has been shown to involve the SRY gene, but specific downstream events have eluded researchers for decades. The current study identifies one of the first direct downstream targets of the male sex determining factor SRY as the basic-helix-loop-helix (bHLH transcription factor TCF21. SRY was found to bind to the Tcf21 promoter and activate gene expression. Mutagenesis of SRY/SOX9 response elements in the Tcf21 promoter eliminated the actions of SRY. SRY was found to directly associate with the Tcf21 promoter SRY/SOX9 response elements in vivo during fetal rat testis development. TCF21 was found to promote an in vitro sex reversal of embryonic ovarian cells to induce precursor Sertoli cell differentiation. TCF21 and SRY had similar effects on the in vitro sex reversal gonadal cell transcriptomes. Therefore, SRY acts directly on the Tcf21 promoter to in part initiate a cascade of events associated with Sertoli cell differentiation and embryonic testis development.

  1. Functional diversity of human basic helix-loop-helix transcription factor TCF4 isoforms generated by alternative 5' exon usage and splicing.

    Directory of Open Access Journals (Sweden)

    Mari Sepp

    Full Text Available BACKGROUND: Transcription factor 4 (TCF4 alias ITF2, E2-2, ME2 or SEF2 is a ubiquitous class A basic helix-loop-helix protein that binds to E-box DNA sequences (CANNTG. While involved in the development and functioning of many different cell types, recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and TCF4 haploinsufficiency is the cause of the Pitt-Hopkins mental retardation syndrome. However, the structure, expression and coding potential of the human TCF4 gene have not been described in detail. PRINCIPAL FINDINGS: In the present study we used human tissue samples to characterize human TCF4 gene structure and TCF4 expression at mRNA and protein level. We report that although widely expressed, human TCF4 mRNA expression is particularly high in the brain. We demonstrate that usage of numerous 5' exons of the human TCF4 gene potentially yields in TCF4 protein isoforms with 18 different N-termini. In addition, the diversity of isoforms is increased by alternative splicing of several internal exons. For functional characterization of TCF4 isoforms, we overexpressed individual isoforms in cultured human cells. Our analysis revealed that subcellular distribution of TCF4 isoforms is differentially regulated: Some isoforms contain a bipartite nuclear localization signal and are exclusively nuclear, whereas distribution of other isoforms relies on heterodimerization partners. Furthermore, the ability of different TCF4 isoforms to regulate E-box controlled reporter gene transcription is varied depending on whether one or both of the two TCF4 transcription activation domains are present in the protein. Both TCF4 activation domains are able to activate transcription independently, but act synergistically in combination. CONCLUSIONS: Altogether, in this study we have described the inter-tissue variability of TCF4 expression in human and provided evidence

  2. Coordinated movement of cytoplasmic and transmembrane domains of RyR1 upon gating.

    Directory of Open Access Journals (Sweden)

    Montserrat Samsó

    2009-04-01

    Full Text Available Ryanodine receptor type 1 (RyR1 produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices". Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right

  3. Molecular Mechanism of Disease-Associated Mutations in the Pre-M1 Helix of NMDA Receptors and Potential Rescue Pharmacology.

    Directory of Open Access Journals (Sweden)

    Kevin K Ogden

    2017-01-01

    Full Text Available N-methyl-D-aspartate receptors (NMDARs, ligand-gated ionotropic glutamate receptors, play key roles in normal brain development and various neurological disorders. Here we use standing variation data from the human population to assess which protein domains within NMDAR GluN1, GluN2A and GluN2B subunits show the strongest signal for being depleted of missense variants. We find that this includes the GluN2 pre-M1 helix and linker between the agonist-binding domain (ABD and first transmembrane domain (M1. We then evaluate the functional changes of multiple missense mutations in the NMDAR pre-M1 helix found in children with epilepsy and developmental delay. We find mutant GluN1/GluN2A receptors exhibit prolonged glutamate response time course for channels containing 1 or 2 GluN2A-P552R subunits, and a slow rise time only for receptors with 2 mutant subunits, suggesting rearrangement of one GluN2A pre-M1 helix is sufficient for rapid activation. GluN2A-P552R and analogous mutations in other GluN subunits increased the agonist potency and slowed response time course, suggesting a functionally conserved role for this residue. Although there is no detectable change in surface expression or open probability for GluN2A-P552R, the prolonged response time course for receptors that contained GluN2A-P552R increased charge transfer for synaptic-like activation, which should promote excitotoxic damage. Transfection of cultured neurons with GluN2A-P552R prolonged EPSPs, and triggered pronounced dendritic swelling in addition to excitotoxicity, which were both attenuated by memantine. These data implicate the pre-M1 region in gating, provide insight into how different subunits contribute to gating, and suggest that mutations in the pre-M1 helix can compromise neuronal health. Evaluation of FDA-approved NMDAR inhibitors on the mutant NMDAR-mediated current response and neuronal damage provides a potential clinical path to treat individuals harboring similar

  4. Stability analysis of the inverse transmembrane potential problem in electrocardiography

    Science.gov (United States)

    Burger, Martin; Mardal, Kent-André; Nielsen, Bjørn Fredrik

    2010-10-01

    In this paper we study some mathematical properties of an inverse problem arising in connection with electrocardiograms (ECGs). More specifically, we analyze the possibility for recovering the transmembrane potential in the heart from ECG recordings, a challenge currently investigated by a growing number of groups. Our approach is based on the bidomain model for the electrical activity in the myocardium, and leads to a parameter identification problem for elliptic partial differential equations (PDEs). It turns out that this challenge can be split into two subproblems: the task of recovering the potential at the heart surface from body surface recordings; the problem of computing the transmembrane potential inside the heart from the potential determined at the heart surface. Problem (1), which can be formulated as the Cauchy problem for an elliptic PDE, has been extensively studied and is well known to be severely ill-posed. The main purpose of this paper is to prove that problem (2) is stable and well posed if a suitable prior is available. Moreover, our theoretical findings are illuminated by a series of numerical experiments. Finally, we discuss some aspects of uniqueness related to the anisotropy in the heart.

  5. Comparative Study on the Adaptation and Growth Dynamics of the Helix pomatia and Helix aspersa Muller Terrestrial Snails Under Different Feeding Regimes

    Directory of Open Access Journals (Sweden)

    Adrian Toader-Williams

    2010-05-01

    Full Text Available We used Helix pomatia and Helix aspersa species and measure their growth as the snails were approaching the hibernation season. Helix pomatia 2yo shown a decrease in weight while being raised in enclosed parcels of 4sqm the younger Helix pomatia 1yo as well as Helix aspersa Muller demonstrated the ability to adapt relatively fast to the same conditions. We established 5 experimental lots in a Helix pomatia farm, GPS coordinates N46.606040 E23.599950. Control lot contained Taraxacum officinales, Sonchus oleraceus, Equisetum arvense and Atriplex hortensis, wild flora found within the farm. The other lots contained the same plants as the control lot plus different combinations of imported plants from other areals. The H. pomatia 2yo weight decreased in the control lot by a mean of -3.86% while H. aspersa 1yo marked an increase of +16.89% in the same lot during the same period. The lot containing lupinus polyphyllus delivered snails with weight gain of +24.66% for H. pomatia 2yo and an increase of only +1.98% for H. aspersa 1yo. As a contrast, H. pomatia 2yo gained only +7.72% while H. aspersa 1yo gained +28.89%, in the lot containing Lavanda officinalis, Foeniculum vulgare and Hyssopus officinalis among the other plants.

  6. Inactivation of colicin Y by intramembrane helix–helix interaction with its immunity protein

    Czech Academy of Sciences Publication Activity Database

    Šmajs, D.; Doležalová, M.; Macek, Pavel; Žídek, L.

    2008-01-01

    Roč. 275, č. 21 (2008), s. 5325-5331 ISSN 1742-464X Institutional research plan: CEZ:AV0Z50200510 Keywords : colicin immunity * colicin y * helix-helix interaction Subject RIV: CE - Biochemistry Impact factor: 3.139, year: 2008

  7. Assembly of Liposomes Controlled by Triple Helix Formation

    DEFF Research Database (Denmark)

    Vogel, Stefan; Jakobsen, Ulla

    2013-01-01

    Attachment of DNA to the surface of different solid nanoparticles (e.g. gold- and silica nanoparticles) is well established and a number of DNA-modified solid nanoparticle systems have been applied to thermal denaturation analysis of oligonucleotides. We report herein the non...... sequences (G or C-rich) to explore the applicability of the method for different triple helical assembly modes. We demonstrate advantages and limitations of the approach and proof the reversible and reproducible formation of liposome aggregates during thermal denaturation cycles. Nanoparticle tracking......-covalent immobilization of oligonucleotides on the surface of soft nanoparticles (e.g. liposomes) and the subsequent controlled assembly by DNA triple helix formation. The non-covalent approach avoids tedious surface chemistry and necessary purification procedures and can simplify and extend the available methodology...

  8. Associative learning phenomena in the snail (Helix aspersa): conditioned inhibition.

    Science.gov (United States)

    Acebes, Félix; Solar, Patricia; Moris, Joaquín; Loy, Ignacio

    2012-03-01

    Two experiments using garden snails (Helix aspersa) showed conditioned inhibition using both retardation and summation tests. Conditioned inhibition is a procedure by which a stimulus becomes a predictor of the absence of a relevant event--the unconditioned stimulus (US). Typically, conditioned inhibition consists of pairings between an initially neutral conditioned stimulus, CS(2), and an effective excitatory conditioned stimulus, CS(1), in the absence of the US. Retardation and summation tests are required in order to confirm that CS(2) has acquired inhibitory properties. Conditioned inhibition has previously been found in invertebrates; however, these demonstrations did not use the retardation and summation tests required for an unambiguous demonstration of inhibition, allowing for alternative explanations. The implications of our results for the fields of comparative cognition and invertebrate physiological models of learning are discussed.

  9. Significantly enhanced mechanical properties in AlN helix

    Science.gov (United States)

    Zhang, Xinghong; Zhao, Chaoliang; Yao, Tai; Zhou, Shanbao; Han, Jiecai; Li, Jiajie; Gao, Tangling; Wang, Xianjie; Zheng, Kun; Song, Bo

    2017-07-01

    To safely and reliably use aluminum nitride (AlN) helices in the fabrication of novel micro/nanodevices, it is very important to know their mechanical properties. Herein, we investigate the mechanical properties of individual AlN helices using an in situ tensile-bending test. Tensile tests reveal that an AlN helix has an average ε of ∼4.7 ± 0.8% elastic deformation before a typical brittle fracture occurs. The bending test shows a two-step mechanical feature—linear-elastic followed by an elastic-plastic process—with an average ε bent of ∼54.5 ± 0.6%. Our results provide direct cognition about the mechanical properties of AlN helices and their benefit to the design of AlN-based flexible micro/nanodevices.

  10. Computational analysis of the 3-D structure of human GPR87 protein: implications for structure-based drug design.

    Science.gov (United States)

    Rani, Mukta; Nischal, Anuradha; Sahoo, Ganesh Chandra; Khattri, Sanjay

    2013-01-01

    The G-protein coupled receptor 87 (GPR87) is a recently discovered orphan GPCR which means that the search of their endogenous ligands has been a novel challenge. GPR87 has been shown to be overexpressed in squamous cell carcinomas (SCCs) or adenocarcinomas in lungs and bladder. The 3D structure of GPR87 was here modeled using two templates (2VT4 and 2ZIY) by a threading method. Functional assignment of GPR87 by SVM revealed that along with transporter activity, various novel functions were predicted. The 3D structure was further validated by comparison with structural features of the templates through Verify-3D, ProSA and ERRAT for determining correct stereochemical parameters. The resulting model was evaluated by Ramachandran plot and good 3D structure compatibility was evidenced by DOPE score. Molecular dynamics simulation and solvation of protein were studied through explicit spherical boundaries with a harmonic restraint membrane water system. A DRY-motif (Asp-Arg-Tyr sequence) was found at the end of transmembrane helix3, where GPCR binds and thus activation of signals is transduced. In a search for better inhibitors of GPR87, in silico modification of some substrate ligands was carried out to form polar interactions with Arg115 and Lys296. Thus, this study provides early insights into the structure of a major drug target for SCCs.

  11. The double helix revisited: a paradox of science and a paradigm of human behaviour

    Directory of Open Access Journals (Sweden)

    Argüelles, Juan Carlos

    2007-06-01

    Full Text Available In the modern history of Science, few breakthroughs have caused an impact comparative to the Double Helix, the three-dimensional structure of DNA proposed by Watson & Crick in 1953, an event whose 50th anniversary was widely celebrated in the non-specialist media, three years ago. Although the discovery had little transcendence at the time, it has unquestionably been of great importance ever since. The Double Helix has underlined the true biological value of nucleic acids compared with proteins, demonstrating that genes are not amorphous entities but have a specific chemical composition and adopt an ordered spatial folding pattern. Elucidation of this key configuration made it possible to establish a direct relationship between the structure and the function of macromolecules, a relationship which is not so clear in the case of proteins. During these last fifty years much has been written and argued about the circumstances surrounding the discovery and about the behaviour and attitudes of many of the protagonists. Besides Watson & Crick, other scientists, whose contribution has not been adequately recognised, played an important part in solving the Double Helix mystery. This article contains some ethical and scientific reflections which revise some of these essential contributions and throws light on the role played in history by these comparatively «unknown soldiers» of science. The Double Helix story is undoubtedly a manifestation of the human side of science and many scientists believe that the available evidence taken as a whole permits an alternative story to be written.

    En la desarrollo histórico de la Ciencia moderna, pocos descubrimientos han causado un impacto comparativo a las repercusiones de la Doble Hélice, la estructura tridimensional del ADN, propuesta por Watson y Crick en 1953. El 50º aniversario de aquel evento fue ampliamente celebrado hace tres años, incluso por los medios no especializados en informaci

  12. After the double helix: Rosalind Franklin's research on Tobacco mosaic virus.

    Science.gov (United States)

    Creager, Angela N H; Morgan, Gregory J

    2008-06-01

    Rosalind Franklin is best known for her informative X-ray diffraction patterns of DNA that provided vital clues for James Watson and Francis Crick's double-stranded helical model. Her scientific career did not end when she left the DNA work at King's College, however. In 1953 Franklin moved to J. D. Bernal's crystallography laboratory at Birkbeck College, where she shifted her focus to the three-dimensional structure of viruses, obtaining diffraction patterns of Tobacco mosaic virus (TMV) of unprecedented detail and clarity. During the next five years, while making significant headway on the structural determination of TMV, Franklin maintained an active correspondence with both Watson and Crick, who were also studying aspects of virus structure. Developments in TMV research during the 1950s illustrate the connections in the emerging field of molecular biology between structural studies of nucleic acids and of proteins and viruses. They also reveal how the protagonists of the "race for the double helix" continued to interact personally and professionally during the years when Watson and Crick's model for the double-helical structure of DNA was debated and confirmed.

  13. Beluga (Delphinapterus leucas Novel Bubble Helix Play Behavior

    Directory of Open Access Journals (Sweden)

    Brittany L. Jones

    2014-05-01

    Full Text Available Cetaceans demonstrate considerable ingenuity in their play with bubbles. Both wild and captive cetaceans have been reported to manipulate self-produced bubbles (Delfour & Aulagnier, 1997; Gewalt, 1989; Kuczaj, Makecha, Trone, Paulos, & Ramos, 2006; Kuczaj & Walker, 2006; McCowan, Marino, Vance, Walke, & Reiss, 2000; Pace, 2000; Paulos, Trone, & Kuczaj, 2010; Tizzi, Castellano, & Pace, 2000. The spread of unique and novel play behaviors across a group may involve social learning as well as trial and error learning (Kuczaj et al., 2006; Kuczaj, Yeater, & Highfill, 2012; McCowan et al., 2000; Pace, 2000. We report on a form of bubble play in belugas (Delphinapterus leucas that has not been previously reported. Four belugas at the Shedd Aquarium were videotaped producing bubble helices, smooth, long “helical tubes” that were created by the animal producing a pressure vortex that caused bubbles to merge and elongate based on pressure variation in the vortex (Marten, Shariff, Psarakos, & White, 1996. These observations revealed that belugas create novel bubble play behaviors that are transmitted among members of the group through social learning. When a beluga engaged in bubble helix play following the play of another beluga, it often acted on the bubble in the same manner as the most recent player, consistent with the notion that the second beluga was mimicking the behavior of the first beluga. Kimalu, a calf, was more likely to both observe and interact with Miki, his older brother, during bubble helix play bouts than with Naya (no relation, or Mauyak, his mother. Dolphin calves have also been found to be more likely to imitate the play behaviors of older more competent peers (Kuczaj et al., 2006; Kuczaj et al., 2012. Consistent with previous analyses of cetacean play (Kuczaj et al., 2006, belugas also varied the complexity of the play behavior in order to keep the game stimulating.

  14. Preparation and evaluation of appertized from snail Helix aspersa M

    Directory of Open Access Journals (Sweden)

    Nelson Loyola López

    2015-01-01

    Full Text Available This study includes the development and evaluation of snails (Helix aspersa M. appertized, collected at a heliciculture breeding center, located in Los Niches sector, Curico, Maule region, South-central of Chile. The test was conducted at the Laboratory of Sciences of the Catholic University of Maule, Nuestra Señora del Carmen Campus, Curico. The main objective of this work was to study the influence of appertized on sensory attributes and commercial durability of snail Helix aspersa M. Additionally, some specific objectives were proposed as follow: to provide this mollusc with a commercial alternative for it consume, to evaluate its organoleptic characteristics and guarantee the product from both the microbiological and nutritional points of view. Three media cover were used (T0: water + NaCl 2%; T1: Water + NaCl 2% + citric acid 0.5% + kilol and T2: extra virgin olive oil + spices + tocopherol. The product was assessed at two different times, after 30 and 90 days of storage. Two sensory evaluations were conducted to measure various organoleptic attributes and acceptability of the appertized by 14 trained panelists. Amino acid, vitamins, cholesterol, acidity, heavy metals, phosphorus and organochlorines analysis were performed. The presence of both total and fecal contaminant microorganisms was determined. Attributes such as color, flavor, aroma, texture and overall acceptability were also measured. Preserves made by T0 and T1 treatments were equally accepted by the panelists. However, preserve from treatment T2 was rejected because of the detection in them of a very dark color, odor and mealy texture. Positive results regarding the content of amino acids, vitamin C and low cholesterol, as well as the absence of pathogenic microorganisms were obtained for the three treatments.

  15. Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

    Science.gov (United States)

    Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

    2018-01-23

    Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

  16. DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

    Science.gov (United States)

    Northall, Sarah J; Buckley, Ryan; Jones, Nathan; Penedo, J Carlos; Soultanas, Panos; Bolt, Edward L

    2017-09-01

    Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. NMR studies of abasic sites in DNA duplexes: Deoxyadenosine stacks into the helix opposite acyclic lesions

    International Nuclear Information System (INIS)

    Kalnik, M.W.; Chang, Chienneng; Johnson, F.; Grollman, A.P.; Patel, D.J.

    1989-01-01

    Proton and phosphorus NMR studies are reported for two complementary nonanucleotide duplexes containing acyclic abasic sites. The first duplex, d(C-A-T-G-A-G-T-A-C)·d(G-T-A-C-P-C-A-T-G), contains an acyclic propanyl moiety, P, located opposite a deoxyadenosine at the center of the helix (designated AP P 9-mer duplex). The second duplex, d(C-A-T-G-A-G-T-A-C-)·d(G-T-A-C-E-C-A-T-G), contains a similarly located acyclic ethanyl moiety, E (designated AP E 9-mer duplex). The ethanyl moiety is one carbon shorter than the natural carbon-phosphodiester backbone of a single nucleotide unit of DNA. The majority of the exchangeable and nonexchangeable base and sugar protons in both the AP P 9-mer and AP E 9-mer duplexes, including those at the abasic site, have been assigned by recording and analyzing two-dimensional phase-sensitive NOESY data sets in H 2 O and D 2 O solution between -5 and 5 degree C. These spectroscopic observations establish that A5 inserts into the helix opposite the abasic site (P14 and El14) and stacks between the flanking G4·C15 and G6·C13 Watson-Crick base pairs in both the AP P 9-mer and AP E 9-mer duplexes. Proton NMR parameters for the Ap P 9-mer and AP E 9-mer duplexes are similar to those reported previously. These proton NMR experiments demonstrate that the structures at abasic sites are very similar whether the five-membered ring is open or closed or whether the phosphodiester backbone is shortened by one carbon atom. Phosphorus spectra of the AP P 9-mer and AP E 9-mer duplexes (5 degree C) indicate that the backbone conformation is similarly perturbed at three phosphodiester backbone torsion angles

  18. An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity.

    Science.gov (United States)

    Pacheco, Sabino; Gómez, Isabel; Sánchez, Jorge; García-Gómez, Blanca-Ines; Soberón, Mario; Bravo, Alejandra

    2017-10-15

    Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity. Copyright © 2017 American Society for Microbiology.

  19. Purification and crystallization of the cystic fibrosis transmembrane conductance regulator (CFTR).

    Science.gov (United States)

    Rosenberg, Mark F; Kamis, Alhaji Bukar; Aleksandrov, Luba A; Ford, Robert C; Riordan, John R

    2004-09-10

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein that is mutated in patients suffering from cystic fibrosis. Here we report the purification and first crystallization of wild-type human CFTR. Functional characterization of the material showed it to be highly active. Electron crystallography of negatively stained two-dimensional crystals of CFTR has revealed the overall architecture of this channel for two different conformational states. These show a strong structural homology to two conformational states of another eukaryotic ATP-binding cassette transporter, P-glycoprotein. In contrast to P-glycoprotein, however, both conformational states can be observed in the presence of a nucleotide, which may be related to the role of CFTR as an ion channel rather than a transporter. The hypothesis that the two conformations could represent the "open" and "closed" states of the channel is considered.

  20. The role of transmembrane segment II in 7TM receptor activation

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Rosenkilde, M M

    2009-01-01

    During the two past decades tremendous effort has been put into uncovering the activation mechanism of 7TM receptors. The majority of such studies have focused on the major binding pocket, comprised of transmembrane segments (TM) -III through -VII, as most non-peptide and peptide ligands......, in addition to biogenic amines and retinal a.m.o. bind to residues in this region. Consequently the major helical movements occur here during activation, as described recently in the Global Toggle Switch Model for Family A (also known as rhodopsin-like) members of the 7TM receptors. As a result, the minor......, accumulating evidence emphasize that this is not the case. In this review, we focus on TM-II with an emphasis on position II:20/2.60, and present data from structure-activity studies on a range of Family A 7TM receptors including chemokine, ghrelin and melanocortin receptors in addition to the orphan EBI2...

  1. Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol.

    Science.gov (United States)

    Hacke, Moritz; Björkholm, Patrik; Hellwig, Andrea; Himmels, Patricia; Ruiz de Almodóvar, Carmen; Brügger, Britta; Wieland, Felix; Ernst, Andreas M

    2015-07-09

    The high pathogenicity of the Ebola virus reflects multiple concurrent processes on infection. Among other important determinants, Ebola fusogenic glycoprotein (GP) has been associated with the detachment of infected cells and eventually leads to vascular leakage and haemorrhagic fever. Here we report that the membrane-anchored GP is sufficient to induce the detachment of adherent cells. The results show that the detachment induced through either full-length GP1,2 or the subunit GP2 depends on cholesterol and the structure of the transmembrane domain. These data reveal a novel molecular mechanism in which GP regulates Ebola virus assembly and suggest that cholesterol-reducing agents could be useful as therapeutics to counteract GP-mediated cell detachment.

  2. The Nup62 Coiled-Coil Motif Provides Plasticity for Triple-Helix Bundle Formation.

    Science.gov (United States)

    Dewangan, Pravin S; Sonawane, Parshuram J; Chouksey, Ankita R; Chauhan, Radha

    2017-06-06

    The central transport channel of the vertebrate nuclear pore complex (NPC) consists of nucleoporins: Nup62, Nup54, and Nup58. The coiled-coil domains in α-helical regions of these nucleoporins are thought to be crucial for several protein-protein interactions in the NPC subcomplexes. In this study, we determined the crystal structure of the coiled-coil domain of rat Nup62 fragment (residues 362-425) to 2.4 Å resolution. The crystal structure shows the conserved coiled-coil domain as a parallel three-helix bundle for the Nup62(362-425) fragment. On the basis of our size exclusion chromatography coupled to multiangle light scattering analysis and glutaraldehyde cross-linking experiments, we conclude that the Nup62(362-425) fragment displays dynamic behavior in solution and can also exist in either homodimeric or homotrimeric states. Our comparative analysis of the rat Nup62(362-425) homotrimeric structure with previously reported heterotrimeric structures [rat Nup62(362-425)·Nup54(346-407) and Xenopus Nup62(358-485)·Nup54(315-450)·Nup58(283-406) complexes] demonstrates the structural basis for parallel triple-helix bundle formation for Nup62 with different partners. Moreover, we show that the coiled-coil domain of Nup62 is sufficient for interaction with the coiled-coil domain of rat Exo70, a protein in an exocyst complex. On the basis of these observations, we suggest the plausible chain replacement mechanism that yields to diverse protein assemblies with Nup62. In summary, the coiled-coil motif present in Nup62 imparts the ability to form a homotrimer and heterotrimers either with Nup54 or with Nup54-Nup58 within the NPCs as well as with Exo70 beyond the NPCs. These complexes of Nup62 suggest the crucial role of the coiled-coil motifs in providing plasticity to various modular assemblies.

  3. Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix.

    Science.gov (United States)

    Horton, John R; Wang, Hua; Mabuchi, Megumu Yamada; Zhang, Xing; Roberts, Richard J; Zheng, Yu; Wilson, Geoffrey G; Cheng, Xiaodong

    2014-10-29

    MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. High-resolution structures of a heterochiral coiled coil.

    Science.gov (United States)

    Mortenson, David E; Steinkruger, Jay D; Kreitler, Dale F; Perroni, Dominic V; Sorenson, Gregory P; Huang, Lijun; Mittal, Ritesh; Yun, Hyun Gi; Travis, Benjamin R; Mahanthappa, Mahesh K; Forest, Katrina T; Gellman, Samuel H

    2015-10-27

    Interactions between polypeptide chains containing amino acid residues with opposite absolute configurations have long been a source of interest and speculation, but there is very little structural information for such heterochiral associations. The need to address this lacuna has grown in recent years because of increasing interest in the use of peptides generated from d amino acids (d peptides) as specific ligands for natural proteins, e.g., to inhibit deleterious protein-protein interactions. Coiled-coil interactions, between or among α-helices, represent the most common tertiary and quaternary packing motif in proteins. Heterochiral coiled-coil interactions were predicted over 50 years ago by Crick, and limited experimental data obtained in solution suggest that such interactions can indeed occur. To address the dearth of atomic-level structural characterization of heterochiral helix pairings, we report two independent crystal structures that elucidate coiled-coil packing between l- and d-peptide helices. Both structures resulted from racemic crystallization of a peptide corresponding to the transmembrane segment of the influenza M2 protein. Networks of canonical knobs-into-holes side-chain packing interactions are observed at each helical interface. However, the underlying patterns for these heterochiral coiled coils seem to deviate from the heptad sequence repeat that is characteristic of most homochiral analogs, with an apparent preference for a hendecad repeat pattern.

  5. Glycosylation and the cystic fibrosis transmembrane conductance regulator

    Science.gov (United States)

    Scanlin, Thomas F; Glick, Mary Catherine

    2001-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wt)CFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF), and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined. PMID:11686896

  6. Glycosylation and the cystic fibrosis transmembrane conductance regulator

    Directory of Open Access Journals (Sweden)

    Glick Mary Catherine

    2001-08-01

    Full Text Available Abstract The cystic fibrosis transmembrane conductance regulator (CFTR has been known for the past 11 years to be a membrane glycoprotein with chloride channel activity. Only recently has the glycosylation of CFTR been examined in detail, by O'Riordan et al in Glycobiology. Using cells that overexpress wild-type (wtCFTR, the presence of polylactosamine was noted on the fully glycosylated form of CFTR. In the present commentary the results of that work are discussed in relation to the glycosylation phenotype of cystic fibrosis (CF, and the cellular localization and processing of ΔF508 CFTR. The significance of the glycosylation will be known when endogenous CFTR from primary human tissue is examined.

  7. The transmembrane channel-like protein family and human papillomaviruses

    Science.gov (United States)

    Horton, Jaime S; Stokes, Alexander J

    2014-01-01

    Epidermodysplasia verruciformis (EV) is a rare genodermatosis characterized by increased sensitivity to infection by the β-subtype of human papillomaviruses (β-HPVs), causing persistent, tinea versicolor-like dermal lesions. In a majority of affected individuals, these macular lesions progress to invasive cutaneous squamous cell carcinoma (CSCC) in sun-exposed areas. While mutations in transmembrane channel-like 6 (TMC6 / EVER1) and 8 (TMC8 / EVER2) have been causally linked to EV, their molecular functions are unclear. It is likely that their protective effects involve regulation of the β-HPV life cycle, host keratinocyte apoptosis vs. survival balance and/or T-cell interaction with infected host cells. PMID:24800179

  8. 21 CFR 866.5900 - Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation detection system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cystic fibrosis transmembrane conductance... DEVICES Immunological Test Systems § 866.5900 Cystic fibrosis transmembrane conductance regulator (CFTR... intended as an aid in confirmatory diagnostic testing of individuals with suspected cystic fibrosis (CF...

  9. Hidden markov model for the prediction of transmembrane proteins using MATLAB.

    Science.gov (United States)

    Chaturvedi, Navaneet; Shanker, Sudhanshu; Singh, Vinay Kumar; Sinha, Dhiraj; Pandey, Paras Nath

    2011-01-01

    Since membranous proteins play a key role in drug targeting therefore transmembrane proteins prediction is active and challenging area of biological sciences. Location based prediction of transmembrane proteins are significant for functional annotation of protein sequences. Hidden markov model based method was widely applied for transmembrane topology prediction. Here we have presented a revised and a better understanding model than an existing one for transmembrane protein prediction. Scripting on MATLAB was built and compiled for parameter estimation of model and applied this model on amino acid sequence to know the transmembrane and its adjacent locations. Estimated model of transmembrane topology was based on TMHMM model architecture. Only 7 super states are defined in the given dataset, which were converted to 96 states on the basis of their length in sequence. Accuracy of the prediction of model was observed about 74 %, is a good enough in the area of transmembrane topology prediction. Therefore we have concluded the hidden markov model plays crucial role in transmembrane helices prediction on MATLAB platform and it could also be useful for drug discovery strategy. The database is available for free at bioinfonavneet@gmail.comvinaysingh@bhu.ac.in.

  10. Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

    Science.gov (United States)

    Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

    2018-05-01

    We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

  11. Thermal helix-coil transition in UV irradiated collagen from rat tail tendon.

    Science.gov (United States)

    Sionkowska, A; Kamińska, A

    1999-05-01

    The thermal helix-coil transition in UV irradiated collagen solution, collagen film and pieces of rat tail tendon (RTT) were compared. Their thermal stability's were determined by differential scanning calorimeter (DSC) and by viscometric measurements. The denaturation temperatures of collagen solution, film and pieces of RTT were different. The helix-coil transition occur near 40 degrees C in collagen solution, near 112 degrees C in collagen film, and near 101 degrees C in pieces of RTT. After UV irradiation the thermal helix-coil transition of collagen samples were changed. These changes depend on the degree of hydratation.

  12. Characterization of a crosslinked nucleic acid - helix destabilizing protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Karpel, R.L.; Levin, V.Y.; Haley, B.E.

    1986-05-01

    They have enzymatically synthesized /sup 3/H- and /sup 32/P-poly(A,8N/sub 3/A) from 8-N/sub 3/ADP and radiolabeled ADP, and have used this polynucleotide to photoaffinity label T4 gene 32 protein, as well as several other helix-destabilizing proteins (HDPs). Irradiation of /sup 32/P-/sup 3/H-poly(A,N/sub 3/A) mixtures for short durations produces a covalent complex, seen as a high molecular weight, radioactive band on SDS-polyacrylamide gels. Preliminary experiments on other HDPs, from prokaryotic, eukaryotic and animal viral sources, show analogous results. Several successful control experiments indicate that this system is suitable for binding site localization on /sup 32/P. Single-stranded nucleic acids competitively inhibit photolabeling. The effect of NaCl on photolabeling parallels the salt-dependence of /sup 32/P-poly(A,N/sub 3/A) binding. Photolabeling reaches a plateau after approx.1 min, and the formation of the high molecular weight complex parallels the reduction of free /sup 32/P on SDS gels. Staph. nuclease digestion of crosslinked complexes produces a diffuse, radioactive band on SDS gels, migrating just behind free /sup 32/P. When these digested complexes are subjected to reverse-phase HPLC on a C3 Ultrapore column, the nucleic acid /sup 32/P-label is seen to coelute with protein. They are currently employing RP-HPLC methods to locate the label on tryptic peptides of nuclease-digested complexes.

  13. Calorimetric Study of Helix aspersa Maxima Hemocyanin Isoforms

    Directory of Open Access Journals (Sweden)

    Svetla Todinova

    2018-01-01

    Full Text Available The thermal unfolding of hemocyanin isoforms, β-HaH and αD+N-HaH, isolated from the hemolymph of garden snails Helix aspersa maxima, was studied by means of differential scanning calorimetry (DSC. One transition, with an apparent transition temperature (Tm at 79.88°C, was detected in the thermogram of β-HaH in 20 mM HEPES buffer, containing 0.1 M NaCl, 5 mM CaCl2, and 5 mM MgCl2, pH 7.0, at scan rate of 1.0°C min−1. By means of successive annealing procedure, two individual transitions were identified in the thermogram of αD+N-HaH. Denaturation of both hemocyanins was found to be an irreversible process. The scan-rate dependence of the calorimetric profiles indicated that the thermal unfolding of investigated hemocyanins was kinetically controlled. The thermal denaturation of the isoforms β-HaH and αD+N-HaH was described by the two-state irreversible model, and parameters of the Arrhenius equation were calculated.

  14. Double helix boron-10 powder thermal neutron detector

    Science.gov (United States)

    Wang, Zhehui; Morris, Christopher L.; Bacon, Jeffrey D.

    2015-06-02

    A double-helix Boron-10 powder detector having intrinsic thermal neutron detection efficiency comparable to 36'' long, 2-in diameter, 2-bar Helium-3 detectors, and which can be used to replace such detectors for use in portal monitoring, is described. An embodiment of the detector includes a metallic plate coated with Boron-10 powder for generating alpha and Lithium-7 particles responsive to neutrons impinging thereon supported by insulators affixed to at least two opposing edges; a grounded first wire wound in a helical manner around two opposing insulators; and a second wire having a smaller diameter than that of the first wire, wound in a helical manner around the same insulators and spaced apart from the first wire, the second wire being positively biased. A gas, disposed within a gas-tight container enclosing the plate, insulators and wires, and capable of stopping alpha and Lithium-7 particles and generating electrons produces a signal on the second wire which is detected and subsequently related to the number of neutrons impinging on the plate.

  15. Helix Nebula: sunshine and clouds on the CERN computing horizon

    CERN Multimedia

    Joannah Caborn Wengler

    2012-01-01

    23 petabytes is how much data CERN recorded during 2011, and this number will rise in 2012. In order to respond to the challenge, the IT department is upping its game, amongst other things by participating in the Helix Nebula project, a public-private partnership to create a European cloud-computing platform, as announced in a recent CERN press release.   “We’re not replacing the Grid,” clarifies Bob Jones, responsible for CERN openlab who is also responsible for EC-funded projects in IT, “but looking at three complementary ways of increasing CERN’s computing capacity, so that as demand goes up we can continue to satisfy our users.” “First we are upgrading the electrical and cooling infrastructure of the computer centre in order to increase the availability of critical IT services needed for the Laboratory. This will also provide more floor space in the area called The Barn, allowing for more servers to fit in.”...

  16. RFID Tag Helix Antenna Sensors for Wireless Drug Dosage Monitoring

    Science.gov (United States)

    Huang, Haiyu; Zhao, Peisen; Chen, Pai-Yen; Ren, Yong; Liu, Xuewu; Ferrari, Mauro; Hu, Ye; Akinwande, Deji

    2014-01-01

    Miniaturized helix antennas are integrated with drug reservoirs to function as RFID wireless tag sensors for real-time drug dosage monitoring. The general design procedure of this type of biomedical antenna sensors is proposed based on electromagnetic theory and finite element simulation. A cost effective fabrication process is utilized to encapsulate the antenna sensor within a biocompatible package layer using PDMS material, and at the same time form a drug storage or drug delivery unit inside the sensor. The in vitro experiment on two prototypes of antenna sensor-drug reservoir assembly have shown the ability to monitor the drug dosage by tracking antenna resonant frequency shift from 2.4–2.5-GHz ISM band with realized sensitivity of 1.27 \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$\\mu~{\\rm l}/{\\rm MHz}$\\end{document} for transdermal drug delivery monitoring and 2.76-\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$\\mu~{\\rm l}/{\\rm MHz}$\\end{document} sensitivity for implanted drug delivery monitoring. PMID:27170865

  17. A double-helix and cross-patterned solenoid used as a wirelessly powered receiver for medical implants

    Science.gov (United States)

    Mao, Shitong; Wang, Hao; Mao, Zhi-Hong; Sun, Mingui

    2018-05-01

    Many medical implants need to be designed in the shape of a cylinder (rod), a cuboid or a capsule in order to adapt to a specific site within the human body or facilitate the implantation procedure. In order to wirelessly power these types of implants, a pair of coils, one is located inside the human body and one is outside, is often used. Since most organs such as major muscles, blood vessels, and nerve bundles are anatomically parallel to the body surface, the most desired wireless power transfer (WPT) direction is from the external power transmission pad (a planar coil) to the lateral surface of the implant. However, to obtain optimal coupling, the currently used solenoid coil requires being positioned perpendicular to the body surface, which is often medically or anatomically unacceptable. In this research, a concentric double-helix (DH) coil with an air core is presented for use in implantable devices. Two helical coils are tilted at opposite angles (±45 degrees) to form a cross pattern. The WPT system is designed using the magnetic resonance concept for wireless power transfer (MR-WPT). The power transfer efficiency (PTE) relies on the near-field magnetic coupling which is closely related to the location and orientation of the DH coil. We explain how the novel structure of the DH solenoid magnifies the mutual inductance with the widely adopted circular planner coil and how the PTE is improved in comparison to the case of the conventional solenoid coil. We also study an important case where the double-helix power reception coil is laterally and angularly misaligned with the transmitter. Finally, our computational study using the finite element method and experimental study with actually constructed prototypes are presented which have proven our new double-helix coil design.

  18. Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18?

    DEFF Research Database (Denmark)

    Christiansen, J; Douthwaite, S R; Christensen, A

    1985-01-01

    Adenosine-66 is unpaired within helix II of Escherichia coli 5S RNA and lies in the binding site of ribosomal protein L18. It has been proposed as a recognition site for protein L18. We have investigated further the structural importance of this nucleotide by deleting it. The 5S RNA gene of the r...... adenosine and the adjacent guanosine-67 of the RNA and glutamine-19 of the protein L18.......Adenosine-66 is unpaired within helix II of Escherichia coli 5S RNA and lies in the binding site of ribosomal protein L18. It has been proposed as a recognition site for protein L18. We have investigated further the structural importance of this nucleotide by deleting it. The 5S RNA gene of the rrn...... plasmid derived from pKK3535. Binding studies with protein L18 revealed that the protein bound much more weakly to the mutated 5S RNA. We consider the most likely explanation of this result is that L18 interacts with adenosine-66, and we present a tentative model for an interaction between the unpaired...

  19. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    International Nuclear Information System (INIS)

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-01-01

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed

  20. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jimin, E-mail: jimin.wang@yale.edu; Li, Yue; Modis, Yorgo, E-mail: yorgo.modis@yale.edu

    2014-04-15

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

  1. Non-linearity of the collagen triple helix in solution and implications for collagen function.

    Science.gov (United States)

    Walker, Kenneth T; Nan, Ruodan; Wright, David W; Gor, Jayesh; Bishop, Anthony C; Makhatadze, George I; Brodsky, Barbara; Perkins, Stephen J

    2017-06-16

    Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly) n sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units. In addition, one unblocked peptide, (POG) 10unblocked , was compared with the blocked (POG) 10 as a control for the significance of end effects. Complementary analytical ultracentrifugation and synchrotron small angle X-ray scattering data showed that the conformations of the longer triple helical peptides were not well explained by a linear structure derived from crystallography. To interpret these data, molecular dynamics simulations were used to generate 50 000 physically realistic collagen structures for each of the helices. These structures were fitted against their respective scattering data to reveal the best fitting structures from this large ensemble of possible helix structures. This curve fitting confirmed a small degree of non-linearity to exist in these best fit triple helices, with the degree of bending approximated as 4-17° from linearity. Our results open the way for further studies of other collagen triple helices with different sequences and stabilities in order to clarify the role of molecular rigidity and flexibility in collagen extracellular and immune function and disease. © 2017 The Author(s).

  2. Probing membrane protein structure using water polarization transfer solid-state NMR.

    Science.gov (United States)

    Williams, Jonathan K; Hong, Mei

    2014-10-01

    Water plays an essential role in the structure and function of proteins, lipid membranes and other biological macromolecules. Solid-state NMR heteronuclear-detected (1)H polarization transfer from water to biomolecules is a versatile approach for studying water-protein, water-membrane, and water-carbohydrate interactions in biology. We review radiofrequency pulse sequences for measuring water polarization transfer to biomolecules, the mechanisms of polarization transfer, and the application of this method to various biological systems. Three polarization transfer mechanisms, chemical exchange, spin diffusion and NOE, manifest themselves at different temperatures, magic-angle-spinning frequencies, and pulse irradiations. Chemical exchange is ubiquitous in all systems examined so far, and spin diffusion plays the key role in polarization transfer within the macromolecule. Tightly bound water molecules with long residence times are rare in proteins at ambient temperature. The water polarization-transfer technique has been used to study the hydration of microcrystalline proteins, lipid membranes, and plant cell wall polysaccharides, and to derive atomic-resolution details of the kinetics and mechanism of ion conduction in channels and pumps. Using this approach, we have measured the water polarization transfer to the transmembrane domain of the influenza M2 protein to obtain information on the structure of this tetrameric proton channel. At short mixing times, the polarization transfer rates are site-specific and depend on the pH, labile protons, sidechain conformation, as well as the radial position of the residues in this four-helix bundle. Despite the multiple dependences, the initial transfer rates reflect the periodic nature of the residue positions from the water-filled pore, thus this technique provides a way of gleaning secondary structure information, helix tilt angle, and the oligomeric structure of membrane proteins. Copyright © 2014 Elsevier Inc. All

  3. One motif to bind them: A small-XXX-small motif affects transmembrane domain 1 oligomerization, function, localization, and cross-talk between two yeast GPCRs.

    Science.gov (United States)

    Lock, Antonia; Forfar, Rachel; Weston, Cathryn; Bowsher, Leo; Upton, Graham J G; Reynolds, Christopher A; Ladds, Graham; Dixon, Ann M

    2014-12-01

    G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors in mammals and facilitate a range of physiological responses triggered by a variety of ligands. GPCRs were thought to function as monomers, however it is now accepted that GPCR homo- and hetero-oligomers also exist and influence receptor properties. The Schizosaccharomyces pombe GPCR Mam2 is a pheromone-sensing receptor involved in mating and has previously been shown to form oligomers in vivo. The first transmembrane domain (TMD) of Mam2 contains a small-XXX-small motif, overrepresented in membrane proteins and well-known for promoting helix-helix interactions. An ortholog of Mam2 in Saccharomyces cerevisiae, Ste2, contains an analogous small-XXX-small motif which has been shown to contribute to receptor homo-oligomerization, localization and function. Here we have used experimental and computational techniques to characterize the role of the small-XXX-small motif in function and assembly of Mam2 for the first time. We find that disruption of the motif via mutagenesis leads to reduction of Mam2 TMD1 homo-oligomerization and pheromone-responsive cellular signaling of the full-length protein. It also impairs correct targeting to the plasma membrane. Mutation of the analogous motif in Ste2 yielded similar results, suggesting a conserved mechanism for assembly. Using co-expression of the two fungal receptors in conjunction with computational models, we demonstrate a functional change in G protein specificity and propose that this is brought about through hetero-dimeric interactions of Mam2 with Ste2 via the complementary small-XXX-small motifs. This highlights the potential of these motifs to affect a range of properties that can be investigated in other GPCRs. Copyright © 2014. Published by Elsevier B.V.

  4. Orientations and proximities of the extracellular ends of transmembrane helices S0 and S4 in open and closed BK potassium channels.

    Directory of Open Access Journals (Sweden)

    Xiaowei Niu

    Full Text Available The large-conductance potassium channel (BK α subunit contains a transmembrane (TM helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory β1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in β1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the β1 subunit than in its absence, consistent with β1 acting through S0 to stabilize its immobilization relative to α S4.

  5. SREBP-2, a second basic-helix-loop-helix-leucine zipper protein that stimulates transcription by binding to a sterol regulatory element.

    OpenAIRE

    Hua, X; Yokoyama, C; Wu, J; Briggs, M R; Brown, M S; Goldstein, J L; Wang, X

    1993-01-01

    We report the cDNA cloning of SREBP-2, the second member of a family of basic-helix-loop-helix-leucine zipper (bHLH-Zip) transcription factors that recognize sterol regulatory element 1 (SRE-1). SRE-1, a conditional enhancer in the promoters for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A synthase genes, increases transcription in the absence of sterols and is inactivated when sterols accumulate. Human SREBP-2 contains 1141 amino acids and is 47% identical t...

  6. Peptides derived from nucleoside beta-amino acids form an unusual 8-helix.

    Science.gov (United States)

    Threlfall, Richard; Davies, Andrew; Howarth, Nicola M; Fisher, Julie; Cosstick, Richard

    2008-02-07

    Peptides of varying length (dimers to octamers) were prepared from nucleoside beta-amino acids and conformational studies, based on NOE observations, show that the beta-peptides form an unusual 8-helix.

  7. Helix Nebula: Enabling federation of existing data infrastructures and data services to an overarching cross-domain e-infrastructure

    Science.gov (United States)

    Lengert, Wolfgang; Farres, Jordi; Lanari, Riccardo; Casu, Francesco; Manunta, Michele; Lassalle-Balier, Gerard

    2014-05-01

    Helix Nebula has established a growing public private partnership of more than 30 commercial cloud providers, SMEs, and publicly funded research organisations and e-infrastructures. The Helix Nebula strategy is to establish a federated cloud service across Europe. Three high-profile flagships, sponsored by CERN (high energy physics), EMBL (life sciences) and ESA/DLR/CNES/CNR (earth science), have been deployed and extensively tested within this federated environment. The commitments behind these initial flagships have created a critical mass that attracts suppliers and users to the initiative, to work together towards an "Information as a Service" market place. Significant progress in implementing the following 4 programmatic goals (as outlined in the strategic Plan Ref.1) has been achieved: × Goal #1 Establish a Cloud Computing Infrastructure for the European Research Area (ERA) serving as a platform for innovation and evolution of the overall infrastructure. × Goal #2 Identify and adopt suitable policies for trust, security and privacy on a European-level can be provided by the European Cloud Computing framework and infrastructure. × Goal #3 Create a light-weight governance structure for the future European Cloud Computing Infrastructure that involves all the stakeholders and can evolve over time as the infrastructure, services and user-base grows. × Goal #4 Define a funding scheme involving the three stake-holder groups (service suppliers, users, EC and national funding agencies) into a Public-Private-Partnership model to implement a Cloud Computing Infrastructure that delivers a sustainable business environment adhering to European level policies. Now in 2014 a first version of this generic cross-domain e-infrastructure is ready to go into operations building on federation of European industry and contributors (data, tools, knowledge, ...). This presentation describes how Helix Nebula is being used in the domain of earth science focusing on geohazards. The

  8. Extended and structurally supported insights into extracellular hormone binding, signal transduction and organization of the thyrotropin receptor.

    Directory of Open Access Journals (Sweden)

    Gerd Krause

    Full Text Available The hormone thyrotropin (TSH and its receptor (TSHR are crucial for the growth and function of the thyroid gland. The TSHR is evolutionary linked with the receptors of follitropin (FSHR and lutropin/choriogonadotropin (LHR and their sequences and structures are similar. The extracellular region of TSHR contains more than 350 amino acids and binds hormone and antibodies. Several important questions related to functions and mechanisms of TSHR are still not comprehensively understood. One major reason for these open questions is the lack of any structural information about the extracellular segment of TSHR that connects the N-terminal leucine-rich repeat domain (LRRD with the transmembrane helix (TMH 1, the hinge region. It has been shown experimentally that this segment is important for fine tuning of signaling and ligand interactions. A new crystal structure containing most of the extracellular hFSHR region in complex with hFSH has recently been published. Now, we have applied these new structural insights to the homologous TSHR and have generated a structural model of the TSHR LRRD/hinge-region/TSH complex. This structural model is combined and evaluated with experimental data including hormone binding (bTSH, hTSH, thyrostimulin, super-agonistic effects, antibody interactions and signaling regulation. These studies and consideration of significant and non-significant amino acids have led to a new description of mechanisms at the TSHR, including ligand-induced displacements of specific hinge region fragments. This event triggers conformational changes at a convergent center of the LRRD and the hinge region, activating an "intramolecular agonistic unit" close to the transmembrane domain.

  9. Extended and structurally supported insights into extracellular hormone binding, signal transduction and organization of the thyrotropin receptor.

    Science.gov (United States)

    Krause, Gerd; Kreuchwig, Annika; Kleinau, Gunnar

    2012-01-01

    The hormone thyrotropin (TSH) and its receptor (TSHR) are crucial for the growth and function of the thyroid gland. The TSHR is evolutionary linked with the receptors of follitropin (FSHR) and lutropin/choriogonadotropin (LHR) and their sequences and structures are similar. The extracellular region of TSHR contains more than 350 amino acids and binds hormone and antibodies. Several important questions related to functions and mechanisms of TSHR are still not comprehensively understood. One major reason for these open questions is the lack of any structural information about the extracellular segment of TSHR that connects the N-terminal leucine-rich repeat domain (LRRD) with the transmembrane helix (TMH) 1, the hinge region. It has been shown experimentally that this segment is important for fine tuning of signaling and ligand interactions. A new crystal structure containing most of the extracellular hFSHR region in complex with hFSH has recently been published. Now, we have applied these new structural insights to the homologous TSHR and have generated a structural model of the TSHR LRRD/hinge-region/TSH complex. This structural model is combined and evaluated with experimental data including hormone binding (bTSH, hTSH, thyrostimulin), super-agonistic effects, antibody interactions and signaling regulation. These studies and consideration of significant and non-significant amino acids have led to a new description of mechanisms at the TSHR, including ligand-induced displacements of specific hinge region fragments. This event triggers conformational changes at a convergent center of the LRRD and the hinge region, activating an "intramolecular agonistic unit" close to the transmembrane domain.

  10. Transmembrane protein OSTA-1 shapes sensory cilia morphology via regulation of intracellular membrane trafficking in C. elegans.

    Science.gov (United States)

    Olivier-Mason, Anique; Wojtyniak, Martin; Bowie, Rachel V; Nechipurenko, Inna V; Blacque, Oliver E; Sengupta, Piali

    2013-04-01

    The structure and function of primary cilia are critically dependent on intracellular trafficking pathways that transport ciliary membrane and protein components. The mechanisms by which these trafficking pathways are regulated are not fully characterized. Here we identify the transmembrane protein OSTA-1 as a new regulator of the trafficking pathways that shape the morphology and protein composition of sensory cilia in C. elegans. osta-1 encodes an organic solute transporter alpha-like protein, mammalian homologs of which have been implicated in membrane trafficking and solute transport, although a role in regulating cilia structure has not previously been demonstrated. We show that mutations in osta-1 result in altered ciliary membrane volume, branch length and complexity, as well as defects in localization of a subset of ciliary transmembrane proteins in different sensory cilia types. OSTA-1 is associated with transport vesicles, localizes to a ciliary compartment shown to house trafficking proteins, and regulates both retrograde and anterograde flux of the endosome-associated RAB-5 small GTPase. Genetic epistasis experiments with sensory signaling, exocytic and endocytic proteins further implicate OSTA-1 as a crucial regulator of ciliary architecture via regulation of cilia-destined trafficking. Our findings suggest that regulation of transport pathways in a cell type-specific manner contributes to diversity in sensory cilia structure and might allow dynamic remodeling of ciliary architecture via multiple inputs.

  11. Emergence of the persistent spin helix in semiconductor quantum wells

    International Nuclear Information System (INIS)

    Koralek, Jake; Weber, Chris; Orenstein, Joe; Bernevig, Andrei; Zhang, Shoucheng; Mack, Shawn; Awschalom, David

    2008-01-01

    According to Noether's theorem, for every symmetry in nature there is a corresponding conservation law. For example, invariance with respect to spatial translation corresponds to conservation of momentum. In another well-known example, invariance with respect to rotation of the electron's spin, or SU(2) symmetry, leads to conservation of spin polarization. For electrons in a solid, this symmetry is ordinarily broken by spin-orbit (SO) coupling, allowing spin angular momentum to flow to orbital angular momentum. However, it has recently been predicted that SU(2) can be recovered in a two-dimensional electron gas (2DEG), despite the presence of SO coupling. The corresponding conserved quantities include the amplitude and phase of a helical spin density wave termed the 'persistent spin helix' (PSH) .2 SU(2) is restored, in principle, when the strength of two dominant SO interactions, the Rashba (alpha) and linear Dresselhaus (beta 1), are equal. This symmetry is predicted to be robust against all forms of spin-independent scattering, including electron-electron interactions, but is broken by the cubic Dresselhaus term (beta 3) and spin-dependent scattering. When these terms are negligible, the distance over which spin information can propagate is predicted to diverge as alpha approaches beta 1. Here we observe experimentally the emergence of the PSH in GaAs quantum wells (QW's) by independently tuning alpha and beta 1. Using transient spin-grating spectroscopy (TSG), we find a spin-lifetime enhancement of two orders of magnitude near the symmetry point. Excellent quantitative agreement with theory across a wide range of sample parameters allows us to obtain an absolute measure of all relevant SO terms, identifying beta 3 as the main SU(2) violating term in our samples. The tunable suppression of spin-relaxation demonstrated in this work is well-suited for application to spintronics

  12. Emergence of the Persistent Spin Helix in Semiconductor Quantum Wells

    International Nuclear Information System (INIS)

    Koralek, Jake

    2011-01-01

    According to Noether's theorem, for every symmetry in nature there is a corresponding conservation law. For example, invariance with respect to spatial translation corresponds to conservation of momentum. In another well-known example, invariance with respect to rotation of the electron's spin, or SU(2) symmetry, leads to conservation of spin polarization. For electrons in a solid, this symmetry is ordinarily broken by spin-orbit (SO) coupling, allowing spin angular momentum to flow to orbital angular momentum. However, it has recently been predicted that SU(2) can be recovered in a two-dimensional electron gas (2DEG), despite the presence of SO coupling. The corresponding conserved quantities include the amplitude and phase of a helical spin density wave termed the 'persistent spin helix' (PSH). SU(2) is restored, in principle, when the strength of two dominant SO interactions, the Rashba (α) and linear Dresselhaus (β 1 ), are equal. This symmetry is predicted to be robust against all forms of spin-independent scattering, including electron-electron interactions, but is broken by the cubic Dresselhaus term (β 3 ) and spin-dependent scattering. When these terms are negligible, the distance over which spin information can propagate is predicted to diverge as α → β 1 . Here we observe experimentally the emergence of the PSH in GaAs quantum wells (QW's) by independently tuning α and β 1 . Using transient spin-grating spectroscopy (TSG), we find a spin-lifetime enhancement of two orders of magnitude near the symmetry point. Excellent quantitative agreement with theory across a wide range of sample parameters allows us to obtain an absolute measure of all relevant SO terms, identifying β 3 as the main SU(2) violating term in our samples. The tunable suppression of spin-relaxation demonstrated in this work is well-suited for application to spintronics.

  13. Defect-facilitated buckling in supercoiled double-helix DNA

    Science.gov (United States)

    Brahmachari, Sumitabha; Dittmore, Andrew; Takagi, Yasuharu; Neuman, Keir C.; Marko, John F.

    2018-02-01

    We present a statistical-mechanical model for stretched twisted double-helix DNA, where thermal fluctuations are treated explicitly from a Hamiltonian without using any scaling hypotheses. Our model applied to defect-free supercoiled DNA describes the coexistence of multiple plectoneme domains in long DNA molecules at physiological salt concentrations (≈0.1 M Na+) and stretching forces (≈1 pN ) . We find a higher (lower) number of domains at lower (higher) ionic strengths and stretching forces, in accord with experimental observations. We use our model to study the effect of an immobile point defect on the DNA contour that allows a localized kink. The degree of the kink is controlled by the defect size, such that a larger defect further reduces the bending energy of the defect-facilitated kinked end loop. We find that a defect can spatially pin a plectoneme domain via nucleation of a kinked end loop, in accord with experiments and simulations. Our model explains previously reported magnetic tweezer experiments [A. Dittmore et al., Phys. Rev. Lett. 119, 147801 (2017), 10.1103/PhysRevLett.119.147801] showing two buckling signatures: buckling and "rebuckling" in supercoiled DNA with a base-unpaired region. Comparing with experiments, we find that under 1 pN force, a kinked end loop nucleated at a base-mismatched site reduces the bending energy by ≈0.7 kBT per unpaired base. Our model predicts the coexistence of three states at the buckling and rebuckling transitions, which warrants new experiments.

  14. A Novel Type III Endosome Transmembrane Protein, TEMP

    Directory of Open Access Journals (Sweden)

    Rohan D. Teasdale

    2012-11-01

    Full Text Available As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP's plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport.

  15. Transmembrane topology of the acetylcholine receptor examined in reconstituted vesicles

    International Nuclear Information System (INIS)

    McCrea, P.D.

    1987-01-01

    Each of the five acetylcholine receptor (AChR) subunits, α 2 β-γδ, is believed to have the same number of transmembrane crossing and to share the same general folding pattern. AChR isolated from the electric organ of electric fish is predominantly dimeric. We have used this bridge as a marker for the C-terminus of the δ subunit, and presumably that of the other subunits in addition. The disulfide's accessibility to hydrophilic reductants, principally glutathione (GSH), was tested in a reconstituted vesicle system. The reduction of the δ-δ desulfide, as evidenced by the transition of AChrR dimers to monomers, was quantitatively monitored on velocity sedimentation sucrose gradients. Alternatively, the reduction of δ 2 to δ was followed by employing non-reducing SDS-PAGE. Reductants such as GSH were able to access the bridge in intact right-side-out vesicles. No acceleration of this process was evident when the vesicles were disrupted by freeze-thaw or by detergents. Control experiments which determined the rate of reduction of entrapped diphtheria toxin, or that of 3 H-GSH efflux, demonstrated that intact reconstituted vesicles provide an adequate permeability barrier to GSH access of their intravesicular space

  16. Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion.

    Science.gov (United States)

    Guo, Yanmeng; Wang, Yuping; Zhang, Wei; Meltzer, Shan; Zanini, Damiano; Yu, Yue; Li, Jiefu; Cheng, Tong; Guo, Zhenhao; Wang, Qingxiu; Jacobs, Julie S; Sharma, Yashoda; Eberl, Daniel F; Göpfert, Martin C; Jan, Lily Yeh; Jan, Yuh Nung; Wang, Zuoren

    2016-06-28

    Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, we found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs.

  17. Requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by NMR.

    Science.gov (United States)

    Gottstein, Daniel; Reckel, Sina; Dötsch, Volker; Güntert, Peter

    2012-06-06

    Nuclear magnetic resonance (NMR) structure calculations of the α-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary for obtaining enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables α-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Crystal structure of a bacterial homologue of glucose transporters GLUT1-4.

    Science.gov (United States)

    Sun, Linfeng; Zeng, Xin; Yan, Chuangye; Sun, Xiuyun; Gong, Xinqi; Rao, Yu; Yan, Nieng

    2012-10-18

    Glucose transporters are essential for metabolism of glucose in cells of diverse organisms from microbes to humans, exemplified by the disease-related human proteins GLUT1, 2, 3 and 4. Despite rigorous efforts, the structural information for GLUT1-4 or their homologues remains largely unknown. Here we report three related crystal structures of XylE, an Escherichia coli homologue of GLUT1-4, in complex with d-xylose, d-glucose and 6-bromo-6-deoxy-D-glucose, at resolutions of 2.8, 2.9 and 2.6 Å, respectively. The structure consists of a typical major facilitator superfamily fold of 12 transmembrane segments and a unique intracellular four-helix domain. XylE was captured in an outward-facing, partly occluded conformation. Most of the important amino acids responsible for recognition of D-xylose or d-glucose are invariant in GLUT1-4, suggesting functional and mechanistic conservations. Structure-based modelling of GLUT1-4 allows mapping and interpretation of disease-related mutations. The structural and biochemical information reported here constitutes an important framework for mechanistic understanding of glucose transporters and sugar porters in general.

  19. Inactivation of Mechanically Activated Piezo1 Ion Channels Is Determined by the C-Terminal Extracellular Domain and the Inner Pore Helix

    Directory of Open Access Journals (Sweden)

    Jason Wu

    2017-11-01

    Full Text Available Piezo proteins form mechanically activated ion channels that are responsible for our sense of light touch, proprioception, and vascular blood flow. Upon activation by mechanical stimuli, Piezo channels rapidly inactivate in a voltage-dependent manner through an unknown mechanism. Inactivation of Piezo channels is physiologically important, as it modulates overall mechanical sensitivity, gives rise to frequency filtering of repetitive mechanical stimuli, and is itself the target of numerous human disease-related channelopathies that are not well understood mechanistically. Here, we identify the globular C-terminal extracellular domain as a structure that is sufficient to confer the time course of inactivation and a single positively charged lysine residue at the adjacent inner pore helix as being required for its voltage dependence. Our results are consistent with a mechanism for inactivation that is mediated through voltage-dependent conformations of the inner pore helix and allosteric coupling with the C-terminal extracellular domain.

  20. Optics of short-pitch deformed-helix ferroelectric liquid crystals: Symmetries, exceptional points, and polarization-resolved angular patterns

    Science.gov (United States)

    Kiselev, Alexei D.; Chigrinov, Vladimir G.

    2014-10-01

    In order to explore electric-field-induced transformations of polarization singularities in the polarization-resolved angular (conoscopic) patterns emerging after deformed-helix ferroelectric liquid crystal (DHFLC) cells with subwavelength helix pitch, we combine the transfer matrix formalism with the results for the effective dielectric tensor of biaxial FLCs evaluated using an improved technique of averaging over distorted helical structures. Within the framework of the transfer matrix method, we deduce a number of symmetry relations and show that the symmetry axis of L lines (curves of linear polarization) is directed along the major in-plane optical axis which rotates under the action of the electric field. When the angle between this axis and the polarization plane of incident linearly polarized light is above its critical value, the C points (points of circular polarization) appear in the form of symmetrically arranged chains of densely packed star-monstar pairs. We also emphasize the role of phase singularities of a different kind and discuss the enhanced electro-optic response of DHFLCs near the exceptional point where the condition of zero-field isotropy is fulfilled.

  1. Changes in the reproductive system of the snail Helix aspersa caused by mucus from the love dart.

    Science.gov (United States)

    Koene, J M; Chase, R

    1998-08-01

    The function of the love dart in certain species of terrestrial snails is unknown. In Helix aspersa, the dart is a sharp calcareous structure that is used to pierce the partner's skin during courtship. When expelled, the dart is covered with a thick mucus. The hypothesis tested here is that the mucus contains a biologically active substance. Extracts of the digitiform glands that produce this mucus were applied to parts of the reproductive system in vitro. The extracts triggered an initial reconfiguration of the copulatory canal that caused the bursa tract diverticulum to become more accessible to the spermatophore. The reconfiguration of the copulatory canal also closed off the tract leading to the bursa copulatrix, a sperm-digesting organ. A few minutes after the initial contraction, the peristaltic contractions in the diverticulum became significantly more frequent. This latter effect continued for at least 1 h, provided that the mucus extract remained in the saline bath. The minimum effective dosage was less than the 2.2 mg of mucus transferred with the dart. Sperm competition is expected in Helix aspersa since multiple matings occur before eggs are laid. By influencing the female organs involved in the processing of foreign sperm, the dart shooter may increase the chance that his sperm will fertilise eggs.

  2. Analysis of α-helix unfolding in the pine nut peptide Lys-Cys-His-Lys-Pro induced by pulsed electric field.

    Science.gov (United States)

    Xing, Jie; Zhang, Sitian; Zhang, Mingdi; Lin, Songyi

    2017-09-01

    A variety of analytical techniques were applied to explore the effects of pulsed electric field (PEF) on α-helix structural changes in the novel antioxidant peptide Lys-Cys-His-Lys-Pro (KCHKP, 611.76 Da). The relative α-helix content of the KCHKP peptide was significantly altered from 100% to 89.91 ± 0.97% when the electric pulse frequency was 1800 Hz and the field intensity was 10 kV cm -1 . Moreover, the 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) and 2,2-azinobis diammonium salt (ABTS) radical-scavenging activities of PEF-treated KCHKP were increased from 56.31% ± 0.74% to 84.33% ± 1.23% and from 40.56% ± 0.78% to 51.33% ± 0.27%, respectively. PEF treatment increased peptide linkage stretch vibration and altered hydrogen bonding of KCHKP. The stability of the α-helix structure was influenced by hydrogen bonds within the peptide linkage of KCHKP induced by PEF and was related to changes in antioxidant activity. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  3. Aβ1-25-Derived Sphingolipid-Domain Tracer Peptide SBD Interacts with Membrane Ganglioside Clusters via a Coil-Helix-Coil Motif

    Directory of Open Access Journals (Sweden)

    Yaofeng Wang

    2015-11-01

    Full Text Available The Amyloid-β (Aβ-derived, sphingolipid binding domain (SBD peptide is a fluorescently tagged probe used to trace the diffusion behavior of sphingolipid-containing microdomains in cell membranes through binding to a constellation of glycosphingolipids, sphingomyelin, and cholesterol. However, the molecular details of the binding mechanism between SBD and plasma membrane domains remain unclear. Here, to investigate how the peptide recognizes the lipid surface at an atomically detailed level, SBD peptides in the environment of raft-like bilayers were examined in micro-seconds-long molecular dynamics simulations. We found that SBD adopted a coil-helix-coil structural motif, which binds to multiple GT1b gangliosides via salt bridges and CH–π interactions. Our simulation results demonstrate that the CH–π and electrostatic forces between SBD monomers and GT1b gangliosides clusters are the main driving forces in the binding process. The presence of the fluorescent dye and linker molecules do not change the binding mechanism of SBD probes with gangliosides, which involves the helix-turn-helix structural motif that was suggested to constitute a glycolipid binding domain common to some sphingolipid interacting proteins, including HIV gp120, prion, and Aβ.

  4. Overlapping effector interfaces define the multiple functions of the HIV-1 Nef polyproline helix

    Directory of Open Access Journals (Sweden)

    Kuo Lillian S

    2012-05-01

    Full Text Available Abstract Background HIV-1 Nef is a multifunctional protein required for full pathogenicity of the virus. As Nef has no known enzymatic activity, it necessarily functions through protein-protein interaction interfaces. A critical Nef protein interaction interface is centered on its polyproline segment (P69VRPQVPLRP78 which contains the helical SH3 domain binding protein motif, PXXPXR. We hypothesized that any Nef-SH3 domain interactions would be lost upon mutation of the prolines or arginine of PXXPXR. Further, mutation of the non-motif “X” residues, (Q73, V74, and L75 would give altered patterns of inhibition for different Nef/SH3 domain protein interactions. Results We found that mutations of either of the prolines or the arginine of PXXPXR are defective for Nef-Hck binding, Nef/activated PAK2 complex formation and enhancement of virion infectivity (EVI. Mutation of the non-motif “X” residues (Q, V and L gave similar patterns of inhibition for Nef/activated PAK2 complex formation and EVI which were distinct from the pattern for Hck binding. These results implicate an SH3 domain containing protein other than Hck for Nef/activated PAK2 complex formation and EVI. We have also mutated Nef residues at the N-and C-terminal ends of the polyproline segment to explore interactions outside of PXXPXR. We discovered a new locus GFP/F (G67, F68, P69 and F90 that is required for Nef/activated PAK2 complex formation and EVI. MHC Class I (MHCI downregulation was only partially inhibited by mutating the PXXPXR motif residues, but was fully inhibited by mutating the C-terminal P78. Further, we observed that MHCI downregulation strictly requires G67 and F68. Our mutational analysis confirms the recently reported structure of the complex between Nef, AP-1 μ1 and the cytoplasmic tail of MHCI, but does not support involvement of an SH3 domain protein in MHCI downregulation. Conclusion Nef has evolved to be dependent on interactions with multiple SH3 domain

  5. Intramolecular cross-linking in a bacterial homolog of mammalian SLC6 neurotransmitter transporters suggests an evolutionary conserved role of transmembrane segments 7 and 8

    DEFF Research Database (Denmark)

    Kniazeff, Julie; Loland, Claus Juul; Goldberg, Naomi

    2005-01-01

    transporters has recently been identified in prokaryotes. Here we have probed structural relationships in a 'microdoman' corresponding to the extracellular ends of transmembrane segments (TM) 7 and 8 in one of these homologs, the tryptophan transporter TnaT from Symbiobacterium thermophilum. We found...... that simultaneous - but not individual - substitution of Ala286 at the top of TM7 and Met311 at the top of TM8 with cysteines conferred sensitivity to submicromolar concentrations of Hg(2+) as assessed in a [(3)H]tryptophan uptake assay. Because Hg(2+) can cross-link pairs of cysteines, this suggests close...... proximity between TM 7 and 8 in the tertiary structure of TnaT as previously suggested for the mammalian counterparts. Furthermore, the inhibition of uptake upon cross-linking the two cysteines provides indirect support for a conserved conformational role of these transmembrane domains in the transport...

  6. The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum.

    Science.gov (United States)

    De Smet, Lina; Dimitrov, Ivan; Debyser, Griet; Dolashka-Angelova, Pavlina; Dolashki, Aleksandar; Van Beeumen, Jozef; Devreese, Bart

    2011-11-10

    Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α(D)-HlH, α(N)-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α(D)-HlH and β-HlH was obtained, including the 5' and 3' UTR, 180bp of the 5' end and around 900bp at the 3' end are missing for the third subunit. The subunits α(D)-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α(N)-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α(D)-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. CFD analysis and flow model reduction for surfactant production in helix reactor = CFD analiza i redukcija modela strujanja za proizvodnju surfaktanta u helix reaktoru

    NARCIS (Netherlands)

    Nikačević, N.M.; Thielen, L.; Twerda, A.; Hof, P.M.J. van den

    2015-01-01

    Flow pattern analysis in a spiral Helix reactor is conducted, for the application in commercial surfactant production. Step change response curves (SCR) were obtained from numerical tracer experiments by three-dimensional computational fluid dynamics (CFD) simulations. Non-reactive flow is

  8. Importance of the alphaC-helix in the cyclic nucleotide binding domain for the stable channel regulation and function of cyclic nucleotide gated ion channels in Arabidopsis.

    Science.gov (United States)

    Chin, Kimberley; Moeder, Wolfgang; Abdel-Hamid, Huda; Shahinas, Dea; Gupta, Deepali; Yoshioka, Keiko

    2010-05-01

    The involvement of cyclic nucleotide gated ion channels (CNGCs) in the signal transduction of animal light and odorant perception is well documented. Although plant CNGCs have recently been revealed to mediate multiple stress responses and developmental pathways, studies that aim to elucidate their structural and regulatory properties are still very much in their infancy. The structure-function relationship of plant CNGCs was investigated here by using the chimeric Arabidopsis AtCNGC11/12 gene that induces multiple defence responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22) for the identification of functionally essential residues. A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12. One of these mutants, suppressor S58 possesses a single amino acid substitution, arginine 557 to cysteine, in the alphaC-helix of the cyclic nucleotide-binding domain (CNBD). The suppressor S58 lost all cpr22 related phenotypes, such as spontaneous cell death formation under ambient temperature conditions. However, these phenotypes were recovered at 16 degrees C suggesting that the stability of channel function is affected by temperature. In silico modelling and site-directed mutagenesis analyses suggest that arginine 557 in the alphaC-helix of the CNBD is important for channel regulation, but not for basic function. Furthermore, another suppressor mutant, S136 that lacks the entire alphaC-helix due to a premature stop codon, lost channel function completely. Our data presented here indicate that the alphaC-helix is functionally important in plant CNGCs.

  9. Mechanisms of triggering H1 helix in prion proteins unfolding revealed by molecular dynamic simulation

    Science.gov (United States)

    Tseng, Chih-Yuan; Lee, H. C.

    2006-03-01

    In template-assistance model, normal Prion protein (PrP^C), the pathogen to cause several prion diseases such as Creutzfeldt-Jakob (CJD) in human, Bovine Spongiform Encephalopathy (BSE) in cow, and scrapie in sheep, converts to infectious prion (PrP^Sc) through a transient interaction with PrP^Sc. Furthermore, conventional studies showed S1-H1-S2 region in PrP^C to be the template of S1-S2 β-sheet in PrP^Sc, and Prion protein's conformational conversion may involve an unfolding of H1 and refolding into β-sheet. Here we prepare several mouse prion peptides that contain S1-H1-S2 region with specific different structures, which are corresponding to specific interactions, to investigate possible mechanisms to trigger H1 α-helix unfolding process via molecular dynamic simulation. Three properties, conformational transition, salt-bridge in H1, and hydrophobic solvent accessible surface (SAS) are analyzed. From these studies, we found the interaction that triggers H1 unfolding to be the one that causes dihedral angle at residue Asn^143 changes. Whereas interactions that cause S1 segment's conformational changes play a minor in this process. These studies offers an additional evidence for template-assistance model.

  10. Flexoelectro-optic properties of chiral nematic liquid crystals in the uniform standing helix configuration

    Science.gov (United States)

    Castles, F.; Morris, S. M.; Coles, H. J.

    2009-09-01

    The flexoelectro-optic effect describes the rotation of the optic axis of a short-pitch chiral nematic liquid crystal under the application of an electric field. We investigate the effect in the uniform standing helix, or “Grandjean” configuration. An in-plane electric field is applied. The director profile is determined numerically using a static one-dimensional continuum model with strong surface anchoring. The Berreman method is used to solve for plane-wave solutions to Maxwell’s equations, and predict the optical properties of the resulting structure in general cases. By using a chiral nematic with short pitch between crossed polarizers an optical switch may be generated. With no applied field the configuration is nontransmissive at normal incidence, but becomes transmissive with an applied field. For this case, numerical results using the Berreman method are supplemented with an analytic theory and found to be in good agreement. The transmitted intensity as a function of tilt, the contrast ratio, and the tilt required for full intensity modulation are presented. The angular dependence of the transmission is calculated and the isocontrast curves are plotted. For typical material and cell parameters a switching speed of 0.017 ms and contrast ratio of 1500:1 at normal incidence are predicted, at a switch-on tilt of 41.5 degrees. Experimental verification of the analytic and numerical models is provided.

  11. DISCOVERY OF A HALO AROUND THE HELIX NEBULA NGC 7293 IN THE WISE ALL-SKY SURVEY

    International Nuclear Information System (INIS)

    Zhang Yong; Hsia, Chih-Hao; Kwok, Sun

    2012-01-01

    We report the discovery of an extended halo (∼40' in diameter) around the planetary nebula NGC 7293 (the Helix Nebula) observed in the 12 μm band from the Wide-field Infrared Survey Explorer all-sky survey. The mid-infrared halo has an axisymmetric structure with a sharp boundary to the northeast and a more diffuse boundary to the southwest, suggesting an interaction between the stellar wind and the interstellar medium (ISM). The symmetry axis of the halo is well aligned with that of a northeast arc, suggesting that the two structures are physically associated. We have attempted to fit the observed geometry with a model of a moving steady-state stellar wind interacting with the ISM. Possible combinations of the ISM density and the stellar velocity are derived from these fittings. The discrepancies between the model and the observations suggest that the stellar mass loss has a more complicated history, including possible time and angle dependences.

  12. Amino acid duplication in the coiled-coil structure of collagen XVII alters its maturation and trimerization causing mild junctional epidermolysis bullosa.

    Science.gov (United States)

    Kroeger, Jasmin K; Hofmann, Silke C; Leppert, Juna; Has, Cristina; Franzke, Claus-Werner

    2017-02-01

    The function and stability of collagens depend on the accurate triple helix formation of three distinct polypeptide chains. Disruption of this triple-helical structure can result in connective-tissue disorders. Triple helix formation is thought to depend on three-stranded coiled-coil oligomerization sites within non-collagenous domains. However, only little is known about the physiological relevance of these coiled-coil structures. Transmembrane collagen XVII, also known as 180 kDa bullous pemphigoid antigen provides mechanical stability through the anchorage of epithelial cells to the basement membrane. Mutations in the collagen XVII gene, COL17A1, cause junctional epidermolysis bullosa (JEB), characterized by chronic trauma-induced skin blistering. Here we exploited a novel naturally occurring COL17A1 mutation, leading to an in-frame lysine duplication within the coiled-coil structure of the juxtamembranous NC16A domain of collagen XVII, which resulted in a mild phenotype of JEB due to reduced membrane-anchored collagen XVII molecules. This mutation causes structural changes in the mutant molecule and interferes with its maturation. The destabilized coiled-coil structure of the mutant collagen XVII unmasks a furin cleavage site that results in excessive and non-physiological ectodomain shedding during its maturation. Furthermore, it decreases its triple-helical stability due to defective coiled-coil oligomerization, which makes it highly susceptible to proteolytic degradation. As a consequence of altered maturation and decreased stability of collagen XVII trimers, reduced collagen XVII is incorporated into the cell membrane, resulting in compromised dermal-epidermal adhesion. Taken together, using this genetic model, we provide the first proof that alteration of the coiled-coil structure destabilizes oligomerization and impairs physiological shedding of collagen XVII in vivo. © The Author 2016. Published by Oxford University Press. All rights reserved. For

  13. Structural mechanism of ligand activation in human calcium-sensing receptor

    Energy Technology Data Exchange (ETDEWEB)

    Geng, Yong; Mosyak, Lidia; Kurinov, Igor; Zuo, Hao; Sturchler, Emmanuel; Cheng, Tat Cheung; Subramanyam, Prakash; Brown, Alice P.; Brennan, Sarah C.; Mun, Hee-chang; Bush, Martin; Chen, Yan; Nguyen, Trang X.; Cao, Baohua; Chang, Donald D.; Quick, Matthias; Conigrave, Arthur D.; Colecraft, Henry M.; McDonald, Patricia; Fan, Qing R.

    2016-07-19

    Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+and PO43-ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ions stabilize the active state, PO43-ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

  14. Crystal structure of the potassium-importing KdpFABC membrane complex

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Ching-Shin; Pedersen, Bjørn Panyella; Stokes, David L.

    2017-06-21

    Cellular potassium import systems play a fundamental role in osmoregulation, pH homeostasis and membrane potential in all domains of life. In bacteria, the kdp operon encodes a four-subunit potassium pump that maintains intracellular homeostasis, cell shape and turgor under conditions in which potassium is limiting1. This membrane complex, called KdpFABC, has one channel-like subunit (KdpA) belonging to the superfamily of potassium transporters and another pump-like subunit (KdpB) belonging to the superfamily of P-type ATPases. Although there is considerable structural and functional information about members of both superfamilies, the mechanism by which uphill potassium transport through KdpA is coupled with ATP hydrolysis by KdpB remains poorly understood. Here we report the 2.9 Å X-ray structure of the complete Escherichia coli KdpFABC complex with a potassium ion within the selectivity filter of KdpA and a water molecule at a canonical cation site in the transmembrane domain of KdpB. The structure also reveals two structural elements that appear to mediate the coupling between these two subunits. Specifically, a protein-embedded tunnel runs between these potassium and water sites and a helix controlling the cytoplasmic gate of KdpA is linked to the phosphorylation domain of KdpB. On the basis of these observations, we propose a mechanism that repurposes protein channel architecture for active transport across biomembranes.

  15. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    Science.gov (United States)

    Wang, Jimin; Li, Yue; Modis, Yorgo

    2014-01-01

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. PMID:24725935

  16. Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel

    Science.gov (United States)

    Carrasquel-Ursulaez, Willy; Contreras, Gustavo F.; Sepúlveda, Romina V.; Aguayo, Daniel; González-Nilo, Fernando

    2015-01-01

    Large-conductance Ca2+- and voltage-activated K+ channel (BK) open probability is enhanced by depolarization, increasing Ca2+ concentration, or both. These stimuli activate modular voltage and Ca2+ sensors that are allosterically coupled to channel gating. Here, we report a point mutation of a phenylalanine (F380A) in the S6 transmembrane helix that, in the absence of internal Ca2+, profoundly hinders channel opening while showing only minor effects on the voltage sensor active–resting equilibrium. Interpretation of these results using an allosteric model suggests that the F380A mutation greatly increases the free energy difference between open and closed states and uncouples Ca2+ binding from voltage sensor activation and voltage sensor activation from channel opening. However, the presence of a bulky and more hydrophobic amino acid in the F380 position (F380W) increases the intrinsic open–closed equilibrium, weakening the coupling between both sensors with the pore domain. Based on these functional experiments and molecular dynamics simulations, we propose that F380 interacts with another S6 hydrophobic residue (L377) in contiguous subunits. This pair forms a hydrophobic ring important in determining the open–closed equilibrium and, like an integration node, participates in the communication between sensors and between the sensors and pore. Moreover, because of its effects on open probabilities, the F380A mutant can be used for detailed voltage sensor experiments in the presence of permeant cations. PMID:25548136

  17. On the Use of Cross-Correlation between Volume Scattering and Helix Scattering from Polarimetric SAR Data for the Improvement of Ship Detection

    Directory of Open Access Journals (Sweden)

    Jujie Wei

    2016-01-01

    Full Text Available Synthetic Aperture Radar (SAR ship detection is an important maritime application. However, azimuth ambiguities caused by the finite sampling of the Doppler spectrum are often visible in SAR images and are always mistaken as ships by classic detection techniques, like the Constant False Alarm Rate (CFAR. It is known that radar targets and azimuth ambiguities have different characteristics in polarimetric SAR (PolSAR data, i.e., first ambiguities usually have strong odd- or double-bounce scattering and the maximum amplitude of the first ambiguity in SHV is always considerably smaller than that of the corresponding target for zero or high velocity. On the basis of this characteristics, this paper finds that first ambiguities usually have low volume scattering power relative to ships and almost have no helix scattering by Yamaguchi decomposition. But some residual ambiguities still exit in the volume scattering power and have similar scattering intensity to small ships, and some parts of a ship also have zero helix scattering owing to some physical factors (e.g., ship structure, radar incidence angle, etc.. Thus, for high-precision ship detection, a new ship detection method based on cross-correlation between the volume and helix scattering mechanisms derived from Yamaguchi decomposition is proposed to avoid false alarms caused by azimuth ambiguities and enhance Target-to-Clutter Ratio (TCR for improving the miss detection rate of small ships. By experiments, it is proved that our method can work effectively and has high detection accuracy.

  18. A comparison of three-dimensional stress distribution and displacement of naso-maxillary complex on application of forces using quad-helix and nickel titanium palatal expander 2 (NPE2: a FEM study

    Directory of Open Access Journals (Sweden)

    Avinash Kumar

    2016-06-01

    Full Text Available Abstract Background Our objectives are to analyse and to compare the stress distribution and displacement of the craniofacial structures, following the application of forces from quad-helix and Nickel Titanium Palatal Expander-2 (NPE2 using finite element analysis. Methods Three-dimensional finite element models of young dried human skull, quad-helix appliance and NPE2 were constructed, and the initial activation of the expanders was stimulated to carry out the analysis and to evaluate the Von Misses stresses and displacement. Results Both the models demonstrated the highest stresses at the mid-palatal suture, with maximum posterior dislocation. The second highest stress was recorded at the fronto-zygomatic suture. The pattern of stress distribution was almost similar in both the groups, but NPE2 revealed lower magnitude stresses than quad-helix. The only exception being quad-helix model showed high stress levels around pterygo-maxillary suture whereas minimal stress around pterygo-maxillary suture was noticed after NPE2 activation. The cusp of the erupting canine and the erupting mesiobuccal cusp of the second molar showed outward, backward and downward displacement signifying increase in their eruption pattern following maxillary expansion. Conclusions Maxillary expansion using quad-helix and NPE2 can be used in posterior crossbite correction in cases where maximum skeletal changes are desirable at a younger age; it is furthermore effective in treating young patients with impacted or displaced teeth. Quad-helix and NPE2 produced acceptable forces for orthopaedic treatment even after being orthodontic appliances; their clinical application should be correctly planned as the effects of these appliances are largely age dependent.

  19. Design, synthesis, and evaluation of an alpha-helix mimetic library targeting protein-protein interactions.

    Science.gov (United States)

    Shaginian, Alex; Whitby, Landon R; Hong, Sukwon; Hwang, Inkyu; Farooqi, Bilal; Searcey, Mark; Chen, Jiandong; Vogt, Peter K; Boger, Dale L

    2009-04-22

    The design and solution-phase synthesis of an alpha-helix mimetic library as an integral component of a small-molecule library targeting protein-protein interactions are described. The iterative design, synthesis, and evaluation of the candidate alpha-helix mimetic was initiated from a precedented triaryl template and refined by screening the designs for inhibition of MDM2/p53 binding. Upon identifying a chemically and biologically satisfactory design and consistent with the screening capabilities of academic collaborators, the corresponding complete library was assembled as 400 mixtures of 20 compounds (20 x 20 x 20-mix), where the added subunits are designed to mimic all possible permutations of the naturally occurring i, i + 4, i + 7 amino acid side chains of an alpha-helix. The library (8000 compounds) was prepared using a solution-phase synthetic protocol enlisting acid/base liquid-liquid extractions for purification on a scale that insures its long-term availability for screening campaigns. Screening of the library for inhibition of MDM2/p53 binding not only identified the lead alpha-helix mimetic upon which the library was based, but also suggests that a digestion of the initial screening results that accompany the use of such a comprehensive library can provide insights into the nature of the interaction (e.g., an alpha-helix mediated protein-protein interaction) and define the key residues and their characteristics responsible for recognition.

  20. An Organellar Nα-Acetyltransferase, Naa60, Acetylates Cytosolic N Termini of Transmembrane Proteins and Maintains Golgi Integrity

    Directory of Open Access Journals (Sweden)

    Henriette Aksnes

    2015-03-01

    Full Text Available N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs. NatF, or Nα-acetyltransferase 60 (Naa60, was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi’s structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.

  1. A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER

    Directory of Open Access Journals (Sweden)

    Haga Christopher L

    2007-09-01

    Full Text Available Abstract Background In mouse the cytokine interleukin-7 (IL-7 is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER. The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR, a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules.

  2. Comparative exploration of hydrogen sulfide and water transmembrane free energy surfaces via orthogonal space tempering free energy sampling.

    Science.gov (United States)

    Lv, Chao; Aitchison, Erick W; Wu, Dongsheng; Zheng, Lianqing; Cheng, Xiaolin; Yang, Wei

    2016-03-05

    Hydrogen sulfide (H2 S), a commonly known toxic gas compound, possesses unique chemical features that allow this small solute molecule to quickly diffuse through cell membranes. Taking advantage of the recent orthogonal space tempering (OST) method, we comparatively mapped the transmembrane free energy landscapes of H2 S and its structural analogue, water (H2 O), seeking to decipher the molecular determinants that govern their drastically different permeabilities. As revealed by our OST sampling results, in contrast to the highly polar water solute, hydrogen sulfide is evidently amphipathic, and thus inside membrane is favorably localized at the interfacial region, that is, the interface between the polar head-group and nonpolar acyl chain regions. Because the membrane binding affinity of H2 S is mainly governed by its small hydrophobic moiety and the barrier height inbetween the interfacial region and the membrane center is largely determined by its moderate polarity, the transmembrane free energy barriers to encounter by this toxic molecule are very small. Moreover when H2 S diffuses from the bulk solution to the membrane center, the above two effects nearly cancel each other, so as to lead to a negligible free energy difference. This study not only explains why H2 S can quickly pass through cell membranes but also provides a practical illustration on how to use the OST free energy sampling method to conveniently analyze complex molecular processes. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  3. Regulation of Exocytotic Fusion Pores by SNARE Protein Transmembrane Domains

    Directory of Open Access Journals (Sweden)

    Zhenyong Wu

    2017-10-01

    Full Text Available Calcium-triggered exocytotic release of neurotransmitters and hormones from neurons and neuroendocrine cells underlies neuronal communication, motor activity and endocrine functions. The core of the neuronal exocytotic machinery is composed of soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs. Formation of complexes between vesicle-attached v- and plasma-membrane anchored t-SNAREs in a highly regulated fashion brings the membranes into close apposition. Small, soluble proteins called Complexins (Cpx and calcium-sensing Synaptotagmins cooperate to block fusion at low resting calcium concentrations, but trigger release upon calcium increase. A growing body of evidence suggests that the transmembrane domains (TMDs of SNARE proteins play important roles in regulating the processes of fusion and release, but the mechanisms involved are only starting to be uncovered. Here we review recent evidence that SNARE TMDs exert influence by regulating the dynamics of the fusion pore, the initial aqueous connection between the vesicular lumen and the extracellular space. Even after the fusion pore is established, hormone release by neuroendocrine cells is tightly controlled, and the same may be true of neurotransmitter release by neurons. The dynamics of the fusion pore can regulate the kinetics of cargo release and the net amount released, and can determine the mode of vesicle recycling. Manipulations of SNARE TMDs were found to affect fusion pore properties profoundly, both during exocytosis and in biochemical reconstitutions. To explain these effects, TMD flexibility, and interactions among TMDs or between TMDs and lipids have been invoked. Exocytosis has provided the best setting in which to unravel the underlying mechanisms, being unique among membrane fusion reactions in that single fusion pores can be probed using high-resolution methods. An important role will likely be played by methods that can probe single fusion pores

  4. Transmembrane TNF-dependent uptake of anti-TNF antibodies.

    Science.gov (United States)

    Deora, Arun; Hegde, Subramanya; Lee, Jacqueline; Choi, Chee-Ho; Chang, Qing; Lee, Cheryl; Eaton, Lucia; Tang, Hua; Wang, Dongdong; Lee, David; Michalak, Mark; Tomlinson, Medha; Tao, Qingfeng; Gaur, Nidhi; Harvey, Bohdan; McLoughlin, Shaun; Labkovsky, Boris; Ghayur, Tariq

    TNF-α (TNF), a pro-inflammatory cytokine is synthesized as a 26 kDa protein, anchors in the plasma membrane as transmembrane TNF (TmTNF), and is subjected to proteolysis by the TNF-α converting enzyme (TACE) to release the 15 kDa form of soluble TNF (sTNF). TmTNF and sTNF interact with 2 distinct receptors, TNF-R1 (p55) and TNF-R2 (p75), to mediate the multiple biologic effects of TNF described to date. Several anti-TNF biologics that bind to both forms of TNF and block their interactions with the TNF receptors are now approved for the treatment of a variety of immune-mediated diseases. Several reports suggest that binding of anti-TNFs to TmTNF delivers an outside-to-inside 'reverse' signal that may also contribute to the efficacy of anti-TNFs. Some patients, however, develop anti-TNF drug antibody responses (ADA or immunogenicity). Here, we demonstrate biochemically that TmTNF is transiently expressed on the surface of lipopolysaccharide-stimulated primary human monocytes, macrophages, and monocyte-derived dendritic cells (DCs) and expression of TmTNF on the cell surface is enhanced following treatment of cells with TAPI-2, a TACE inhibitor. Importantly, binding of anti-TNFs to TmTNF on DCs results in rapid internalization of the anti-TNF/TmTNF complex first into early endosomes and then lysosomes. The internalized anti-TNF is processed and anti-TNF peptides can be eluted from the surface of DCs. Finally, tetanus toxin peptides fused to anti-TNFs are presented by DCs to initiate T cell recall proliferation response. Collectively, these observations may provide new insights into understanding the biology of TmTNF, mode of action of anti-TNFs, biology of ADA response to anti-TNFs, and may help with the design of the next generation of anti-TNFs.

  5. Cystic fibrosis transmembrane conductance regulator intracellular processing, trafficking, and opportunities for mutation-specific treatment.

    LENUS (Irish Health Repository)

    Rogan, Mark P

    2012-02-01

    Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease?

  6. Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

    Science.gov (United States)

    Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David

    2015-06-01

    Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia.

    Science.gov (United States)

    Yaffe, Yakey; Shepshelovitch, Jeanne; Nevo-Yassaf, Inbar; Yeheskel, Adva; Shmerling, Hedva; Kwiatek, Joanna M; Gaus, Katharina; Pasmanik-Chor, Metsada; Hirschberg, Koret

    2012-08-01

    Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

  8. Self-Assembling Organic Nanopores as Synthetic Transmembrane Channels with Tunable Functions

    Science.gov (United States)

    Wei, Xiaoxi

    A long-standing goal in the area of supramolecular self-assembly involves the development of synthetic ion/water channels capable of mimicking the mass-transport characteristics of biological channels and pores. Few examples of artificial transmembrane channels with large lumen, high conductivity and selectivity are known. A review of pronounced biological transmembrane protein channels and some representative synthetic models have been provided in Chapter 1, followed by our discovery and initial investigation of shape-persistent oligoamide and phenylene ethynylene macrocycles as synthetic ion/water channels. In Chapter 2, the systematic structural modification of oligoamide macrocycles 1, the so-called first-generation of these shape-persistent macrocycles, has led to third-generation macrocycles 3. The third generation was found to exhibit unprecedented, strong intermolecular association in both the solid state and solution via multiple techniques including X-ray diffraction (XRD), SEM, and 1H NMR. Fluorescence spectroscopy paired with dynamic light scattering (DLS) revealed that macrocycles 3 can assemble into a singly dispersed nanotubular structure in solution. The resultant self-assembling pores consisting of 3 were examined by HPTS-LUVs assays and BLM studies (Chapter 3) and found to form cation-selective (PK+/PCl- = 69:1) transmembrane ion channels with large conductance (200 ˜ 2000 pS for alkali cations) and high stability with open times reaching to 103 seconds. Tuning the aggregation state of macrocycles by choosing an appropriate polar solvent mixture (i.e., 3:1, THF:DMF, v/v) and concentration led to the formation of ion channels with well-defined square top behavior. A parallel study using DLS to examine the size of aggregates was used in conjunction with channel activity assays (LUVs/BLM) to reveal the effects of the aggregation state on channel activity. Empirical evidence now clearly indicates that a preassembled state, perhaps that of a

  9. The PEST sequence does not contribute to the stability of the cystic fibrosis transmembrane conductance regulator.

    Science.gov (United States)

    Chen, Eva Y; Clarke, David M

    2002-10-02

    Endoplasmic reticulum retention of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) mutants and their rapid degradation is the major cause of cystic fibrosis (CF). An important goal is to understand the mechanism of how the misfolded proteins are recognized, retained, and targeted for degradation. Using a web-based algorithm, PESTFind, we found a PEST sequence in the regulatory (R) domain of CFTR. The PEST sequence is found in many short-lived eukaryotic proteins and plays a role in their degradation. To determine its role in the stability and degradation of misprocessed CFTR, we introduced a number of site-directed mutations into the PEST sequence in the cDNA of DeltaF508 CFTR, the most prevalent misprocessed mutation found in CF patients. Analysis of these mutants showed that the disruption of the PEST sequence plays a minor role in the degradation of the CFTR mutants. Multiple mutations to the PEST sequence within the R domain of CFTR inhibit maturation of CFTR and prevent the formation of a 100 kDa degradation product. The mutations, however, do not improve the stability of the mutant DeltaF508 CFTR. These observations show that disruption of the structure of the R domain of CFTR can inhibit maturation of the protein and that the predicted PEST sequence plays no significant role in the degradation of CFTR.

  10. The PEST sequence does not contribute to the stability of the cystic fibrosis transmembrane conductance regulator

    Directory of Open Access Journals (Sweden)

    Clarke David M

    2002-10-01

    Full Text Available Abstract Background Endoplasmic reticulum retention of misfolded cystic fibrosis transmembrane conductance regulator (CFTR mutants and their rapid degradation is the major cause of cystic fibrosis (CF. An important goal is to understand the mechanism of how the misfolded proteins are recognized, retained, and targeted for degradation. Results Using a web-based algorithm, PESTFind, we found a PEST sequence in the regulatory (R domain of CFTR. The PEST sequence is found in many short-lived eukaryotic proteins and plays a role in their degradation. To determine its role in the stability and degradation of misprocessed CFTR, we introduced a number of site-directed mutations into the PEST sequence in the cDNA of ΔF508 CFTR, the most prevalent misprocessed mutation found in CF patients. Analysis of these mutants showed that the disruption of the PEST sequence plays a minor role in the degradation of the CFTR mutants. Multiple mutations to the PEST sequence within the R domain of CFTR inhibit maturation of CFTR and prevent the formation of a 100 kDa degradation product. The mutations, however, do not improve the stability of the mutant ΔF508 CFTR. Conclusion These observations show that disruption of the structure of the R domain of CFTR can inhibit maturation of the protein and that the predicted PEST sequence plays no significant role in the degradation of CFTR.

  11. Transmembrane prostatic acid phosphatase (TMPAP interacts with snapin and deficient mice develop prostate adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Ileana B Quintero

    Full Text Available The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP, a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930's. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP and a transmembrane type-I (TMPAP. The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP(-/- with C57BL/6J background. The PAP(-/- mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.

  12. Regulation of KV channel voltage-dependent activation by transmembrane β subunits

    Directory of Open Access Journals (Sweden)

    Xiaohui eSun

    2012-04-01

    Full Text Available Voltage-activated K+ (KV channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD surrounded by four voltage-sensing domains (VSD. The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

  13. BuD, a helix–loop–helix DNA-binding domain for genome modification

    Energy Technology Data Exchange (ETDEWEB)

    Stella, Stefano [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark); Molina, Rafael; López-Méndez, Blanca [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Juillerat, Alexandre; Bertonati, Claudia; Daboussi, Fayza [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Campos-Olivas, Ramon [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); Duchateau, Phillippe [Cellectis, 8 Rue de la Croix Jarry, 75013 Paris (France); Montoya, Guillermo, E-mail: guillermo.montoya@cpr.ku.dk [Spanish National Cancer Research Centre (CNIO), Calle de Melchor Fernández Almagro 3, 28029 Madrid (Spain); University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen (Denmark)

    2014-07-01

    Crystal structures of BurrH and the BurrH–DNA complex are reported. DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

  14. Does unpaired adenosine-66 from helix II of Escherichia coli 5S RNA bind to protein L18?

    DEFF Research Database (Denmark)

    Christiansen, J; Douthwaite, S R; Christensen, A

    1985-01-01

    plasmid derived from pKK3535. Binding studies with protein L18 revealed that the protein bound much more weakly to the mutated 5S RNA. We consider the most likely explanation of this result is that L18 interacts with adenosine-66, and we present a tentative model for an interaction between the unpaired......Adenosine-66 is unpaired within helix II of Escherichia coli 5S RNA and lies in the binding site of ribosomal protein L18. It has been proposed as a recognition site for protein L18. We have investigated further the structural importance of this nucleotide by deleting it. The 5S RNA gene of the rrn...... adenosine and the adjacent guanosine-67 of the RNA and glutamine-19 of the protein L18....

  15. Golden Helix Institute of Biomedical Research: Interdisciplinary research and educational activities in pharmacogenomics and personalized medicine

    Science.gov (United States)

    Mitropoulos, Konstantinos; Innocenti, Federico; van Schaik, Ron H.; Lezhava, Alexander; Tzimas, Giannis; Kollia, Panagoula; Macek, Milan; Fortina, Paolo; Patrinos, George P.

    2013-01-01

    The Golden Helix Institute of Biomedical Research is an international non-profit scientific organization with interdisciplinary research and educational activities in the field of genome medicine in Europe, Asia and Latin America. These activities are supervised by an international scientific advisory council, consisting of world leaders in the field of genomics and translational medicine. Research activities include the regional coordination of the Pharmacogenomics for Every Nation Initiative in Europe, in an effort to integrate pharmacogenomics in developing countries, the development of several National/Ethnic Genetic databases and related web services and the critical assessment of the impact of genetics and genomic medicine to society in various countries. Also, educational activities include the organization of the Golden Helix Symposia®, which are high profile scientific research symposia in the field of personalized medicine, and the Golden Helix Pharmacogenomics Days, an international educational activity focused on pharmacogenomics, as part of its international pharmacogenomics education and outreach efforts. PMID:22379996

  16. Crystal structure of the[mu]-opioid receptor bound to a morphinan antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Mathiesen, Jesper M.; Sunahara, Roger K.; Pardo, Leonardo; Weis, William I.; Kobilka, Brian K.; Granier, Sébastien (Michigan-Med); (Stanford-MED); (UAB, Spain)

    2012-06-27

    Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled {mu}-opioid receptor ({mu}-OR) in the central nervous system. Here we describe the 2.8 {angstrom} crystal structure of the mouse {mu}-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the {mu}-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

  17. Structural basis for KCNE3 modulation of potassium recycling in epithelia

    Science.gov (United States)

    Kroncke, Brett M.; Van Horn, Wade D.; Smith, Jarrod; Kang, CongBao; Welch, Richard C.; Song, Yuanli; Nannemann, David P.; Taylor, Keenan C.; Sisco, Nicholas J.; George, Alfred L.; Meiler, Jens; Vanoye, Carlos G.; Sanders, Charles R.

    2016-01-01

    The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K+) channel to enable K+ recycling coupled to transepithelial chloride ion (Cl−) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K+ recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated “up” state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the “CF gender gap.” PMID:27626070

  18. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction

    Science.gov (United States)

    Höög, Johanna L.; Lacomble, Sylvain; Bouchet-Marquis, Cedric; Briggs, Laura; Park, Kristin; Hoenger, Andreas; Gull, Keith

    2016-01-01

    Background Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC) is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility. Methodology/Principal Findings We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum. Conclusions/Significance The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life

  19. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

    Directory of Open Access Journals (Sweden)

    Johanna L Höög

    2016-01-01

    Full Text Available Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

  20. Transmembrane-sequence-dependent overexpression and secretion of glycoproteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Schuster, M; Wasserbauer, E; Aversa, G; Jungbauer, A

    2001-02-01

    Protein expression using the secretory pathway in Saccharomyces cerevisiae can lead to high amounts of overexpressed and secreted proteins in culture supernatants in a short period of time. These post-translational modified expression products can be purified up to >90% in a single step. The overexpression and secretion of the transmembrane glycoprotein signaling lymphocytic activation molecule (SLAM) was studied. SLAM belongs to the immunoglobulin superfamily and its engagement results in T-cell expansion and INF-gamma production. The molecule is composed of an extracellular, a single-span transmembrane and a cytoplasmatic domain. The extracellular part may be relevant for stimulation studies in vitro since SLAM is a high-affinity self-ligand. Therefore several fragments of this region have been expressed as Flag-fusions in S. cerevisiae: a full-length fragment containing the transmembrane region and the autologous signal sequence, another without the transmembrane region, and two fragments without the autologous signal sequence with and without the transmembrane region. By molecular cloning, the different deletion mutants of the cDNA encoding the full-length construct have been inserted in a yeast episomal plasmid. Upstream of the cDNA, the alpha-leader sequence of a yeast mating pheromone has been cloned to direct the fusion proteins into the secretory protein maturation pathway. All four fragments were expressed but yield, location, and maturation were highly influenced by the transmembrane domain and the autologous signal sequence. Only the fragment without autologous signal sequence and transmembrane domain could be efficiently secreted. High-mannose glycosylation was analyzed by lectin mapping and digestion with specific glycosidases. After enzyme treatment, a single band product with the theoretical size could be detected and identified as SLAM by a specific monoclonal antibody. The fusion protein concentration in the supernatant was 30 microg/ml. The

  1. Effects of hydrophobic helix length and side chain chemistry on biomimicry in peptoid analogues of SP-C.

    Science.gov (United States)

    Brown, Nathan J; Wu, Cindy W; Seurynck-Servoss, Shannon L; Barron, Annelise E

    2008-02-12

    The hydrophobic proteins of lung surfactant (LS), SP-B and SP-C, are critical constituents of an effective surfactant replacement therapy for the treatment of respiratory distress syndrome. Because of concerns and difficulties associated with animal-derived surfactants, recent investigations have focused on the creation of synthetic analogues of the LS proteins. However, creating an accurate mimic of SP-C that retains its biophysical surface activity is extraordinarily challenging given the lipopeptide's extreme hydrophobicity and propensity to misfold and aggregate. One successful approach that overcomes these difficulties is the use of poly-N-substituted glycines, or peptoids, to mimic SP-C. To develop a non-natural, bioactive mimic of SP-C and to investigate the effects of side chain chemistry and length of the helical hydrophobic region, we synthesized, purified, and performed in vitro testing of two classes of peptoid SP-C mimics: those having a rigid alpha-chiral aromatic helix and those having a biomimetic alpha-chiral aliphatic helix. The length of the two classes of mimics was also systematically altered. Circular dichroism spectroscopy gave evidence that all of the peptoid-based mimics studied here emulated SP-C's secondary structure, forming stable helical structures in solution. Langmuir-Wilhelmy surface balance, fluorescence microscopy, and pulsating bubble surfactometry experiments provide evidence that the aromatic-based SP-C peptoid mimics, in conjunction with a synthetic lipid mixture, have superior surface activity and biomimetic film morphology in comparison to the aliphatic-based mimics and that there is an increase in surface activity corresponding to increasing helical length.

  2. The Quadruple Helix Model Enhancing Innovative Performance Of Indonesian Creative Industry

    Directory of Open Access Journals (Sweden)

    Sri Wahyu Lelly Hana Setyanti

    2017-11-01

    Full Text Available The creative industry in Indonesia has contributed positively to the national economic growth. Creative industry grows from the creativity and innovation performance of the business actors. The challenge of creative industry is how to completely understand the creative and innovative processes in business management. Therefore it requires an approach that combines the synergy between academicians entrepreneurs government and society in a quadruple helix model. The objective of this research is to develop a creativity model through a quadruple helix model in improving innovation performance of the creative industry.

  3. Neurotoxic and Lipidic peroxidation effect of metal dust and Cadmium on Helix aspersa

    Directory of Open Access Journals (Sweden)

    GRARA Nedjoud

    2017-04-01

    Full Text Available In this study we were interested in the evaluation of the impact of the metal dust collected on the level of the iron and steel complex of EL-Hadjar and the Cadmium which is regarded as the most toxic pollutant, most widespread in the environment of the zones to strong human activities and their effects on organizations bioaccumulator and bio indicator of pollution Helix aspersa. With regard to the bio markers we highlighted a reduction in the AChE activity on the level of the head. In addition, the exposure of Helix aspersa to metal dust and Cadmium induces a lipidic peroxidation with release of (MDA.

  4. Gate-controlled switching between persistent and inverse persistent spin helix states

    International Nuclear Information System (INIS)

    Yoshizumi, K.; Sasaki, A.; Kohda, M.; Nitta, J.

    2016-01-01

    We demonstrate gate-controlled switching between persistent spin helix (PSH) state and inverse PSH state, which are detected by quantum interference effect on magneto-conductance. These special symmetric spin states showing weak localization effect give rise to a long spin coherence when the strength of Rashba spin-orbit interaction (SOI) is close to that of Dresselhaus SOI. Furthermore, in the middle of two persistent spin helix states, where the Rashba SOI can be negligible, the bulk Dresselhaus SOI parameter in a modulation doped InGaAs/InAlAs quantum well is determined.

  5. Gate-controlled switching between persistent and inverse persistent spin helix states

    Energy Technology Data Exchange (ETDEWEB)

    Yoshizumi, K.; Sasaki, A.; Kohda, M.; Nitta, J. [Department of Materials Science, Tohoku University, Sendai 980-8579 (Japan)

    2016-03-28

    We demonstrate gate-controlled switching between persistent spin helix (PSH) state and inverse PSH state, which are detected by quantum interference effect on magneto-conductance. These special symmetric spin states showing weak localization effect give rise to a long spin coherence when the strength of Rashba spin-orbit interaction (SOI) is close to that of Dresselhaus SOI. Furthermore, in the middle of two persistent spin helix states, where the Rashba SOI can be negligible, the bulk Dresselhaus SOI parameter in a modulation doped InGaAs/InAlAs quantum well is determined.

  6. The signaling helix: a common functional theme in diverse signaling proteins

    Directory of Open Access Journals (Sweden)

    Aravind L

    2006-09-01

    Full Text Available Abstract Background The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood. Results Using sensitive sequence analysis methods we identify a conserved helical segment of around 40 residues in a wide range of signaling proteins, including numerous sensor histidine kinases such as Sln1p, and receptor guanylyl cyclases such as the atrial natriuretic peptide receptor and nitric oxide receptors. We term this helical segment the signaling (S-helix and present evidence that it forms a novel parallel coiled-coil element, distinct from previously known helical segments in signaling proteins, such as the Dimerization-Histidine phosphotransfer module of histidine kinases, the intra-cellular domains of the chemotaxis receptors, inter-GAF domain helical linkers and the α-helical HAMP module. Analysis of domain architectures allowed us to reconstruct the domain-neighborhood graph for the S-helix, which showed that the S-helix almost always occurs between two signaling domains. Several striking patterns in the domain neighborhood of the S-helix also became evident from the graph. It most often separates diverse N-terminal sensory domains from various C-terminal catalytic signaling domains such as histidine kinases, cNMP cyclase, PP2C phosphatases, NtrC-like AAA+ ATPases and diguanylate cyclases. It might also occur between two sensory domains such as PAS domains and occasionally between a DNA-binding HTH domain and a sensory domain. The sequence conservation pattern of the S-helix revealed the presence of a unique constellation of polar residues in the dimer-interface positions within the central heptad of the coiled-coil formed by the S-helix. Conclusion Combining these observations with previously reported mutagenesis studies on different S-helix-containing proteins we suggest that it functions as a switch that prevents constitutive activation of linked downstream

  7. PH4 of petunia is an R2R3-MYB protein that activates vacuolar acidification through interactions with Basic-Helix-Loop-Helix transcription factors of the anthocyanin pathway.

    NARCIS (Netherlands)

    Quattrocchio, F.M.; Verweij, C.W.; Spelt, C.E.; Mol, J.N.M.; Koes, R.E.

    2007-01-01

    The Petunia hybrids genes ANTHOCYANIN1 (AN1) and AN2 encode transcription factors with a basic-helix-loop-helix (BHLH) and a MYB domain, respectively, that are required for anthocyanin synthesis and acidification of the vacuole in petal cells. Mutation of PH4 results in a bluer flower color,

  8. Effect of temperature on DNA double helix: An insight from ...

    Indian Academy of Sciences (India)

    The three-dimensional structure of DNA contains various sequence-dependent structural information, which control many cellular processes in life, such as replication, transcription, DNA repair, etc. For the above functions, DNA double helices need to unwind or melt locally, which is different from terminal melting, as often ...

  9. Identification of SNARE complex modulators that inhibit exocytosis from an alpha-helix-constrained combinatorial library.

    Science.gov (United States)

    Blanes-Mira, Clara; Pastor, Maria T; Valera, Elvira; Fernández-Ballester, Gregorio; Merino, Jaime M; Gutierrez, Luis M; Perez-Payá, Enrique; Ferrer-Montiel, Antonio

    2003-01-01

    Synthetic peptides patterned after the proteins involved in vesicle fusion [the so-called SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins] are potent inhibitors of SNARE complex assembly and neuronal exocytosis. It is noteworthy that the identification of peptide sequences not related to the SNARE proteins has not been accomplished yet; this is due, in part, to the structural constraints and the specificity of the protein interactions that govern the formation of the SNARE complex. Here we have addressed this question and used a combinatorial approach to identify peptides that modulate the assembly of the SNARE core complex and inhibit neuronal exocytosis. An alpha-helix-constrained, mixture-based, 17-mer combinatorial peptide library composed of 137180 sequences was synthesized in a positional scanning format. Peptide mixtures were assayed for their ability to prevent the formation of the in vitro -reconstituted SDS-resistant SNARE core complex. Library deconvolution identified eight peptides that inhibited the assembly of the SNARE core complex. Notably, the most potent 17-mer peptide (acetyl-SAAEAFAKLYAEAFAKG-NH2) abolished both Ca2+-evoked catecholamine secretion from detergent-permeabilized chromaffin cells and L-glutamate release from intact hippocampal primary cultures. Collectively, these findings indicate that amino acid sequences that prevent SNARE complex formation are not restricted to those that mimic domains of SNARE proteins, thus expanding the diversity of molecules that target neuronal exocytosis. Because of the implication of neurosecretion in the aetiology of several human neurological disorders, these newly identified peptides may be considered hits for the development of novel anti-spasmodic drugs. PMID:12852787

  10. HMMpTM: improving transmembrane protein topology prediction using phosphorylation and glycosylation site prediction.

    Science.gov (United States)

    Tsaousis, Georgios N; Bagos, Pantelis G; Hamodrakas, Stavros J

    2014-02-01

    During the last two decades a large number of computational methods have been developed for predicting transmembrane protein topology. Current predictors rely on topogenic signals in the protein sequence, such as the distribution of positively charged residues in extra-membrane loops and the existence of N-terminal signals. However, phosphorylation and glycosylation are post-translational modifications (PTMs) that occur in a compartment-specific manner and therefore the presence of a phosphorylation or glycosylation site in a transmembrane protein provides topological information. We examine the combination of phosphorylation and glycosylation site prediction with transmembrane protein topology prediction. We report the development of a Hidden Markov Model based method, capable of predicting the topology of transmembrane proteins and the existence of kinase specific phosphorylation and N/O-linked glycosylation sites along the protein sequence. Our method integrates a novel feature in transmembrane protein topology prediction, which results in improved performance for topology prediction and reliable prediction of phosphorylation and glycosylation sites. The method is freely available at http://bioinformatics.biol.uoa.gr/HMMpTM. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Expression of Trans-Membrane Proteins in vitro Using a Cell Free System

    Science.gov (United States)

    Weisse, Natalie; Noireaux, Vincent; Chalmeau, Jerome

    2010-10-01

    Trans-membrane proteins represent a significant portion of the proteins expressed by cells. The expression of proteins in vitro, however, remains a challenge. Numerous expression approaches have been developed with cell free expression (CFE) being one of the most promising. CFE is based on a transcription-translation system that has been extracted from E. coli bacteria. Adding the desired DNA allows expression of a selected protein, and in the presence of phospholipids the expression of trans-membrane proteins becomes possible. In order to express trans-membrane proteins in a closed native environment, the cell free system (CFS) is encapsulated with a phospholipid bilayer, creating an artificial cell. To verify protein expression, AquaporinZ (AqpZ), a well-known trans-membrane protein tagged with a green fluorescent protein (eGFP), was used so the expressed proteins could be seen under a fluorescent microscope. These artificial cells will serve as an experimental platform for testing the viability of the expressed trans-membrane proteins. Results from the manipulation of these artificial cells by attaching them to the slide surface through streptavidin-biotin bonding will be presented.

  12. Transmembrane domain quality control systems operate at the endoplasmic reticulum and Golgi apparatus.

    Science.gov (United States)

    Briant, Kit; Johnson, Nicholas; Swanton, Eileithyia

    2017-01-01

    Multiple protein quality control systems operate to ensure that misfolded proteins are efficiently cleared from the cell. While quality control systems that assess the folding status of soluble domains have been extensively studied, transmembrane domain (TMD) quality control mechanisms are poorly understood. Here, we have used chimeras based on the type I plasma membrane protein CD8 in which the endogenous TMD was substituted with transmembrane sequences derived from different polytopic membrane proteins as a mode to investigate the quality control of unassembled TMDs along the secretory pathway. We find that the three TMDs examined prevent trafficking of CD8 to the cell surface via potentially distinct mechanisms. CD8 containing two distinct non-native transmembrane sequences escape the ER and are subsequently retrieved from the Golgi, possibly via Rer1, leading to ER localisation at steady state. A third chimera, containing an altered transmembrane domain, was predominantly localised to the Golgi at steady state, indicating the existence of an additional quality control checkpoint that identifies non-native transmembrane domains that have escaped ER retention and retrieval. Preliminary experiments indicate that protein retained by quality control mechanisms at the Golgi are targeted to lysosomes for degradation.

  13. DNA binding domains and nuclear localization signal of LEDGF: contribution of two helix-turn-helix (HTH)-like domains and a stretch of 58 amino acids of the N-terminal to the trans-activation potential of LEDGF.

    Science.gov (United States)

    Singh, Dhirendra P; Kubo, E; Takamura, Y; Shinohara, T; Kumar, A; Chylack, Leo T; Fatma, N

    2006-01-20

    Lens epithelium derived growth factor (LEDGF), a nuclear protein, plays a role in regulating the transcription of stress-associated genes such as heat shock proteins by binding to consensus core DNA sequences nAGGn or nGAAn or their repeats, and in doing so helps to provide cyto-protection. However, additional information is required to identify the specific structural features of LEDGF involved in gene transcription. Here we have investigated the functional domains activating and repressing DNA-binding modules, by using a DNA binding assay and trans-activation experiments performed by analyzing proteins prepared from deletion constructs. The results disclosed the DNA-binding domain of N-terminal LEDGF mapped between amino acid residues 5 and 62, a 58 amino acid residue stretch PWWP domain which binds to stress response elements (STRE; A/TGGGGA/T). C-terminal LEDGF contains activation domains, an extensive loop-region (aa 418-530) with two helix-turn-helix (HTH)-like domains, and binds to a heat shock element (HSE; nGAAn). A trans-activation assay using Hsp27 promoter revealed that both HTH domains contribute in a cooperative manner to the trans-activation potential of LEDGF. Interestingly, removal of N-terminal LEDGF (aa 1-187) significantly enhances the gene activation potential of C-terminal LEDGF (aa 199-530); thus the N-terminal domain (aa 5-62), exhibits auto-transcriptional repression activity. It appears that this domain is involved in stabilizing the LEDGF-DNA binding complex. Collectively, our results demonstrate that LEDGF contains three DNA-binding domains, which regulate gene expression depending on cellular microenvironment and thus modify the physiology of cells to maintain cellular homeostasis.

  14. Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

    Science.gov (United States)

    Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

    2017-04-07

    Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Cystic fibrosis transmembrane conductance regulator chloride channel blockers: Pharmacological, biophysical and physiological relevance.

    Science.gov (United States)

    Linsdell, Paul

    2014-02-26

    Dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel causes cystic fibrosis, while inappropriate activity of this channel occurs in secretory diarrhea and polycystic kidney disease. Drugs that interact directly with CFTR are therefore of interest in the treatment of a number of disease states. This review focuses on one class of small molecules that interacts directly with CFTR, namely inhibitors that act by directly blocking chloride movement through the open channel pore. In theory such compounds could be of use in the treatment of diarrhea and polycystic kidney disease, however in practice all known substances acting by this mechanism to inhibit CFTR function lack either the potency or specificity for in vivo use. Nevertheless, this theoretical pharmacological usefulness set the scene for the development of more potent, specific CFTR inhibitors. Biophysically, open channel blockers have proven most useful as experimental probes of the structure and function of the CFTR chloride channel pore. Most importantly, the use of these blockers has been fundamental in developing a functional model of the pore that includes a wide inner vestibule that uses positively charged amino acid side chains to attract both permeant and blocking anions from the cell cytoplasm. CFTR channels are also subject to this kind of blocking action by endogenous anions present in the cell cytoplasm, and recently this blocking effect has been suggested to play a role in the physiological control of CFTR channel function, in particular as a novel mechanism linking CFTR function dynamically to the composition of epithelial cell secretions. It has also been suggested that future drugs could target this same pathway as a way of pharmacologically increasing CFTR activity in cystic fibrosis. Studying open channel blockers and their mechanisms of action has resulted in significant advances in our understanding of CFTR as a pharmacological target in disease

  16. Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors.

    Science.gov (United States)

    Horani, Suzzane; Stater, Evan P; Corringer, Pierre-Jean; Trudell, James R; Harris, R Adron; Howard, Rebecca J

    2015-06-01

    Mutagenesis and labeling studies have identified amino acids from the human α1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol (EtOH) potentiation. However, limited high-resolution structural data for physiologically relevant receptors in this Cys-loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys-loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand-gated ion channel (GLIC) produced EtOH and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for EtOH binding to GLIC. We directly compared EtOH modulation of the α1 GlyR and GLIC to a chimera containing the TM domain from human α1 GlyRs and the ligand-binding domain of GLIC using 2-electrode voltage-clamp electrophysiology of receptors expressed in Xenopus laevis oocytes. EtOH potentiated α1 GlyRs in a concentration-dependent manner in the presence of zinc-chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the EtOH potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50 μM picrotoxin to the media, a technique that may have applications in expression of other ion channels. Our results are consistent with a TM mechanism of EtOH modulation in Cys-loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species. Copyright © 2015 by the Research Society on Alcoholism.

  17. Delivering Transmembrane Peptide Complexes to the Gas Phase Using Nanodiscs and Electrospray Ionization

    Science.gov (United States)

    Li, Jun; Richards, Michele R.; Kitova, Elena N.; Klassen, John S.

    2017-10-01

    The gas-phase conformations of dimers of the channel-forming membrane peptide gramicidin A (GA), produced from isobutanol or aqueous solutions of GA-containing nanodiscs (NDs), are investigated using electrospray ionization-ion mobility separation-mass spectrometry (ESI-IMS-MS) and molecular dynamics (MD) simulations. The IMS arrival times measured for (2GA + 2Na)2+ ions from isobutanol reveal three different conformations, with collision cross-sections (Ω) of 683 Å2 (conformation 1, C1), 708 Å2 (C2), and 737 Å2 (C3). The addition of NH4CH3CO2 produced (2GA + 2Na)2+ and (2GA + H + Na)2+ ions, with Ω similar to those of C1, C2, and C3, as well as (2GA + 2H)2+, (2GA + 2NH4)2+, and (2GA + H + NH4)2+ ions, which adopt a single conformation with a Ω similar to that of C2. These results suggest that the nature of the charging agents, imparted by the ESI process, can influence dimer conformation in the gas phase. Notably, the POPC NDs produced exclusively (2GA + 2NH4)2+ dimer ions; the DMPC NDs produced both (2GA + 2H)2+ and (2GA + 2NH4)2+ dimer ions. While the Ω of (2GA + 2H)2+ is similar to that of C2, the (2GA + 2NH4)2+ ions from NDs adopt a more compact structure, with a Ω of 656 Å2. It is proposed that this compact structure corresponds to the ion conducting single stranded head-to-head helical GA dimer. These findings highlight the potential of NDs, combined with ESI, for transferring transmembrane peptide complexes directly from lipid bilayers to the gas phase. [Figure not available: see fulltext.

  18. SAXS analysis of a soluble cytosolic NgBR construct including extracellular and transmembrane domains.

    Directory of Open Access Journals (Sweden)

    Joshua Holcomb

    Full Text Available The Nogo-B receptor (NgBR is involved in oncogenic Ras signaling through directly binding to farnesylated Ras. It recruits farnesylated Ras to the non-lipid-raft membrane for interaction with downstream effectors. However, the cytosolic domain of NgBR itself is only partially folded. The lack of several conserved secondary structural elements makes this domain unlikely to form a complete farnesyl binding pocket. We find that inclusion of the extracellular and transmembrane domains that contain additional conserved residues to the cytosolic region results in a well folded protein with a similar size and shape to the E.coli cis-isoprenyl transferase (UPPs. Small Angle X-ray Scattering (SAXS analysis reveals the radius of gyration (Rg of our NgBR construct to be 18.2 Å with a maximum particle dimension (Dmax of 61.0 Å. Ab initio shape modeling returns a globular molecular envelope with an estimated molecular weight of 23.0 kD closely correlated with the calculated molecular weight. Both Kratky plot and pair distribution function of NgBR scattering reveal a bell shaped peak which is characteristic of a single globularly folded protein. In addition, circular dichroism (CD analysis reveals that our construct has the secondary structure contents similar to the UPPs. However, this result does not agree with the currently accepted topological orientation of NgBR which might partition this construct into three separate domains. This discrepancy suggests another possible NgBR topology and lends insight into a potential molecular basis of how NgBR facilitates farnesylated Ras recruitment.

  19. Rotational symmetry and the transformation of innovation systems in a Triple Helix of university-industry-government relations

    NARCIS (Netherlands)

    Ivanova, I.A.; Leydesdorff, L.

    2014-01-01

    Using a mathematical model, we show that a Triple Helix (TH) system contains self-interaction, and therefore self-organization of innovations can be expected in waves, whereas a Double Helix (DH) remains determined by its linear constituents. (The mathematical model is fully elaborated in the

  20. Secreted and Transmembrane αKlotho Isoforms Have Different Spatio-Temporal Profiles in the Brain during Aging and Alzheimer's Disease Progression.

    Directory of Open Access Journals (Sweden)

    Anna Massó

    Full Text Available The Klotho protein is a β-glucuronidase, and its overexpression is associated with life extension. Its mechanism of action is not fully understood, although it has been recently reported that αKlotho improves synaptic and cognitive functions, and it may also influence a variety of structures and functions during CNS maturation and aging. The αKlotho gene has two transcripts, one encoding a transmembrane isoform (m-KL, and the other a putative secreted isoform (s-KL. Unfortunately, little is known about the secreted αKlotho isoform, since available antibodies cannot discriminate s-KL from the KL1 domain cleaved from the transmembrane isoform. This study shows, for the first time, that the klotho transcript produced by alternative splicing generates a stable protein (70 kDa, and that in contrast to the transmembrane Klotho isoform, it is ten times more abundant in the brain than in the kidney suggesting that the two isoforms may have different functions. We also studied whether klotho expression in the CNS was influenced by aging, Alzheimer's disease (AD, or a healthy lifestyle, such as voluntary moderate continuous exercise. We observed a strong correlation between high expression levels of the two klotho transcripts and the healthy status of the animals. Expression of Klotho in brain areas decayed more rapidly in the 3xTg-AD model of AD than in healthy animals, whilst moderate continuous exercise in adulthood prevents the decline in expression of both klotho transcripts.

  1. Secreted and Transmembrane αKlotho Isoforms Have Different Spatio-Temporal Profiles in the Brain during Aging and Alzheimer's Disease Progression

    Science.gov (United States)

    Massó, Anna; Sánchez, Angela; Gimenez-Llort, Lydia; Lizcano, Jose Miguel; Cañete, Manuel; García, Belen; Torres-Lista, Virginia; Puig, Meritxell; Bosch, Assumpció; Chillon, Miguel

    2015-01-01

    The Klotho protein is a β-glucuronidase, and its overexpression is associated with life extension. Its mechanism of action is not fully understood, although it has been recently reported that αKlotho improves synaptic and cognitive functions, and it may also influence a variety of structures and functions during CNS maturation and aging. The αKlotho gene has two transcripts, one encoding a transmembrane isoform (m-KL), and the other a putative secreted isoform (s-KL). Unfortunately, little is known about the secreted αKlotho isoform, since available antibodies cannot discriminate s-KL from the KL1 domain cleaved from the transmembrane isoform. This study shows, for the first time, that the klotho transcript produced by alternative splicing generates a stable protein (70 kDa), and that in contrast to the transmembrane Klotho isoform, it is ten times more abundant in the brain than in the kidney suggesting that the two isoforms may have different functions. We also studied whether klotho expression in the CNS was influenced by aging, Alzheimer's disease (AD), or a healthy lifestyle, such as voluntary moderate continuous exercise. We observed a strong correlation between high expression levels of the two klotho transcripts and the healthy status of the animals. Expression of Klotho in brain areas decayed more rapidly in the 3xTg-AD model of AD than in healthy animals, whilst moderate continuous exercise in adulthood prevents the decline in expression of both klotho transcripts. PMID:26599613

  2. Poisson-Nernst-Planck models of nonequilibrium ion electrodiffusion through a protegrin transmembrane pore.

    Directory of Open Access Journals (Sweden)

    Dan S Bolintineanu

    2009-01-01

    Full Text Available Protegrin peptides are potent antimicrobial agents believed to act against a variety of pathogens by forming nonselective transmembrane pores in the bacterial cell membrane. We have employed 3D Poisson-Nernst-Planck (PNP calculations to determine the steady-state ion conduction characteristics of such pores at applied voltages in the range of -100 to +100 mV in 0.1 M KCl bath solutions. We have tested a variety of pore structures extracted from molecular dynamics (MD simulations based on an experimentally proposed octomeric pore structure. The computed single-channel conductance values were in the range of 290-680 pS. Better agreement with the experimental range of 40-360 pS was obtained using structures from the last 40 ns of the MD simulation, where conductance values range from 280 to 430 pS. We observed no significant variation of the conductance with applied voltage in any of the structures that we tested, suggesting that the voltage dependence observed experimentally is a result of voltage-dependent channel formation rather than an inherent feature of the open pore structure. We have found the pore to be highly selective for anions, with anionic to cationic current ratios (I(Cl-/I(K+ on the order of 10(3. This is consistent with the highly cationic nature of the pore but surprisingly in disagreement with the experimental finding of only slight anionic selectivity. We have additionally tested the sensitivity of our PNP model to several parameters and found the ion diffusion coefficients to have a significant influence on conductance characteristics. The best agreement with experimental data was obtained using a diffusion coefficient for each ion set to 10% of the bulk literature value everywhere inside the channel, a scaling used by several other studies employing PNP calculations. Overall, this work presents a useful link between previous work focused on the structure of protegrin pores and experimental efforts aimed at investigating their

  3. Control of transmembrane protein diffusion within the postsynaptic density assessed by simultaneous single-molecule tracking and localization microscopy

    Directory of Open Access Journals (Sweden)

    Thomas A Blanpied

    2016-07-01

    Full Text Available Postsynaptic transmembrane proteins are critical elements of synapses, mediating trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules, and flux of Ca and other ions. Positioning and mobility of each member of this large class of proteins is critical to their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out of the synapse is critical for activity-dependent plasticity. However, factors that control receptor mobility within the boundaries of the synapse are not well understood. Notably, PSD scaffold molecules accumulate in domains much smaller than the synapse. Within these nanodomains, the density of proteins is considerably higher than that of the synapse as a whole, so high that steric hindrance is expected to reduce receptor mobility substantially. However, while numerical modeling has demonstrated several features of how the varying protein density across the face of a single PSD may modulate receptor motion, there is little experimental information about the extent of this influence. To address this critical aspect of synaptic organizational dynamics, we performed single-molecule tracking of transmembrane proteins using uPAINT over PSDs whose internal structure was simultaneously resolved using PALM. The results provide important experimental confirmation that PSD scaffold density protein strongly influences the mobility of transmembrane proteins. Tracking a protein with a cytosolic domain that does not bind PSD-95 still was slowed in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize receptors even in the absence of binding. Because numerous proteins thought to be

  4. Equilibrium shift in solution: molecular shape recognition and precipitation of a synthetic double helix using helicene-grafted silica nanoparticles.

    Science.gov (United States)

    Miyagawa, Masamichi; Ichinose, Wataru; Yamaguchi, Masahiko

    2014-01-27

    Chiral silica nanoparticles (70 nm) grafted with (P)-helicene recognized the molecular shape of double helix and random coil (P)-ethynylhelicene oligomers in solution. A mixture of the (P)-nanoparticles and double helix precipitated much faster than a mixture of the (P)-nanoparticles and random coil, and the precipitate contained only the double helix. The mixture of the (P)-nanoparticles and (P)-ethynylhelicene pentamer reversibly dispersed in trifluoromethylbenzene upon heating at 70 °C and precipitated upon cooling at 25 °C. When a 10:90 equilibrium mixture of the double helix and random coil in solution was treated with the (P)-nanoparticles, the double helix was precipitated in 53% yield and was accompanied by equilibrium shift. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. α2 and α3 helices of dystrophin R16 and R17 frame a microdomain in the α1 helix of dystrophin R17 for neuronal NOS binding

    Science.gov (United States)

    Lai, Yi; Zhao, Junling; Yue, Yongping; Duan, Dongsheng

    2013-01-01

    Homologous spectrin-like repeats can mediate specific protein interaction. The underlying mechanism is poorly understood. Dystrophin contains 24 spectrin-like repeats. However, only repeats 16 and 17 (R16/17) are required for anchoring neuronal NOS (nNOS) to the sarcolemma. Through an adeno-associated virus-based in vivo binding assay, we found that membrane expression of correctly phased R16/17 was sufficient to recruit nNOS to the sarcolemma in mouse muscle. Utrophin R15/16 is homologous to dystrophin R16/17. Substitution of dystrophin R16/17 microdomains with the corresponding regions of utrophin R15/16 suggests that the nNOS binding site is located in a 10-residue fragment in dystrophin R17 α1 helix. Interestingly, swapping this microdomain back into utrophin did not convey the nNOS binding activity. To identify other structural features that are required for nNOS interaction, we replaced an individual α-helix of dystrophin R16/17 with an equivalent α-helix from another dystrophin repeat. In vitro study with yeast two-hybrid suggests that most α-helices of R16/17, except for the R17 α1 helix, were dispensable for nNOS interaction. Surprisingly, in vivo binding assay showed that α2 and α3 helices of both R16 and R17 were essential for nNOS binding in muscle. We concluded that a microdomain in the α1 helix of dystrophin R17 binds to nNOS in a way uniquely defined by two pairs of the flanking helices. Our results provide an explanation for how structurally similar spectrin-like repeats in dystrophin display selective interaction with nNOS. The results also open new therapeutic avenues to restore defective nNOS homeostasis in dystrophin-null Duchenne muscular dystrophy. PMID:23185009