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Sample records for fast protein conformational

  1. A Method for Extracting the Free Energy Surface and Conformational Dynamics of Fast-Folding Proteins from Single Molecule Photon Trajectories

    2015-01-01

    Single molecule fluorescence spectroscopy holds the promise of providing direct measurements of protein folding free energy landscapes and conformational motions. However, fulfilling this promise has been prevented by technical limitations, most notably, the difficulty in analyzing the small packets of photons per millisecond that are typically recorded from individual biomolecules. Such limitation impairs the ability to accurately determine conformational distributions and resolve sub-millisecond processes. Here we develop an analytical procedure for extracting the conformational distribution and dynamics of fast-folding proteins directly from time-stamped photon arrival trajectories produced by single molecule FRET experiments. Our procedure combines the maximum likelihood analysis originally developed by Gopich and Szabo with a statistical mechanical model that describes protein folding as diffusion on a one-dimensional free energy surface. Using stochastic kinetic simulations, we thoroughly tested the performance of the method in identifying diverse fast-folding scenarios, ranging from two-state to one-state downhill folding, as a function of relevant experimental variables such as photon count rate, amount of input data, and background noise. The tests demonstrate that the analysis can accurately retrieve the original one-dimensional free energy surface and microsecond folding dynamics in spite of the sub-megahertz photon count rates and significant background noise levels of current single molecule fluorescence experiments. Therefore, our approach provides a powerful tool for the quantitative analysis of single molecule FRET experiments of fast protein folding that is also potentially extensible to the analysis of any other biomolecular process governed by sub-millisecond conformational dynamics. PMID:25988351

  2. A surprising role for conformational entropy in protein function

    Wand, A. Joshua; Moorman, Veronica R.; Harpole, Kyle W.

    2014-01-01

    Formation of high-affinity complexes is critical for the majority of enzymatic reactions involving proteins. The creation of the family of Michaelis and other intermediate complexes during catalysis clearly involves a complicated manifold of interactions that are diverse and complex. Indeed, computing the energetics of interactions between proteins and small molecule ligands using molecular structure alone remains a grand challenge. One of the most difficult contributions to the free energy of protein-ligand complexes to experimentally access is that due to changes in protein conformational entropy. Fortunately, recent advances in solution nuclear magnetic resonance (NMR) relaxation methods have enabled the use of measures-of-motion between conformational states of a protein as a proxy for conformational entropy. This review briefly summarizes the experimental approaches currently employed to characterize fast internal motion in proteins, how this information is used to gain insight into conformational entropy, what has been learned and what the future may hold for this emerging view of protein function. PMID:23478875

  3. Mitochondrial Dysfunction in Protein Conformational Disorders

    EstherShlomi

    protein misfolding of α-synuclein involves conformational changes in the protein .... upon association with a membrane surface its can adopt a helical form with an 11/3 ... case of α-synuclein electrostatic interactions exist between positively ...

  4. Focused conformational sampling in proteins

    Bacci, Marco; Langini, Cassiano; Vymětal, Jiří; Caflisch, Amedeo; Vitalis, Andreas

    2017-11-01

    A detailed understanding of the conformational dynamics of biological molecules is difficult to obtain by experimental techniques due to resolution limitations in both time and space. Computer simulations avoid these in theory but are often too short to sample rare events reliably. Here we show that the progress index-guided sampling (PIGS) protocol can be used to enhance the sampling of rare events in selected parts of biomolecules without perturbing the remainder of the system. The method is very easy to use as it only requires as essential input a set of several features representing the parts of interest sufficiently. In this feature space, new states are discovered by spontaneous fluctuations alone and in unsupervised fashion. Because there are no energetic biases acting on phase space variables or projections thereof, the trajectories PIGS generates can be analyzed directly in the framework of transition networks. We demonstrate the possibility and usefulness of such focused explorations of biomolecules with two loops that are part of the binding sites of bromodomains, a family of epigenetic "reader" modules. This real-life application uncovers states that are structurally and kinetically far away from the initial crystallographic structures and are also metastable. Representative conformations are intended to be used in future high-throughput virtual screening campaigns.

  5. SCit: web tools for protein side chain conformation analysis

    Gautier, R.; Camproux, A.-C.; Tufféry, P.

    2004-01-01

    SCit is a web server providing services for protein side chain conformation analysis and side chain positioning. Specific services use the dependence of the side chain conformations on the local backbone conformation, which is described using a structural alphabet that describes the conformation of fragments of four-residue length in a limited library of structural prototypes. Based on this concept, SCit uses sets of rotameric conformations dependent on the local backbone conformation of each...

  6. Compact conformations of human protein disulfide isomerase.

    Shang Yang

    Full Text Available Protein disulfide isomerase (PDI composed of four thioredoxin-like domains a, b, b', and a', is a key enzyme catalyzing oxidative protein folding in the endoplasmic reticulum. Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact.

  7. SCit: web tools for protein side chain conformation analysis.

    Gautier, R; Camproux, A-C; Tufféry, P

    2004-07-01

    SCit is a web server providing services for protein side chain conformation analysis and side chain positioning. Specific services use the dependence of the side chain conformations on the local backbone conformation, which is described using a structural alphabet that describes the conformation of fragments of four-residue length in a limited library of structural prototypes. Based on this concept, SCit uses sets of rotameric conformations dependent on the local backbone conformation of each protein for side chain positioning and the identification of side chains with unlikely conformations. The SCit web server is accessible at http://bioserv.rpbs.jussieu.fr/SCit.

  8. Fast, clash-free RNA conformational morphing using molecular junctions.

    Héliou, Amélie; Budday, Dominik; Fonseca, Rasmus; van den Bedem, Henry

    2017-07-15

    Non-coding ribonucleic acids (ncRNA) are functional RNA molecules that are not translated into protein. They are extremely dynamic, adopting diverse conformational substates, which enables them to modulate their interaction with a large number of other molecules. The flexibility of ncRNA provides a challenge for probing their complex 3D conformational landscape, both experimentally and computationally. Despite their conformational diversity, ncRNAs mostly preserve their secondary structure throughout the dynamic ensemble. Here we present a kinematics-based procedure to morph an RNA molecule between conformational substates, while avoiding inter-atomic clashes. We represent an RNA as a kinematic linkage, with fixed groups of atoms as rigid bodies and rotatable bonds as degrees of freedom. Our procedure maintains RNA secondary structure by treating hydrogen bonds between base pairs as constraints. The constraints define a lower-dimensional, secondary-structure constraint manifold in conformation space, where motions are largely governed by molecular junctions of unpaired nucleotides. On a large benchmark set, we show that our morphing procedure compares favorably to peer algorithms, and can approach goal conformations to within a low all-atom RMSD by directing fewer than 1% of its atoms. Our results suggest that molecular junctions can modulate 3D structural rearrangements, while secondary structure elements guide large parts of the molecule along the transition to the correct final conformation. The source code, binaries and data are available at https://simtk.org/home/kgs . amelie.heliou@polytechnique.edu or vdbedem@stanford.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  9. Conformational Entropy as Collective Variable for Proteins.

    Palazzesi, Ferruccio; Valsson, Omar; Parrinello, Michele

    2017-10-05

    Many enhanced sampling methods rely on the identification of appropriate collective variables. For proteins, even small ones, finding appropriate descriptors has proven challenging. Here we suggest that the NMR S 2 order parameter can be used to this effect. We trace the validity of this statement to the suggested relation between S 2 and conformational entropy. Using the S 2 order parameter and a surrogate for the protein enthalpy in conjunction with metadynamics or variationally enhanced sampling, we are able to reversibly fold and unfold a small protein and draw its free energy at a fraction of the time that is needed in unbiased simulations. We also use S 2 in combination with the free energy flooding method to compute the unfolding rate of this peptide. We repeat this calculation at different temperatures to obtain the unfolding activation energy.

  10. Conformational dynamics data bank: a database for conformational dynamics of proteins and supramolecular protein assemblies.

    Kim, Do-Nyun; Altschuler, Josiah; Strong, Campbell; McGill, Gaël; Bathe, Mark

    2011-01-01

    The conformational dynamics data bank (CDDB, http://www.cdyn.org) is a database that aims to provide comprehensive results on the conformational dynamics of high molecular weight proteins and protein assemblies. Analysis is performed using a recently introduced coarse-grained computational approach that is applied to the majority of structures present in the electron microscopy data bank (EMDB). Results include equilibrium thermal fluctuations and elastic strain energy distributions that identify rigid versus flexible protein domains generally, as well as those associated with specific functional transitions, and correlations in molecular motions that identify molecular regions that are highly coupled dynamically, with implications for allosteric mechanisms. A practical web-based search interface enables users to easily collect conformational dynamics data in various formats. The data bank is maintained and updated automatically to include conformational dynamics results for new structural entries as they become available in the EMDB. The CDDB complements static structural information to facilitate the investigation and interpretation of the biological function of proteins and protein assemblies essential to cell function.

  11. Exploring the conformational energy landscape of proteins

    Nienhaus, G.U. [Univ. of Illinois, Urbana, IL (United States)]|[Universitaet Ulm (Germany); Mueller, J.D.; McMahon, B.H. [Univ. of Illinois, Urbana, IL (United States)] [and others

    1997-04-01

    Proteins possess a complex energy landscape with a large number of local minima called conformational substates that are arranged in a hierarchical fashion. Here we discuss experiments aimed at the elucidation of the energy landscape in carbonmonoxy myoglobin (MbCO). In the highest tier of the hierarchy, a few taxonomic substates exist. Because of their small number, these substates are accessible to detailed structural investigations. Spectroscopic experiments are discussed that elucidate the role of protonations of amino acid side chains in creating the substates. The lower tiers of the hierarchy contain a large number of statistical substates. Substate interconversions are observed in the entire temperature range from below 1 K up to the denaturation temperature, indicating a wide spectrum of energy barriers that separate the substates.

  12. An Efficient Null Model for Conformational Fluctuations in Proteins

    Harder, Tim Philipp; Borg, Mikael; Bottaro, Sandro

    2012-01-01

    Protein dynamics play a crucial role in function, catalytic activity, and pathogenesis. Consequently, there is great interest in computational methods that probe the conformational fluctuations of a protein. However, molecular dynamics simulations are computationally costly and therefore are often...... limited to comparatively short timescales. TYPHON is a probabilistic method to explore the conformational space of proteins under the guidance of a sophisticated probabilistic model of local structure and a given set of restraints that represent nonlocal interactions, such as hydrogen bonds or disulfide...... on conformational fluctuations that is in correspondence with experimental measurements. TYPHON provides a flexible, yet computationally efficient, method to explore possible conformational fluctuations in proteins....

  13. Solid state protein monolayers: Morphological, conformational, and functional properties

    Pompa, P. P.; Biasco, A.; Frascerra, V.; Calabi, F.; Cingolani, R.; Rinaldi, R.; Verbeet, M. Ph.; de Waal, E.; Canters, G. W.

    2004-12-01

    We have studied the morphological, conformational, and electron-transfer (ET) function of the metalloprotein azurin in the solid state, by a combination of physical investigation methods, namely atomic force microscopy, intrinsic fluorescence spectroscopy, and scanning tunneling microscopy. We demonstrate that a "solid state protein film" maintains its nativelike conformation and ET function, even after removal of the aqueous solvent.

  14. S-Adenosylmethionine conformations in solution and in protein complexes: Conformational influences of the sulfonium group

    Markham, George D.; Norrby, Per-Ola; Bock, Charles W.

    2002-01-01

    S-Adenosylmethionine (AdoMet) and other sulfonium ions play central roles in the metabolism of all organisms. The conformational preferences of AdoMet and two other biologically important sulfonium ions, S-methylmethionine and dimethylsulfonioproprionic acid, have been investigated by NMR...... and computational studies. Molecular mechanics parameters for the sulfonium center have been developed for the AMBER force field to permit analysis of NMR results and to enable comparison of the relative energies of the different conformations of AdoMet that have been found in crystal structures of complexes...... with proteins. S-Methylmethionine and S-dimethylsulfonioproprionate adopt a variety of conformations in aqueous solution; a conformation with an electrostatic interaction between the sulfonium sulfur and the carboxylate group is not noticeably favored, in contrast to the preferred conformation found by in vacuo...

  15. Conformational plasticity of the Ebola virus matrix protein.

    Radzimanowski, Jens; Effantin, Gregory; Weissenhorn, Winfried

    2014-11-01

    Filoviruses are the causative agents of a severe and often fatal hemorrhagic fever with repeated outbreaks in Africa. They are negative sense single stranded enveloped viruses that can cross species barriers from its natural host bats to primates including humans. The small size of the genome poses limits to viral adaption, which may be partially overcome by conformational plasticity. Here we review the different conformational states of the Ebola virus (EBOV) matrix protein VP40 that range from monomers, to dimers, hexamers, and RNA-bound octamers. This conformational plasticity that is required for the viral life cycle poses a unique opportunity for development of VP40 specific drugs. Furthermore, we compare the structure to homologous matrix protein structures from Paramyxoviruses and Bornaviruses and we predict that they do not only share the fold but also the conformational flexibility of EBOV VP40. © 2014 The Protein Society.

  16. Sampling Realistic Protein Conformations Using Local Structural Bias

    Hamelryck, Thomas Wim; Kent, John T.; Krogh, A.

    2006-01-01

    The prediction of protein structure from sequence remains a major unsolved problem in biology. The most successful protein structure prediction methods make use of a divide-and-conquer strategy to attack the problem: a conformational sampling method generates plausible candidate structures, which...... are subsequently accepted or rejected using an energy function. Conceptually, this often corresponds to separating local structural bias from the long-range interactions that stabilize the compact, native state. However, sampling protein conformations that are compatible with the local structural bias encoded...... in a given protein sequence is a long-standing open problem, especially in continuous space. We describe an elegant and mathematically rigorous method to do this, and show that it readily generates native-like protein conformations simply by enforcing compactness. Our results have far-reaching implications...

  17. Conformational Heterogeneity in Antibody-Protein Antigen Recognition IMPLICATIONS FOR HIGH AFFINITY PROTEIN COMPLEX FORMATION

    Addis, P. W.; Hall, c. J.; Bruton, S.; Veverka, Václav; Wilkinson, I. C.; Muskett, F. W.; Renshaw, P. S.; Prosser, C. E.; Carrington, B.; Lawson, A. D. G.; Griffin, R.; Taylor, R. J.; Waters, L. C.; Henry, A. J.; Carr, M. D.

    2014-01-01

    Roč. 289, č. 10 (2014), s. 7200-7210 ISSN 0021-9258 Institutional support: RVO:61388963 Keywords : NMR * antibody * protein-protein interaction * protein conformation Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  18. Fast dynamics perturbation analysis for prediction of protein functional sites

    Cohn Judith D

    2008-01-01

    Full Text Available Abstract Background We present a fast version of the dynamics perturbation analysis (DPA algorithm to predict functional sites in protein structures. The original DPA algorithm finds regions in proteins where interactions cause a large change in the protein conformational distribution, as measured using the relative entropy Dx. Such regions are associated with functional sites. Results The Fast DPA algorithm, which accelerates DPA calculations, is motivated by an empirical observation that Dx in a normal-modes model is highly correlated with an entropic term that only depends on the eigenvalues of the normal modes. The eigenvalues are accurately estimated using first-order perturbation theory, resulting in a N-fold reduction in the overall computational requirements of the algorithm, where N is the number of residues in the protein. The performance of the original and Fast DPA algorithms was compared using protein structures from a standard small-molecule docking test set. For nominal implementations of each algorithm, top-ranked Fast DPA predictions overlapped the true binding site 94% of the time, compared to 87% of the time for original DPA. In addition, per-protein recall statistics (fraction of binding-site residues that are among predicted residues were slightly better for Fast DPA. On the other hand, per-protein precision statistics (fraction of predicted residues that are among binding-site residues were slightly better using original DPA. Overall, the performance of Fast DPA in predicting ligand-binding-site residues was comparable to that of the original DPA algorithm. Conclusion Compared to the original DPA algorithm, the decreased run time with comparable performance makes Fast DPA well-suited for implementation on a web server and for high-throughput analysis.

  19. Frustration-guided motion planning reveals conformational transitions in proteins.

    Budday, Dominik; Fonseca, Rasmus; Leyendecker, Sigrid; van den Bedem, Henry

    2017-10-01

    Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here, we present a new, robotics-inspired motion planning procedure called dCC-RRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non-native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eight proteins determined in two conformations separated by, on average, 7.5 Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. We then applied dCC-RRT to examine how collective, small-scale motions of four side-chains in the active site of cyclophilin A propagate through the protein. dCC-RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non-canonical capsid binding site 25 Å away, rationalizing NMR and multi-temperature crystallography experiments. In all, dCC-RRT can reveal detailed, all-atom molecular mechanisms for small and large amplitude motions. Source code and binaries are freely available at https://github.com/ExcitedStates/KGS/. © 2017 Wiley Periodicals, Inc.

  20. Mussel glue protein has an open conformation.

    Williams, T; Marumo, K; Waite, J H; Henkens, R W

    1989-03-01

    Both native glue protein from marine mussels and a synthetic nonhydroxylated analog were analyzed by far-uv CD under a variety of conditions. Analysis of the CD spectra using various models strongly suggest a primarily random coil structure for both forms of the protein, a fact also supported by the absence of spectral change for the glue protein upon dilution into 6 M guanidine hydrochloride. The nonhydroxylated analog, which consists of 20 repeats of the peptide sequence Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys, was further characterized by enzyme modification using mushroom tyrosinase. Enzymatic hydroxylation of tyrosines was found to be best fit by a model containing two rate constants, 5.6 (+/- 0.6) X 10(-3) and 7.2 (+/- 0.3) X 10(-2) min-1. At equilibrium, HPLC analysis of digests showed nearly 100% conversion of Tyr-9 and only 15 to 35% conversion of Tyr-5. The Chou and Fasman rules for predicting structure were applied to the repeat sequence listed above. The rules predict the absence of alpha helix and beta pleated sheets in the structure of this peptide. On the other hand, beta turns are predicted to be present with Tyr-5 being in the region of highest probability. These data suggest that the protein in solution has only a small amount of secondary structure.

  1. Conformational transition paths harbor structures useful for aiding drug discovery and understanding enzymatic mechanisms in protein kinases.

    Wong, Chung F

    2016-01-01

    This short article examines the usefulness of fast simulations of conformational transition paths in elucidating enzymatic mechanisms and guiding drug discovery for protein kinases. It applies the transition path method in the MOIL software package to simulate the paths of conformational transitions between six pairs of structures from the Protein Data Bank. The structures along the transition paths were found to resemble experimental structures that mimic transient structures believed to form during enzymatic catalysis or conformational transitions, or structures that have drug candidates bound. These findings suggest that such simulations could provide quick initial insights into the enzymatic mechanisms or pathways of conformational transitions of proteins kinases, or could provide structures useful for aiding structure-based drug design. © 2015 The Protein Society.

  2. Conformational dynamics of amyloid proteins at the aqueous interface

    Armbruster, Matthew; Horst, Nathan; Aoki, Brendy; Malik, Saad; Soto, Patricia

    2013-03-01

    Amyloid proteins is a class of proteins that exhibit distinct monomeric and oligomeric conformational states hallmark of deleterious neurological diseases for which there are not yet cures. Our goal is to examine the extent of which the aqueous/membrane interface modulates the folding energy landscape of amyloid proteins. To this end, we probe the dynamic conformational ensemble of amyloids (monomer prion protein and Alzheimer's Ab protofilaments) interacting with model bilayers. We will present the results of our coarse grain molecular modeling study in terms of the existence of preferential binding spots of the amyloid to the bilayer and the response of the bilayer to the interaction with the amyloid. NSF Nebraska EPSCoR First Award

  3. Effect of External Electric Field Stress on Gliadin Protein Conformation

    Singh, Ashutosh; Munshi, Shirin; Raghavan, Vijaya

    2013-01-01

    A molecular dynamic (MD) modeling approach was applied to evaluate the effect of external electric field on gliadin protein structure and surface properties. Static electric field strengths of 0.001 V/nm and 0.002 V/nm induced conformational changes in the protein but had no significant effect on its surface properties. The study of hydrogen bond evolution during the course of simulation revealed that the root mean square deviation, radius of gyration and secondary structure formation, all de...

  4. Effects of macromolecular crowding on protein conformational changes.

    Hao Dong

    2010-07-01

    Full Text Available Many protein functions can be directly linked to conformational changes. Inside cells, the equilibria and transition rates between different conformations may be affected by macromolecular crowding. We have recently developed a new approach for modeling crowding effects, which enables an atomistic representation of "test" proteins. Here this approach is applied to study how crowding affects the equilibria and transition rates between open and closed conformations of seven proteins: yeast protein disulfide isomerase (yPDI, adenylate kinase (AdK, orotidine phosphate decarboxylase (ODCase, Trp repressor (TrpR, hemoglobin, DNA beta-glucosyltransferase, and Ap(4A hydrolase. For each protein, molecular dynamics simulations of the open and closed states are separately run. Representative open and closed conformations are then used to calculate the crowding-induced changes in chemical potential for the two states. The difference in chemical-potential change between the two states finally predicts the effects of crowding on the population ratio of the two states. Crowding is found to reduce the open population to various extents. In the presence of crowders with a 15 A radius and occupying 35% of volume, the open-to-closed population ratios of yPDI, AdK, ODCase and TrpR are reduced by 79%, 78%, 62% and 55%, respectively. The reductions for the remaining three proteins are 20-44%. As expected, the four proteins experiencing the stronger crowding effects are those with larger conformational changes between open and closed states (e.g., as measured by the change in radius of gyration. Larger proteins also tend to experience stronger crowding effects than smaller ones [e.g., comparing yPDI (480 residues and TrpR (98 residues]. The potentials of mean force along the open-closed reaction coordinate of apo and ligand-bound ODCase are altered by crowding, suggesting that transition rates are also affected. These quantitative results and qualitative trends will

  5. Dissecting Protein Configurational Entropy into Conformational and Vibrational Contributions.

    Chong, Song-Ho; Ham, Sihyun

    2015-10-01

    Quantifying how the rugged nature of the underlying free-energy landscape determines the entropic cost a protein must incur upon folding and ligand binding is a challenging problem. Here, we present a novel computational approach that dissects the protein configurational entropy on the basis of the classification of protein dynamics on the landscape into two separate components: short-term vibrational dynamics related to individual free-energy wells and long-term conformational dynamics associated with transitions between wells. We apply this method to separate the configurational entropy of the protein villin headpiece subdomain into its conformational and vibrational components. We find that the change in configurational entropy upon folding is dominated by the conformational entropy despite the fact that the magnitude of the vibrational entropy is the significantly larger component in each of the folded and unfolded states, which is in accord with the previous empirical estimations. The straightforward applicability of our method to unfolded proteins promises a wide range of applications, including those related to intrinsically disordered proteins.

  6. Universality of fast quenches from the conformal perturbation theory

    Dymarsky, Anatoly; Smolkin, Michael

    2018-01-01

    We consider global quantum quenches, a protocol when a continuous field theoretic system in the ground state is driven by a homogeneous time-dependent external interaction. When the typical inverse time scale of the interaction is much larger than all relevant scales except for the UV-cutoff the system's response exhibits universal scaling behavior. We provide both qualitative and quantitative explanations of this universality and argue that physics of the response during and shortly after the quench is governed by the conformal perturbation theory around the UV fixed point. We proceed to calculate the response of one and two-point correlation functions confirming and generalizing universal scalings found previously. Finally, we discuss late time behavior after the quench and argue that all local quantities will equilibrate to their thermal values specified by an excess energy acquired by the system during the quench.

  7. Fluctuation Flooding Method (FFM) for accelerating conformational transitions of proteins

    Harada, Ryuhei; Takano, Yu; Shigeta, Yasuteru

    2014-03-01

    A powerful conformational sampling method for accelerating structural transitions of proteins, "Fluctuation Flooding Method (FFM)," is proposed. In FFM, cycles of the following steps enhance the transitions: (i) extractions of largely fluctuating snapshots along anisotropic modes obtained from trajectories of multiple independent molecular dynamics (MD) simulations and (ii) conformational re-sampling of the snapshots via re-generations of initial velocities when re-starting MD simulations. In an application to bacteriophage T4 lysozyme, FFM successfully accelerated the open-closed transition with the 6 ns simulation starting solely from the open state, although the 1-μs canonical MD simulation failed to sample such a rare event.

  8. Conformational Fluctuations in G-Protein-Coupled Receptors

    Brown, Michael F.

    2014-03-01

    G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets, where rhodopsin is an important prototype and occurs naturally in a lipid membrane. Rhodopsin photoactivation entails 11-cis to all-trans isomerization of the retinal cofactor, yielding an equilibrium between inactive Meta-I and active Meta-II states. Two important questions are: (1) Is rhodopsin is a simple two-state switch? Or (2) does isomerization of retinal unlock an activated conformational ensemble? For an ensemble-based activation mechanism (EAM) a role for conformational fluctuations is clearly indicated. Solid-state NMR data together with theoretical molecular dynamics (MD) simulations detect increased local mobility of retinal after light activation. Resultant changes in local dynamics of the cofactor initiate large-scale fluctuations of transmembrane helices that expose recognition sites for the signal-transducing G-protein. Time-resolved FTIR studies and electronic spectroscopy further show the conformational ensemble is strongly biased by the membrane lipid composition, as well as pH and osmotic pressure. A new flexible surface model (FSM) describes how the curvature stress field of the membrane governs the energetics of active rhodopsin, due to the spontaneous monolayer curvature of the lipids. Furthermore, influences of osmotic pressure dictate that a large number of bulk water molecules are implicated in rhodopsin activation. Around 60 bulk water molecules activate rhodopsin, which is much larger than the number of structural waters seen in X-ray crystallography, or inferred from studies of bulk hydrostatic pressure. Conformational selection and promoting vibrational motions of rhodopsin lead to activation of the G-protein (transducin). Our biophysical data give a paradigm shift in understanding GPCR activation. The new view is: dynamics and conformational fluctuations involve an ensemble of substates that activate the cognate G-protein in the amplified visual

  9. Conformational dependence of a protein kinase phosphate transfer reaction

    Labute, Montiago; Henkelman, Graeme; Tung, Chang-Shung; Fenimore, Paul; McMahon, Ben

    2007-03-01

    Atomic motions and energetics for a phosphate transfer reaction catalyzed by the cAMP-dependent protein kinase have been calculated using plane-wave density functional theory, starting from structures of proteins crystallized in both the reactant conformation (RC) and the transition-state conformation (TC). In TC, we calculate that the reactants and products are nearly isoenergetic with a 20-kJ/mol barrier, whereas phosphate transfer is unfavorable by 120 kJ/mol in the RC, with an even higher barrier. Our results demonstrate that the phosphate transfer reaction occurs rapidly and reversibly in a particular conformation of the protein, and that the reaction can be gated by changes of a few tenths of an angstrom in the catalytic site [1]. [1] G.H. Henkelman, M.X. LaBute, C.-S. Tung, P.W. Fenimore, B.H. McMahon, Proc. Natl. Acad. Sci. USA vol. 102, no. 43:15347-15351 (2005).

  10. Dissecting the dynamic conformations of the metamorphic protein lymphotactin.

    Harvey, Sophie R; Porrini, Massimiliano; Konijnenberg, Albert; Clarke, David J; Tyler, Robert C; Langridge-Smith, Patrick R R; MacPhee, Cait E; Volkman, Brian F; Barran, Perdita E

    2014-10-30

    A mass spectrometer provides an ideal laboratory to probe the structure and stability of isolated protein ions. Interrogation of each discrete mass/charge-separated species enables the determination of the intrinsic stability of a protein fold, gaining snapshots of unfolding pathways. In solution, the metamorphic protein lymphotactin (Ltn) exists in equilibrium between two distinct conformations, a monomeric (Ltn10) and a dimeric (Ltn40) fold. Here, we use electron capture dissociation (ECD) and drift tube ion mobility-mass spectrometry (DT IM-MS) to analyze both forms and use molecular dynamics (MD) to consider how the solution fold alters in a solvent-free environment. DT IM-MS reveals significant conformational flexibility for the monomer, while the dimer appears more conformationally restricted. These findings are supported by MD calculations, which reveal how salt bridges stabilize the conformers in vacuo. Following ECD experiments, a distinctive fragmentation pattern is obtained for both the monomer and dimer. Monomer fragmentation becomes more pronounced with increasing charge state especially in the disordered regions and C-terminal α-helix in the solution fold. Lower levels of fragmentation are seen in the β-sheet regions and in regions that contain salt bridges, identified by MD simulations. The lowest charge state of the dimer for which we obtain ECD data ([D+9H](9+)) exhibits extensive fragmentation with no relationship to the solution fold and has a smaller collision cross section (CCS) than charge states 10-13+, suggesting a "collapsed" encounter complex. Other charge states of the dimer, as for the monomer, are resistant to fragmentation in regions of β-sheets in the solution fold. This study provides evidence for preservation and loss of global fold and secondary structural elements, providing a tantalizing glimpse into the power of the emerging field of native top-down mass spectrometry.

  11. Time-resolved infrared studies of protein conformational dynamics

    Woodruff, W.H.; Causgrove, T.P.; Dyer, R.B. [Los Alamos National Laboratory, NM (United States); Callender, R.H. [Univ. of New York, NY (United States)

    1994-12-01

    We have demonstrated that TRIR in the amide I region gives structural information regarding protein conformational changes in realtime, both on processes involved in the development of the functional structure (protein folding) and on protein structural changes that accompany the functional dynamics of the native structure. Assignment of many of the amide I peaks to specific amide or sidechain structures will require much additional effort. Specifically, the congestion and complexity of the protein vibrational spectra dictate that isotope studies are an absolute requirement for more than a qualitative notion of the structural interpretation of these measurements. It is clear, however, that enormous potential exists for elucidating structural relaxation dynamics and energetics with a high degree of structural specificity using this approach.

  12. Dynamics of DNA conformations and DNA-protein interaction

    Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen

    2005-01-01

    Optical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA...... denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed...... in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent...

  13. Exploiting conformational ensembles in modeling protein-protein interactions on the proteome scale

    Kuzu, Guray; Gursoy, Attila; Nussinov, Ruth; Keskin, Ozlem

    2013-01-01

    Cellular functions are performed through protein-protein interactions; therefore, identification of these interactions is crucial for understanding biological processes. Recent studies suggest that knowledge-based approaches are more useful than ‘blind’ docking for modeling at large scales. However, a caveat of knowledge-based approaches is that they treat molecules as rigid structures. The Protein Data Bank (PDB) offers a wealth of conformations. Here, we exploited ensemble of the conformations in predictions by a knowledge-based method, PRISM. We tested ‘difficult’ cases in a docking-benchmark dataset, where the unbound and bound protein forms are structurally different. Considering alternative conformations for each protein, the percentage of successfully predicted interactions increased from ~26% to 66%, and 57% of the interactions were successfully predicted in an ‘unbiased’ scenario, in which data related to the bound forms were not utilized. If the appropriate conformation, or relevant template interface, is unavailable in the PDB, PRISM could not predict the interaction successfully. The pace of the growth of the PDB promises a rapid increase of ensemble conformations emphasizing the merit of such knowledge-based ensemble strategies for higher success rates in protein-protein interaction predictions on an interactome-scale. We constructed the structural network of ERK interacting proteins as a case study. PMID:23590674

  14. Prion protein β2-α2 loop conformational landscape.

    Caldarulo, Enrico; Barducci, Alessandro; Wüthrich, Kurt; Parrinello, Michele

    2017-09-05

    In transmissible spongiform encephalopathies (TSEs), which are lethal neurodegenerative diseases that affect humans and a wide range of other mammalian species, the normal "cellular" prion protein ([Formula: see text]) is transformed into amyloid aggregates representing the "scrapie form" of the protein ([Formula: see text]). Continued research on this system is of keen interest, since new information on the physiological function of [Formula: see text] in healthy organisms is emerging, as well as new data on the mechanism of the transformation of [Formula: see text] to [Formula: see text] In this paper we used two different approaches: a combination of the well-tempered ensemble (WTE) and parallel tempering (PT) schemes and metadynamics (MetaD) to characterize the conformational free-energy surface of [Formula: see text] The focus of the data analysis was on an 11-residue polypeptide segment in mouse [Formula: see text](121-231) that includes the [Formula: see text]2-[Formula: see text]2 loop of residues 167-170, for which a correlation between structure and susceptibility to prion disease has previously been described. This study includes wild-type mouse [Formula: see text] and a variant with the single-residue replacement Y169A. The resulting detailed conformational landscapes complement in an integrative manner the available experimental data on [Formula: see text], providing quantitative insights into the nature of the structural transition-related function of the [Formula: see text]2-[Formula: see text]2 loop.

  15. Protein Loop Structure Prediction Using Conformational Space Annealing.

    Heo, Seungryong; Lee, Juyong; Joo, Keehyoung; Shin, Hang-Cheol; Lee, Jooyoung

    2017-05-22

    We have developed a protein loop structure prediction method by combining a new energy function, which we call E PLM (energy for protein loop modeling), with the conformational space annealing (CSA) global optimization algorithm. The energy function includes stereochemistry, dynamic fragment assembly, distance-scaled finite ideal gas reference (DFIRE), and generalized orientation- and distance-dependent terms. For the conformational search of loop structures, we used the CSA algorithm, which has been quite successful in dealing with various hard global optimization problems. We assessed the performance of E PLM with two widely used loop-decoy sets, Jacobson and RAPPER, and compared the results against the DFIRE potential. The accuracy of model selection from a pool of loop decoys as well as de novo loop modeling starting from randomly generated structures was examined separately. For the selection of a nativelike structure from a decoy set, E PLM was more accurate than DFIRE in the case of the Jacobson set and had similar accuracy in the case of the RAPPER set. In terms of sampling more nativelike loop structures, E PLM outperformed E DFIRE for both decoy sets. This new approach equipped with E PLM and CSA can serve as the state-of-the-art de novo loop modeling method.

  16. Generating intrinsically disordered protein conformational ensembles from a Markov chain

    Cukier, Robert I.

    2018-03-01

    Intrinsically disordered proteins (IDPs) sample a diverse conformational space. They are important to signaling and regulatory pathways in cells. An entropy penalty must be payed when an IDP becomes ordered upon interaction with another protein or a ligand. Thus, the degree of conformational disorder of an IDP is of interest. We create a dichotomic Markov model that can explore entropic features of an IDP. The Markov condition introduces local (neighbor residues in a protein sequence) rotamer dependences that arise from van der Waals and other chemical constraints. A protein sequence of length N is characterized by its (information) entropy and mutual information, MIMC, the latter providing a measure of the dependence among the random variables describing the rotamer probabilities of the residues that comprise the sequence. For a Markov chain, the MIMC is proportional to the pair mutual information MI which depends on the singlet and pair probabilities of neighbor residue rotamer sampling. All 2N sequence states are generated, along with their probabilities, and contrasted with the probabilities under the assumption of independent residues. An efficient method to generate realizations of the chain is also provided. The chain entropy, MIMC, and state probabilities provide the ingredients to distinguish different scenarios using the terminologies: MoRF (molecular recognition feature), not-MoRF, and not-IDP. A MoRF corresponds to large entropy and large MIMC (strong dependence among the residues' rotamer sampling), a not-MoRF corresponds to large entropy but small MIMC, and not-IDP corresponds to low entropy irrespective of the MIMC. We show that MorFs are most appropriate as descriptors of IDPs. They provide a reasonable number of high-population states that reflect the dependences between neighbor residues, thus classifying them as IDPs, yet without very large entropy that might lead to a too high entropy penalty.

  17. [Interconnection between architecture of protein globule and disposition of conformational conservative oligopeptides in proteins from one protein family].

    Batianovskiĭ, A V; Filatov, I V; Namiot, V A; Esipova, N G; Volotovskiĭ, I D

    2012-01-01

    It was shown that selective interactions between helical segments of macromolecules can realize in globular proteins in the segments characterized by the same periodicities of charge distribution i.e. between conformationally conservative oligopeptides. It was found that in the macromolecules of alpha-helical proteins conformationally conservative oligopeptides are disposed at a distance being characteristic of direct interactions. For representatives of many structural families of alpha-type proteins specific disposition of conformationally conservative segments is observed. This disposition is inherent to a particular structural family. Disposition of conformationally conservative segments is not related to homology of the amino acid sequence but reflects peculiarities of native 3D-architectures of protein globules.

  18. Conformational dynamics of ATP/Mg:ATP in motor proteins via data mining and molecular simulation

    Bojovschi, A.; Liu, Ming S.; Sadus, Richard J.

    2012-08-01

    The conformational diversity of ATP/Mg:ATP in motor proteins was investigated using molecular dynamics and data mining. Adenosine triphosphate (ATP) conformations were found to be constrained mostly by inter cavity motifs in the motor proteins. It is demonstrated that ATP favors extended conformations in the tight pockets of motor proteins such as F1-ATPase and actin whereas compact structures are favored in motor proteins such as RNA polymerase and DNA helicase. The incorporation of Mg2+ leads to increased flexibility of ATP molecules. The differences in the conformational dynamics of ATP/Mg:ATP in various motor proteins was quantified by the radius of gyration. The relationship between the simulation results and those obtained by data mining of motor proteins available in the protein data bank is analyzed. The data mining analysis of motor proteins supports the conformational diversity of the phosphate group of ATP obtained computationally.

  19. Distribution, transition and thermodynamic stability of protein conformations in the denaturant-induced unfolding of proteins.

    Bian, Liujiao; Ji, Xu

    2014-01-01

    Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

  20. Microscopic insights into the NMR relaxation based protein conformational entropy meter

    Kasinath, Vignesh; Sharp, Kim A.; Wand, A. Joshua

    2013-01-01

    Conformational entropy is a potentially important thermodynamic parameter contributing to protein function. Quantitative measures of conformational entropy are necessary for an understanding of its role but have been difficult to obtain. An empirical method that utilizes changes in conformational dynamics as a proxy for changes in conformational entropy has recently been introduced. Here we probe the microscopic origins of the link between conformational dynamics and conformational entropy using molecular dynamics simulations. Simulation of seven pro! teins gave an excellent correlation with measures of side-chain motion derived from NMR relaxation. The simulations show that the motion of methyl-bearing side-chains are sufficiently coupled to that of other side chains to serve as excellent reporters of the overall side-chain conformational entropy. These results tend to validate the use of experimentally accessible measures of methyl motion - the NMR-derived generalized order parameters - as a proxy from which to derive changes in protein conformational entropy. PMID:24007504

  1. Incorporation of protein flexibility and conformational energy penalties in docking screens to improve ligand discovery

    Fischer, Marcus; Coleman, Ryan G.; Fraser, James S.; Shoichet, Brian K.

    2014-07-01

    Proteins fluctuate between alternative conformations, which presents a challenge for ligand discovery because such flexibility is difficult to treat computationally owing to problems with conformational sampling and energy weighting. Here we describe a flexible docking method that samples and weights protein conformations using experimentally derived conformations as a guide. The crystallographically refined occupancies of these conformations, which are observable in an apo receptor structure, define energy penalties for docking. In a large prospective library screen, we identified new ligands that target specific receptor conformations of a cavity in cytochrome c peroxidase, and we confirm both ligand pose and associated receptor conformation predictions by crystallography. The inclusion of receptor flexibility led to ligands with new chemotypes and physical properties. By exploiting experimental measures of loop and side-chain flexibility, this method can be extended to the discovery of new ligands for hundreds of targets in the Protein Data Bank for which similar experimental information is available.

  2. Self Organizing Maps to efficiently cluster and functionally interpret protein conformational ensembles

    Fabio Stella

    2013-09-01

    Full Text Available An approach that combines Self-Organizing maps, hierarchical clustering and network components is presented, aimed at comparing protein conformational ensembles obtained from multiple Molecular Dynamic simulations. As a first result the original ensembles can be summarized by using only the representative conformations of the clusters obtained. In addition the network components analysis allows to discover and interpret the dynamic behavior of the conformations won by each neuron. The results showed the ability of this approach to efficiently derive a functional interpretation of the protein dynamics described by the original conformational ensemble, highlighting its potential as a support for protein engineering.

  3. Preference of small molecules for local minimum conformations when binding to proteins.

    Qi Wang

    2007-09-01

    Full Text Available It is well known that small molecules (ligands do not necessarily adopt their lowest potential energy conformations when binding to proteins. Analyses of protein-bound ligand crystal structures have reportedly shown that many of them do not even adopt the conformations at local minima of their potential energy surfaces (local minimum conformations. The results of these analyses raise a concern regarding the validity of virtual screening methods that use ligands in local minimum conformations. Here we report a normal-mode-analysis (NMA study of 100 crystal structures of protein-bound ligands. Our data show that the energy minimization of a ligand alone does not automatically stop at a local minimum conformation if the minimum of the potential energy surface is shallow, thus leading to the folding of the ligand. Furthermore, our data show that all 100 ligand conformations in their protein-bound ligand crystal structures are nearly identical to their local minimum conformations obtained from NMA-monitored energy minimization, suggesting that ligands prefer to adopt local minimum conformations when binding to proteins. These results both support virtual screening methods that use ligands in local minimum conformations and caution about possible adverse effect of excessive energy minimization when generating a database of ligand conformations for virtual screening.

  4. Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations

    Sterling, Harry J.; Kintzer, Alexander F.; Feld, Geoffrey K.; Cassou, Catherine A.; Krantz, Bryan A.; Williams, Evan R.

    2012-02-01

    The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol ( m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

  5. When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.

    Muñoz, Victor; Cerminara, Michele

    2016-09-01

    Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. © 2016 The Author(s).

  6. Analyzing Protein Denaturation using Fast Differential Scanning Calorimetry

    Splinter, R.; Van Herwaarden, A.W.; Iervolino, E.; Vanden Poel, G.; Istrate, D.; Sarro, P.M.

    2012-01-01

    This paper investigates the possibility to measure protein denaturation with Fast Differential Scanning Calorimetry (FDSC). Cancer can be diagnosed by measuring protein denaturation in blood plasma using Differential Scanning Calorimetry (DSC). FDSC can reduce diagnosis time from hours to minutes,

  7. Promiscuity and the conformational rearrangement of drug-like molecules: insight from the protein data bank.

    He, Michael W; Lee, Patrick S; Sweeney, Zachary K

    2015-02-01

    Selectivity is a central aspect of lead optimization in the drug discovery process. Medicinal chemists often try to decrease molecular flexibility to improve selectivity, given the common belief that the two are interdependent. To investigate the relationship between polypharmacology and conformational flexibility, we mined the Protein Data Bank and constructed a dataset of pharmaceutically relevant ligands that crystallized in more than one protein target while binding to each co-crystallized receptor with similar in vitro affinities. After analyzing the molecular conformations of these 100 ligands, we found that 59 ligands bound to different protein targets without significantly changing conformation, suggesting that there is no distinct correlation between conformational flexibility and polypharmacology within our dataset. Ligands crystallized in similar proteins and highly ligand-efficient compounds with five or fewer rotatable bonds were less likely to adjust conformation when binding. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A FAST MORPHING-BASED INTERPOLATION FOR MEDICAL IMAGES: APPLICATION TO CONFORMAL RADIOTHERAPY

    Hussein Atoui

    2011-05-01

    Full Text Available A method is presented for fast interpolation between medical images. The method is intended for both slice and projective interpolation. It allows offline interpolation between neighboring slices in tomographic data. Spatial correspondence between adjacent images is established using a block matching algorithm. Interpolation of image intensities is then carried out by morphing between the images. The morphing-based method is compared to standard linear interpolation, block-matching-based interpolation and registrationbased interpolation in 3D tomographic data sets. Results show that the proposed method scored similar performance in comparison to registration-based interpolation, and significantly outperforms both linear and block-matching-based interpolation. This method is applied in the context of conformal radiotherapy for online projective interpolation between Digitally Reconstructed Radiographs (DRRs.

  9. Roles of electrostatics and conformation in protein-crystal interactions.

    Paul V Azzopardi

    2010-02-01

    Full Text Available In vitro studies have shown that the phosphoprotein osteopontin (OPN inhibits the nucleation and growth of hydroxyapatite (HA and other biominerals. In vivo, OPN is believed to prevent the calcification of soft tissues. However, the nature of the interaction between OPN and HA is not understood. In the computational part of the present study, we used molecular dynamics simulations to predict the adsorption of 19 peptides, each 16 amino acids long and collectively covering the entire sequence of OPN, to the {100} face of HA. This analysis showed that there is an inverse relationship between predicted strength of adsorption and peptide isoelectric point (P<0.0001. Analysis of the OPN sequence by PONDR (Predictor of Naturally Disordered Regions indicated that OPN sequences predicted to adsorb well to HA are highly disordered. In the experimental part of the study, we synthesized phosphorylated and non-phosphorylated peptides corresponding to OPN sequences 65-80 (pSHDHMDDDDDDDDDGD and 220-235 (pSHEpSTEQSDAIDpSAEK. In agreement with the PONDR analysis, these were shown by circular dichroism spectroscopy to be largely disordered. A constant-composition/seeded growth assay was used to assess the HA-inhibiting potencies of the synthetic peptides. The phosphorylated versions of OPN65-80 (IC(50 = 1.93 microg/ml and OPN220-235 (IC(50 = 1.48 microg/ml are potent inhibitors of HA growth, as is the nonphosphorylated version of OPN65-80 (IC(50 = 2.97 microg/ml; the nonphosphorylated version of OPN220-235 has no measurable inhibitory activity. These findings suggest that the adsorption of acidic proteins to Ca2+-rich crystal faces of biominerals is governed by electrostatics and is facilitated by conformational flexibility of the polypeptide chain.

  10. The Dynameomics Entropy Dictionary: A Large-Scale Assessment of Conformational Entropy across Protein Fold Space.

    Towse, Clare-Louise; Akke, Mikael; Daggett, Valerie

    2017-04-27

    Molecular dynamics (MD) simulations contain considerable information with regard to the motions and fluctuations of a protein, the magnitude of which can be used to estimate conformational entropy. Here we survey conformational entropy across protein fold space using the Dynameomics database, which represents the largest existing data set of protein MD simulations for representatives of essentially all known protein folds. We provide an overview of MD-derived entropies accounting for all possible degrees of dihedral freedom on an unprecedented scale. Although different side chains might be expected to impose varying restrictions on the conformational space that the backbone can sample, we found that the backbone entropy and side chain size are not strictly coupled. An outcome of these analyses is the Dynameomics Entropy Dictionary, the contents of which have been compared with entropies derived by other theoretical approaches and experiment. As might be expected, the conformational entropies scale linearly with the number of residues, demonstrating that conformational entropy is an extensive property of proteins. The calculated conformational entropies of folding agree well with previous estimates. Detailed analysis of specific cases identifies deviations in conformational entropy from the average values that highlight how conformational entropy varies with sequence, secondary structure, and tertiary fold. Notably, α-helices have lower entropy on average than do β-sheets, and both are lower than coil regions.

  11. Conformational Dynamics of the Receptor Protein Galactose/Glucose Binding Protein

    Messina, Troy; Talaga, David

    2006-03-01

    We have performed time-correlated single photon counting (TCSPC) anisotropy and Stokes Shift measurements on bulk solutions of galactose/glucose binding protein. Site-directed mutagenesis was used to provide a single cysteine amino acid near the sugar-binding center of the protein (glutamine 26 to cysteine -- Q26C). The cysteine was covalently labeled with the environmentally-sensitive fluorophore acrylodan, and a long-lived ruthenium complex was covalently attached to the N-terminus to provide a fluorescent reference. The TCSPC data were analyzed using global convolute-and-compare fitting routines over the entire glucose titration and temperature range to provide minimal reduced chi-squared values and the highest time resolution possible. Using a standard ligand-binding model, the resulting distributions show that the closed (ligand-bound) conformation exists even at zero glucose concentration. At 20^oC, the relative abundance of this conformation is as high as 40%. The temperature dependence of this conformational study will be discussed and related to the ligand-binding free energy surface.

  12. Identification of key residues for protein conformational transition using elastic network model.

    Su, Ji Guo; Xu, Xian Jin; Li, Chun Hua; Chen, Wei Zu; Wang, Cun Xin

    2011-11-07

    Proteins usually undergo conformational transitions between structurally disparate states to fulfill their functions. The large-scale allosteric conformational transitions are believed to involve some key residues that mediate the conformational movements between different regions of the protein. In the present work, a thermodynamic method based on the elastic network model is proposed to predict the key residues involved in protein conformational transitions. In our method, the key functional sites are identified as the residues whose perturbations largely influence the free energy difference between the protein states before and after transition. Two proteins, nucleotide binding domain of the heat shock protein 70 and human/rat DNA polymerase β, are used as case studies to identify the critical residues responsible for their open-closed conformational transitions. The results show that the functionally important residues mainly locate at the following regions for these two proteins: (1) the bridging point at the interface between the subdomains that control the opening and closure of the binding cleft; (2) the hinge region between different subdomains, which mediates the cooperative motions between the corresponding subdomains; and (3) the substrate binding sites. The similarity in the positions of the key residues for these two proteins may indicate a common mechanism in their conformational transitions.

  13. The Protein-Sparing Modified Fast Diet

    Marwan Bakhach MD

    2016-01-01

    Full Text Available Objectives: The protein-sparing modified fast (PSMF is a rigorous way of rapidly losing a large amount of weight. Although adult studies have shown the PSMF to be effective, data in adolescents are lacking. The aim of this study was to determine the efficacy and safety of the PSMF in severely obese adolescents. Methods: 12 subjects who were evaluated in the Obesity Management Program at the Cleveland Clinic from 2011 to 2014 were included. The subjects were initiated on the PSMF after failing other conventional methods of weight loss. Once the goal weight was achieved, subjects were transitioned to the refeeding phase for weight maintenance. Results: Follow-up was scheduled at 3-month (11 patients and 6-month (6 patients intervals. At the 6-month follow-up visit, the average weight loss was 11.19 kg (95% confidence interval = -5.4, -27.8, P = .028, with average of 9.8% from baseline. Fifty percent of subjects had >5% weight loss and 20% had >10% weight loss. Four patients were lost to the follow-up (40%. An improvement was noted in total cholesterol and high-density lipoprotein. Due to a small sample size these results were not statistically significant. Side effects reported by subjects were mild dehydration due to nausea (2 patients, decreased energy (1 patient, and transient labile mood (1 patient. No life-threatening side effects were reported. Conclusion: Our results show that the PSMF diet can be used as an effective and safe method in the outpatient setting for rapid weight loss in adolescents with severe obesity.

  14. Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)

    Esmail Nazari, Zeinab; van de Weert, Marco; Bou-Assaf, George

    2016-01-01

    Hydrogen Deuterium Exchange coupled to Mass Spectrometry (HDX-MS) has become an established method for analysis of protein higher-order structure. Here, we use HDX-MS methodology based on manual Solid-Phase Extraction (SPE) to allow fast and simplified conformational analysis of proteins under...... pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In Mode 1, we used DMSO-containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable...... in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that the SPE-HDX-MS system can...

  15. Energetically Unfavorable Amide Conformations for N6-Acetyllysine Side Chains in Refined Protein Structures

    Genshaft, Alexander; Moser, Joe-Ann S.; D'Antonio, Edward L.; Bowman, Christine M.; Christianson, David W.

    2013-01-01

    The reversible acetylation of lysine to form N6-acetyllysine in the regulation of protein function is a hallmark of epigenetics. Acetylation of the positively charged amino group of the lysine side chain generates a neutral N-alkylacetamide moiety that serves as a molecular “switch” for the modulation of protein function and protein-protein interactions. We now report the analysis of 381 N6-acetyllysine side chain amide conformations as found in 79 protein crystal structures and 11 protein NMR structures deposited in the Protein Data Bank (PDB) of the Research Collaboratory for Structural Bioinformatics. We find that only 74.3% of N6-acetyllysine residues in protein crystal structures and 46.5% in protein NMR structures contain amide groups with energetically preferred trans or generously trans conformations. Surprisingly, 17.6% of N6-acetyllysine residues in protein crystal structures and 5.3% in protein NMR structures contain amide groups with energetically unfavorable cis or generously cis conformations. Even more surprisingly, 8.1% of N6-acetyllysine residues in protein crystal structures and 48.2% in NMR structures contain amide groups with energetically prohibitive twisted conformations that approach the transition state structure for cis-trans isomerization. In contrast, 109 unique N-alkylacetamide groups contained in 84 highly-accurate small molecule crystal structures retrieved from the Cambridge Structural Database exclusively adopt energetically preferred trans conformations. Therefore, we conclude that cis and twisted N6-acetyllysine amides in protein structures deposited in the PDB are erroneously modeled due to their energetically unfavorable or prohibitive conformations. PMID:23401043

  16. Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

    2015-01-01

    Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

  17. Conformational analysis of g protein-coupled receptor signaling by hydrogen/deuterium exchange mass spectrometry.

    Li, Sheng; Lee, Su Youn; Chung, Ka Young

    2015-01-01

    Conformational change and protein-protein interactions are two major mechanisms of membrane protein signal transduction, including G protein-coupled receptors (GPCRs). Upon agonist binding, GPCRs change conformation, resulting in interaction with downstream signaling molecules such as G proteins. To understand the precise signaling mechanism, studies have investigated the structural mechanism of GPCR signaling using X-ray crystallography, nuclear magnetic resonance (NMR), or electron paramagnetic resonance. In addition to these techniques, hydrogen/deuterium exchange mass spectrometry (HDX-MS) has recently been used in GPCR studies. HDX-MS measures the rate at which peptide amide hydrogens exchange with deuterium in the solvent. Exposed or flexible regions have higher exchange rates and excluded or ordered regions have lower exchange rates. Therefore, HDX-MS is a useful tool for studying protein-protein interfaces and conformational changes after protein activation or protein-protein interactions. Although HDX-MS does not give high-resolution structures, it analyzes protein conformations that are difficult to study with X-ray crystallography or NMR. Furthermore, conformational information from HDX-MS can help in the crystallization of X-ray crystallography by suggesting highly flexible regions. Interactions between GPCRs and downstream signaling molecules are not easily analyzed by X-ray crystallography or NMR because of the large size of the GPCR-signaling molecule complexes, hydrophobicity, and flexibility of GPCRs. HDX-MS could be useful for analyzing the conformational mechanism of GPCR signaling. In this chapter, we discuss details of HDX-MS for analyzing GPCRs using the β2AR-G protein complex as a model system. © 2015 Elsevier Inc. All rights reserved.

  18. The role of metals in protein conformational disorders - The case of prion protein and Aβ -peptide

    De Santis, E; Minicozzi, V; Morante, S; Rossi, G C; Stellato, F

    2016-01-01

    Protein conformational disorders are members of a vast class of pathologies in which endogenous proteins or peptides undergo a misfolding process by switching from the physiological soluble configuration to a pathological fibrillar insoluble state. An important, but not yet fully elucidated, role in the process appears to be played by transition metal ions, mainly copper and zinc. X-ray absorption spectroscopy is one of the most suitable techniques for the structural characterization of biological molecules in complex with metal. Owing to its chemical selectivity and sensitivity to the local atomic geometry around the absorber, it can be successfully used to study the environment of metal ions in complex with proteins and peptides in physiological conditions. In this paper we present X-ray absorption spectroscopy studies of the metal ions coordination modes in systems where metals are complexed with specific amyloidogenic proteins and peptides. In particular, we show results concerning the Amyloid β peptide, that is involved in Alzheimer's disease, and the Prion protein, that is responsible for the Transmissible Spongiform Encephalopathy. Our findings suggest that the copper and zinc ions may play a crucial role in the aggregation and fibril formation process of these two biomolecules. Elucidating this kind of interaction could be a key preliminary step before any viable therapy can be conceived or designed. (paper)

  19. The role of metals in protein conformational disorders - The case of prion protein and Aβ -peptide

    De Santis, E.; Minicozzi, V.; Morante, S.; Rossi, G. C.; Stellato, F.

    2016-02-01

    Protein conformational disorders are members of a vast class of pathologies in which endogenous proteins or peptides undergo a misfolding process by switching from the physiological soluble configuration to a pathological fibrillar insoluble state. An important, but not yet fully elucidated, role in the process appears to be played by transition metal ions, mainly copper and zinc. X-ray absorption spectroscopy is one of the most suitable techniques for the structural characterization of biological molecules in complex with metal. Owing to its chemical selectivity and sensitivity to the local atomic geometry around the absorber, it can be successfully used to study the environment of metal ions in complex with proteins and peptides in physiological conditions. In this paper we present X-ray absorption spectroscopy studies of the metal ions coordination modes in systems where metals are complexed with specific amyloidogenic proteins and peptides. In particular, we show results concerning the Amyloid β peptide, that is involved in Alzheimer's disease, and the Prion protein, that is responsible for the Transmissible Spongiform Encephalopathy. Our findings suggest that the copper and zinc ions may play a crucial role in the aggregation and fibril formation process of these two biomolecules. Elucidating this kind of interaction could be a key preliminary step before any viable therapy can be conceived or designed.

  20. How Diverse are the Protein-Bound Conformations of Small-Molecule Drugs and Cofactors?

    Friedrich, Nils-Ole; Simsir, Méliné; Kirchmair, Johannes

    2018-03-01

    Knowledge of the bioactive conformations of small molecules or the ability to predict them with theoretical methods is of key importance to the design of bioactive compounds such as drugs, agrochemicals and cosmetics. Using an elaborate cheminformatics pipeline, which also evaluates the support of individual atom coordinates by the measured electron density, we compiled a complete set (“Sperrylite Dataset”) of high-quality structures of protein-bound ligand conformations from the PDB. The Sperrylite Dataset consists of a total of 10,936 high-quality structures of 4548 unique ligands. Based on this dataset, we assessed the variability of the bioactive conformations of 91 small molecules—each represented by a minimum of ten structures—and found it to be largely independent of the number of rotatable bonds. Sixty-nine molecules had at least two distinct conformations (defined by an RMSD greater than 1 Å). For a representative subset of 17 approved drugs and cofactors we observed a clear trend for the formation of few clusters of highly similar conformers. Even for proteins that share a very low sequence identity, ligands were regularly found to adopt similar conformations. For cofactors, a clear trend for extended conformations was measured, although in few cases also coiled conformers were observed. The Sperrylite Dataset is available for download from http://www.zbh.uni-hamburg.de/sperrylite_dataset.

  1. Asprosin, a fasting-induced glucogenic protein hormone

    Hepatic glucose release into the circulation is vital for brain function and survival during periods of fasting and is modulated by an array of hormones that precisely regulate plasma glucose levels. We have identified a fasting-induced protein hormone that modulates hepatic glucose release. It is t...

  2. Fast mapping of global protein folding states by multivariate NMR:

    Malmendal, Anders; Underhaug, Jarl; Otzen, Daniel

    2010-01-01

    To obtain insight into the functions of proteins and their specific roles, it is important to establish efficient procedures for exploring the states that encapsulate their conformational space. Global Protein folding State mapping by multivariate NMR (GPS NMR) is a powerful high-throughput method......-lactalbumin in the presence of the anionic surfactant sodium dodecyl sulfate, SDS, and compare these with other surfactants, acid, denaturants and heat....

  3. Mechanisms of protein misfolding in conformational lung diseases.

    McElvaney, N G

    2012-08-01

    Genetic or environmentally-induced alterations in protein structure interfere with the correct folding, assembly and trafficking of proteins. In the lung the expression of misfolded proteins can induce a variety of pathogenetic effects. Cystic fibrosis (CF) and alpha-1 antitrypsin (AAT) deficiency are two major clinically relevant pulmonary disorders associated with protein misfolding. Both are genetic diseases the primary causes of which are expression of mutant alleles of the cystic fibrosis transmembrane conductance regulator (CFTR) and SERPINA1, respectively. The most common and best studied mutant forms of CFTR and AAT are ΔF508 CFTR and the Glu342Lys mutant of AAT called ZAAT, respectively. Non-genetic mechanisms can also damage protein structure and induce protein misfolding in the lung. Cigarette-smoke contains oxidants and other factors that can modify a protein\\'s structure, and is one of the most significant environmental causes of protein damage within the lung. Herein we describe the mechanisms controlling the folding of wild type and mutant versions of CFTR and AAT proteins, and explore the consequences of cigarette-smoke-induced effects on the protein folding machinery in the lung.

  4. Enrichment of Druggable Conformations from Apo Protein Structures Using Cosolvent-Accelerated Molecular Dynamics

    Andrew Kalenkiewicz

    2015-04-01

    Full Text Available Here we describe the development of an improved workflow for utilizing experimental and simulated protein conformations in the structure-based design of inhibitors for anti-apoptotic Bcl-2 family proteins. Traditional structure-based approaches on similar targets are often constrained by the sparsity of available structures and difficulties in finding lead compounds that dock against flat, flexible protein-protein interaction surfaces. By employing computational docking of known small molecule inhibitors, we have demonstrated that structural ensembles derived from either accelerated MD (aMD or MD in the presence of an organic cosolvent generally give better scores than those assessed from analogous conventional MD. Furthermore, conformations obtained from combined cosolvent aMD simulations started with the apo-Bcl-xL structure yielded better average and minimum docking scores for known binders than an ensemble of 72 experimental apo- and ligand-bound Bcl-xL structures. A detailed analysis of the simulated conformations indicates that the aMD effectively enhanced conformational sampling of the flexible helices flanking the main Bcl-xL binding groove, permitting the cosolvent acting as small ligands to penetrate more deeply into the binding pocket and shape ligand-bound conformations not evident in conventional simulations. We believe this approach could be useful for identifying inhibitors against other protein-protein interaction systems involving highly flexible binding sites, particularly for targets with less accumulated structural data.

  5. Monitoring Conformational Landscape of Ovine Prion Protein Monomer Using Ion Mobility Coupled to Mass Spectrometry

    Van der Rest, Guillaume; Rezaei, Human; Halgand, Frédéric

    2017-02-01

    Prion protein is involved in deadly neurodegenerative diseases. Its pathogenicity is linked to its structural conversion (α-helix to β-strand transition). However, recent studies suggest that prion protein can follow a plurality of conversion pathways, which hints towards different conformers that might coexist in solution. To gain insights on the plasticity of the ovine prion protein (PrP) monomer, wild type (A136, R154, Q171), mutants and deletions of ARQ were studied by traveling wave ion mobility experiments coupled to mass spectrometry. In order to perform the analysis of a large body of data sets, we designed and evaluated the performance of a processing pipeline based on Driftscope peak detection and a homemade script for automated peak assignment, annotation, and quantification on specific multiply charged protein data. Using this approach, we showed that in the gas phase, PrPs are represented by at least three conformer families differing in both charge state distribution and collisional cross-section, in agreement with the work of Hilton et al. (2010). We also showed that this plasticity is borne both by the N- and C-terminal domains. Effect of protein concentration, pH and temperature were also assessed, showing that (1) pH does not affect conformer distributions, (2) protein concentration modifies the conformational landscape of one mutant (I208M) only, and (3) heating leads to other unfolded species and to a modification of the conformer intensity ratios.

  6. Substantial conformational change mediated by charge-triad residues of the death effector domain in protein-protein interactions.

    Edward C Twomey

    Full Text Available Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15 protein in the complex with a mitogen-activated protein (MAP kinase, extracellular regulated kinase 2 (ERK2, which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.

  7. Molecular modeling of the conformational dynamics of the cellular prion protein

    Nguyen, Charles; Colling, Ian; Bartz, Jason; Soto, Patricia

    2014-03-01

    Prions are infectious agents responsible for transmissible spongiform encephalopathies (TSEs), a type of fatal neurodegenerative disease in mammals. Prions propagate biological information by conversion of the non-pathological version of the prion protein to the infectious conformation, PrPSc. A wealth of knowledge has shed light on the nature and mechanism of prion protein conversion. In spite of the significance of this problem, we are far from fully understanding the conformational dynamics of the cellular isoform. To remedy this situation we employ multiple biomolecular modeling techniques such as docking and molecular dynamics simulations to map the free energy landscape and determine what specific regions of the prion protein are most conductive to binding. The overall goal is to characterize the conformational dynamics of the cell form of the prion protein, PrPc, to gain insight into inhibition pathways against misfolding. NE EPSCoR FIRST Award to Patricia Soto.

  8. Globular conformation of some ribosomal proteins in solution

    Serdyuk, I.N.; Spirin, A.S.

    1978-01-01

    The possibility that such RNA-binding proteins of the 30 S subparticle as S4, S7, S8 and S16 exist in the form of compact globules in solution has been explored experimentally. These proteins have been studied in D 2 O solution by neutron scattering to measure their radii of gyration. This type of radiation using D 2 O as a solvent provides the maximum 'contrast', that is the maximum difference between the scattering of the protein and the solvent. It allowed measurements to be made using protein at <= 1.5 mg/ml. The radii of gyration for the ribosomal proteins S4, S7, S8 and S16 were found to be relatively small corresponding to the radii of gyration of compact globular proteins of the same molecular weights. (Auth.)

  9. Study on protein conformation and adsorption behaviors in nanodiamond particle-protein complexes

    Wang Haidong; Niu, Catherine Hui; Yang Qiaoqin; Badea, Ildiko

    2011-01-01

    In the present research, the conformation of bovine serum albumin (BSA) in the nanodiamond particle (ND)-BSA complex was studied by Fourier transform infrared spectroscopy, fluorescence spectroscopy, UV-vis spectroscopy, and circular dichroism spectroscopy. The spectroscopic study revealed that most BSA structural features could be preserved in the complex though the BSA underwent conformational changes in the complex due to ND-BSA interaction. In addition, BSA adsorption isotherms and zeta-potential measurements were employed to investigate the pH dependence of the ND-BSA interaction. The changes in surface charge of the ND-BSA complex with pH variations indicated that the binding of BSA to ND might lead to not only the adsorption of BSA onto the ND surface but also the partial breakup of ND aggregates into relatively small ND-BSA aggregates because of the strong binding force between ND and BSA. The results show that ND is an excellent platform for protein immobilization with high affinity and holds great potential to be used for biosensor applications.

  10. Study on protein conformation and adsorption behaviors in nanodiamond particle-protein complexes

    Wang Haidong [Department of Biomedical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9 (Canada); Niu, Catherine Hui [Department of Chemical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9 (Canada); Yang Qiaoqin [Department of Mechanical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9 (Canada); Badea, Ildiko, E-mail: catherine.niu@usask.ca [Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, S7N 5C9 (Canada)

    2011-04-08

    In the present research, the conformation of bovine serum albumin (BSA) in the nanodiamond particle (ND)-BSA complex was studied by Fourier transform infrared spectroscopy, fluorescence spectroscopy, UV-vis spectroscopy, and circular dichroism spectroscopy. The spectroscopic study revealed that most BSA structural features could be preserved in the complex though the BSA underwent conformational changes in the complex due to ND-BSA interaction. In addition, BSA adsorption isotherms and zeta-potential measurements were employed to investigate the pH dependence of the ND-BSA interaction. The changes in surface charge of the ND-BSA complex with pH variations indicated that the binding of BSA to ND might lead to not only the adsorption of BSA onto the ND surface but also the partial breakup of ND aggregates into relatively small ND-BSA aggregates because of the strong binding force between ND and BSA. The results show that ND is an excellent platform for protein immobilization with high affinity and holds great potential to be used for biosensor applications.

  11. Conformational changes in the G protein Gs induced by the β2 adrenergic receptor

    Chung, Ka Young; Rasmussen, Søren Gøgsig Faarup; Liu, Tong

    2011-01-01

    and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide...

  12. Guiding exploration in conformational feature space with Lipschitz underestimation for ab-initio protein structure prediction.

    Hao, Xiaohu; Zhang, Guijun; Zhou, Xiaogen

    2018-04-01

    Computing conformations which are essential to associate structural and functional information with gene sequences, is challenging due to the high dimensionality and rugged energy surface of the protein conformational space. Consequently, the dimension of the protein conformational space should be reduced to a proper level, and an effective exploring algorithm should be proposed. In this paper, a plug-in method for guiding exploration in conformational feature space with Lipschitz underestimation (LUE) for ab-initio protein structure prediction is proposed. The conformational space is converted into ultrafast shape recognition (USR) feature space firstly. Based on the USR feature space, the conformational space can be further converted into Underestimation space according to Lipschitz estimation theory for guiding exploration. As a consequence of the use of underestimation model, the tight lower bound estimate information can be used for exploration guidance, the invalid sampling areas can be eliminated in advance, and the number of energy function evaluations can be reduced. The proposed method provides a novel technique to solve the exploring problem of protein conformational space. LUE is applied to differential evolution (DE) algorithm, and metropolis Monte Carlo(MMC) algorithm which is available in the Rosetta; When LUE is applied to DE and MMC, it will be screened by the underestimation method prior to energy calculation and selection. Further, LUE is compared with DE and MMC by testing on 15 small-to-medium structurally diverse proteins. Test results show that near-native protein structures with higher accuracy can be obtained more rapidly and efficiently with the use of LUE. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Fast loop modeling for protein structures

    Zhang, Jiong; Nguyen, Son; Shang, Yi; Xu, Dong; Kosztin, Ioan

    2015-03-01

    X-ray crystallography is the main method for determining 3D protein structures. In many cases, however, flexible loop regions of proteins cannot be resolved by this approach. This leads to incomplete structures in the protein data bank, preventing further computational study and analysis of these proteins. For instance, all-atom molecular dynamics (MD) simulation studies of structure-function relationship require complete protein structures. To address this shortcoming, we have developed and implemented an efficient computational method for building missing protein loops. The method is database driven and uses deep learning and multi-dimensional scaling algorithms. We have implemented the method as a simple stand-alone program, which can also be used as a plugin in existing molecular modeling software, e.g., VMD. The quality and stability of the generated structures are assessed and tested via energy scoring functions and by equilibrium MD simulations. The proposed method can also be used in template-based protein structure prediction. Work supported by the National Institutes of Health [R01 GM100701]. Computer time was provided by the University of Missouri Bioinformatics Consortium.

  14. Protein Conformational Plasticity: the 'off-on' Switching Movement in Cdk5

    Cavalli, Andrea; Recanatini, Maurizio; Berteotti, Anna; Branduardi, Davide; Gervasio, Francesco L.; Parrinello, Michele

    2007-01-01

    Cyclin-dependent kinases (CDKs) are mostly known for their role in the cell cycle regulation. The activation mechanism of all CDKs involves the association with a regulatory protein, generally a cyclin, that binds to the kinase unit and stabilizes a catalytically active conformation. Active and inactive conformations of CDKs are characterized by the different spatial localization of two typical elements, namely the activation loop and an □-helix, whose amino-acid composition varies throughout the family

  15. REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment

    Hao Yang

    2018-01-01

    Full Text Available Bryostatin 1 (henceforth bryostatin is in clinical trials for the treatment of Alzheimer’s disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC–ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

  16. FastBLAST: homology relationships for millions of proteins.

    Morgan N Price

    Full Text Available BACKGROUND: All-versus-all BLAST, which searches for homologous pairs of sequences in a database of proteins, is used to identify potential orthologs, to find new protein families, and to provide rapid access to these homology relationships. As DNA sequencing accelerates and data sets grow, all-versus-all BLAST has become computationally demanding. METHODOLOGY/PRINCIPAL FINDINGS: We present FastBLAST, a heuristic replacement for all-versus-all BLAST that relies on alignments of proteins to known families, obtained from tools such as PSI-BLAST and HMMer. FastBLAST avoids most of the work of all-versus-all BLAST by taking advantage of these alignments and by clustering similar sequences. FastBLAST runs in two stages: the first stage identifies additional families and aligns them, and the second stage quickly identifies the homologs of a query sequence, based on the alignments of the families, before generating pairwise alignments. On 6.53 million proteins from the non-redundant Genbank database ("NR", FastBLAST identifies new families 25 times faster than all-versus-all BLAST. Once the first stage is completed, FastBLAST identifies homologs for the average query in less than 5 seconds (8.6 times faster than BLAST and gives nearly identical results. For hits above 70 bits, FastBLAST identifies 98% of the top 3,250 hits per query. CONCLUSIONS/SIGNIFICANCE: FastBLAST enables research groups that do not have supercomputers to analyze large protein sequence data sets. FastBLAST is open source software and is available at http://microbesonline.org/fastblast.

  17. Visualization of SV2A conformations in situ by the use of Protein Tomography

    Lynch, Berkley A.; Matagne, Alain; Braennstroem, Annika; Euler, Anne von; Jansson, Magnus; Hauzenberger, Elenor; Soederhaell, J. Arvid

    2008-01-01

    The synaptic vesicle protein 2A (SV2A), the brain-binding site of the anti-epileptic drug levetiracetam (LEV), has been characterized by Protein Tomography TM . We identified two major conformations of SV2A in mouse brain tissue: first, a compact, funnel-structure with a pore-like opening towards the cytoplasm; second, a more open, V-shaped structure with a cleft-like opening towards the intravesicular space. The large differences between these conformations suggest a high degree of flexibility and support a valve-like mechanism consistent with the postulated transporter role of SV2A. These two conformations are represented both in samples treated with LEV, and in saline-treated samples, which indicates that LEV binding does not cause a large-scale conformational change of SV2A, or lock a specific conformational state of the protein. This study provides the first direct structural data on SV2A, and supports a transporter function suggested by sequence homology to MFS class of transporter proteins

  18. Driving Calmodulin Protein towards Conformational Shift by Changing Ionization States of Select Residues

    Negi, Sunita; Atilgan, Ali Rana; Atilgan, Canan

    2012-01-01

    Proteins are complex systems made up of many conformational sub-states which are mainly determined by the folded structure. External factors such as solvent type, temperature, pH and ionic strength play a very important role in the conformations sampled by proteins. Here we study the conformational multiplicity of calmodulin (CaM) which is a protein that plays an important role in calcium signaling pathways in the eukaryotic cells. CaM can bind to a variety of other proteins or small organic compounds, and mediates different physiological processes by activating various enzymes. Binding of calcium ions and proteins or small organic molecules to CaM induces large conformational changes that are distinct to each interacting partner. In particular, we discuss the effect of pH variation on the conformations of CaM. By using the pKa values of the charged residues as a basis to assign protonation states, the conformational changes induced in CaM by reducing the pH are studied by molecular dynamics simulations. Our current view suggests that at high pH, barrier crossing to the compact form is prevented by repulsive electrostatic interactions between the two lobes. At reduced pH, not only is barrier crossing facilitated by protonation of residues, but also conformations which are on average more compact are attained. The latter are in accordance with the fluorescence resonance energy transfer experiment results of other workers. The key events leading to the conformational change from the open to the compact conformation are (i) formation of a salt bridge between the N-lobe and the linker, stabilizing their relative motions, (ii) bending of the C-lobe towards the N-lobe, leading to a lowering of the interaction energy between the two-lobes, (iii) formation of a hydrophobic patch between the two lobes, further stabilizing the bent conformation by reducing the entropic cost of the compact form, (iv) sharing of a Ca +2 ion between the two lobes.

  19. Driving Calmodulin Protein towards Conformational Shift by Changing Ionization States of Select Residues

    Negi, Sunita; Rana Atilgan, Ali; Atilgan, Canan

    2012-12-01

    Proteins are complex systems made up of many conformational sub-states which are mainly determined by the folded structure. External factors such as solvent type, temperature, pH and ionic strength play a very important role in the conformations sampled by proteins. Here we study the conformational multiplicity of calmodulin (CaM) which is a protein that plays an important role in calcium signaling pathways in the eukaryotic cells. CaM can bind to a variety of other proteins or small organic compounds, and mediates different physiological processes by activating various enzymes. Binding of calcium ions and proteins or small organic molecules to CaM induces large conformational changes that are distinct to each interacting partner. In particular, we discuss the effect of pH variation on the conformations of CaM. By using the pKa values of the charged residues as a basis to assign protonation states, the conformational changes induced in CaM by reducing the pH are studied by molecular dynamics simulations. Our current view suggests that at high pH, barrier crossing to the compact form is prevented by repulsive electrostatic interactions between the two lobes. At reduced pH, not only is barrier crossing facilitated by protonation of residues, but also conformations which are on average more compact are attained. The latter are in accordance with the fluorescence resonance energy transfer experiment results of other workers. The key events leading to the conformational change from the open to the compact conformation are (i) formation of a salt bridge between the N-lobe and the linker, stabilizing their relative motions, (ii) bending of the C-lobe towards the N-lobe, leading to a lowering of the interaction energy between the two-lobes, (iii) formation of a hydrophobic patch between the two lobes, further stabilizing the bent conformation by reducing the entropic cost of the compact form, (iv) sharing of a Ca+2 ion between the two lobes.

  20. Molecular mechanics work station for protein conformational studies

    Fine, R.; Levinthal, C.; Schoenborn, B.; Dimmier, G.; Rankowitz, C.

    1984-01-01

    Interest in computational problems in Biology has intensified over the last few years, partly due to the development of techniques for the rapid cloning, sequencing, and mutagenesis of genes from organisims ranging from E. coli to Man. The central dogma of molecular biology; that DNA codes for mRNA which codes for protein, has been understood in a linear programming sense since the genetic code was cracked. But what is not understood at present is how a protein, once assembled as a long sequence of amino acids, folds back on itself to produce a three-dimensional structure which is unique to that protein and which dictates its chemical and biological activity. This folding process is purely physics, and involves the time evolution of a system of several thousand atoms which interact with each other and with atoms from the surrounding solvent. Molecular dynamics simulations on smaller molecules suggest that approaches which treat the protein as a classical ensemble of atoms interacting with each other via an empirical Hamiltonian can yield the kind of predictive results one would like when applied to proteins

  1. Conformational targeting of fibrillar polyglutamine proteins in live cells escalates aggregation and cytotoxicity.

    Erik Kvam

    2009-05-01

    Full Text Available Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.Using atomic force microscopy (AFM and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to

  2. Prediction of protein conformational freedom from distance constraints

    de Groot, B.L.; van Aalten, D.M.F.; Scheek, R.M.; Amadei, A; Vriend, G.; Berendsen, H.J.C.

    1997-01-01

    A method is presented that generates random protein structures that fulfil a set of upper and lower interatomic distance limits, These limits depend on distances measured in experimental structures and the strength of the interatomic interaction, Structural differences between generated structures

  3. Binding induced conformational changes of proteins correlate with their intrinsic fluctuations: a case study of antibodies

    Keskin Ozlem

    2007-05-01

    Full Text Available Abstract Background How antibodies recognize and bind to antigens can not be totally explained by rigid shape and electrostatic complimentarity models. Alternatively, pre-existing equilibrium hypothesis states that the native state of an antibody is not defined by a single rigid conformation but instead with an ensemble of similar conformations that co-exist at equilibrium. Antigens bind to one of the preferred conformations making this conformation more abundant shifting the equilibrium. Results Here, two antibodies, a germline antibody of 36–65 Fab and a monoclonal antibody, SPE7 are studied in detail to elucidate the mechanism of antibody-antigen recognition and to understand how a single antibody recognizes different antigens. An elastic network model, Anisotropic Network Model (ANM is used in the calculations. Pre-existing equilibrium is not restricted to apply to antibodies. Intrinsic fluctuations of eight proteins, from different classes of proteins, such as enzymes, binding and transport proteins are investigated to test the suitability of the method. The intrinsic fluctuations are compared with the experimentally observed ligand induced conformational changes of these proteins. The results show that the intrinsic fluctuations obtained by theoretical methods correlate with structural changes observed when a ligand is bound to the protein. The decomposition of the total fluctuations serves to identify the different individual modes of motion, ranging from the most cooperative ones involving the overall structure, to the most localized ones. Conclusion Results suggest that the pre-equilibrium concept holds for antibodies and the promiscuity of antibodies can also be explained this hypothesis: a limited number of conformational states driven by intrinsic motions of an antibody might be adequate to bind to different antigens.

  4. De novo protein structure prediction by dynamic fragment assembly and conformational space annealing.

    Lee, Juyong; Lee, Jinhyuk; Sasaki, Takeshi N; Sasai, Masaki; Seok, Chaok; Lee, Jooyoung

    2011-08-01

    Ab initio protein structure prediction is a challenging problem that requires both an accurate energetic representation of a protein structure and an efficient conformational sampling method for successful protein modeling. In this article, we present an ab initio structure prediction method which combines a recently suggested novel way of fragment assembly, dynamic fragment assembly (DFA) and conformational space annealing (CSA) algorithm. In DFA, model structures are scored by continuous functions constructed based on short- and long-range structural restraint information from a fragment library. Here, DFA is represented by the full-atom model by CHARMM with the addition of the empirical potential of DFIRE. The relative contributions between various energy terms are optimized using linear programming. The conformational sampling was carried out with CSA algorithm, which can find low energy conformations more efficiently than simulated annealing used in the existing DFA study. The newly introduced DFA energy function and CSA sampling algorithm are implemented into CHARMM. Test results on 30 small single-domain proteins and 13 template-free modeling targets of the 8th Critical Assessment of protein Structure Prediction show that the current method provides comparable and complementary prediction results to existing top methods. Copyright © 2011 Wiley-Liss, Inc.

  5. A unified conformational selection and induced fit approach to protein-peptide docking.

    Mikael Trellet

    Full Text Available Protein-peptide interactions are vital for the cell. They mediate, inhibit or serve as structural components in nearly 40% of all macromolecular interactions, and are often associated with diseases, making them interesting leads for protein drug design. In recent years, large-scale technologies have enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. Yet, the paucity of data regarding their molecular binding mechanisms together with their inherent flexibility makes the structural prediction of protein-peptide interactions very challenging. This leaves flexible docking as one of the few amenable computational techniques to model these complexes. We present here an ensemble, flexible protein-peptide docking protocol that combines conformational selection and induced fit mechanisms. Starting from an ensemble of three peptide conformations (extended, a-helix, polyproline-II, flexible docking with HADDOCK generates 79.4% of high quality models for bound/unbound and 69.4% for unbound/unbound docking when tested against the largest protein-peptide complexes benchmark dataset available to date. Conformational selection at the rigid-body docking stage successfully recovers the most relevant conformation for a given protein-peptide complex and the subsequent flexible refinement further improves the interface by up to 4.5 Å interface RMSD. Cluster-based scoring of the models results in a selection of near-native solutions in the top three for ∼75% of the successfully predicted cases. This unified conformational selection and induced fit approach to protein-peptide docking should open the route to the modeling of challenging systems such as disorder-order transitions taking place upon binding, significantly expanding the applicability limit of biomolecular interaction modeling by docking.

  6. Studies of Single Biomolecules, DNA Conformational Dynamics, and Protein Binding

    2008-07-11

    Nucleotide Base pairs Hydrogen bonds FIG. 1: Ladder structure of DNA showing the Watson - Crick bonding of the bases A, T, G, and C which are suspended by a...protected against unwanted action of chemicals and proteins. The three-dimensional structure of DNA is the famed Watson - Crick double-helix, the equilibrium...quantitative analysis [88]. [1] A. Kornberg and T. A. Baker, DNA Replication (W. H. Freeman, New York, 1992). [2] J. D. Watson and F. H. C. Crick

  7. Conformational disorder in folded and intrinsically disordered proteins from nuclear magnetic resonance

    Salmon, Loic

    2010-01-01

    Biological macromolecules are, by essence, dynamical systems. While the importance of this flexibility is nowadays well established, the accurate characterization of the conformational disorder of these systems remains an important challenge. Nuclear magnetic resonance spectroscopy is a unique tool to probe these motions at atomic level, through the analysis of spin relaxation or residual dipolar couplings. The latter allows all motions occurring at timescales faster than the millisecond to be investigated, including physiologically important timescales. The information presents in those couplings is interpreted here using mainly analytical approaches in order to quantify the amounts of dynamics present in folded protein, to determine the direction of those motions and to obtain structural information within this conformational disorder. These analytical approaches are complemented by numerical methods, that allowed the observation of phenomena from a different point of view or the investigation of other systems such as intrinsically disordered proteins. All of these studies demonstrate an important complementarity between structural order and conformational disorder. (author)

  8. Anion induced conformational preference of Cα NN motif residues in functional proteins.

    Patra, Piya; Ghosh, Mahua; Banerjee, Raja; Chakrabarti, Jaydeb

    2017-12-01

    Among different ligand binding motifs, anion binding C α NN motif consisting of peptide backbone atoms of three consecutive residues are observed to be important for recognition of free anions, like sulphate or biphosphate and participate in different key functions. Here we study the interaction of sulphate and biphosphate with C α NN motif present in different proteins. Instead of total protein, a peptide fragment has been studied keeping C α NN motif flanked in between other residues. We use classical force field based molecular dynamics simulations to understand the stability of this motif. Our data indicate fluctuations in conformational preferences of the motif residues in absence of the anion. The anion gives stability to one of these conformations. However, the anion induced conformational preferences are highly sequence dependent and specific to the type of anion. In particular, the polar residues are more favourable compared to the other residues for recognising the anion. © 2017 Wiley Periodicals, Inc.

  9. A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

    Eden, Thomas; Menzel, Stephan; Wesolowski, Janusz; Bergmann, Philine; Nissen, Marion; Dubberke, Gudrun; Seyfried, Fabienne; Albrecht, Birte; Haag, Friedrich; Koch-Nolte, Friedrich

    2018-01-01

    Nanobodies (Nbs) are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic

  10. A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

    Thomas Eden

    2018-01-01

    Full Text Available Nanobodies (Nbs are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy-chain antibodies naturally occurring in camelids. Nbs hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic Nbs because they are accessible to systemically injected biologics. In order to be effective, therapeutic Nbs must recognize their target membrane protein in native conformation. However, raising Nbs against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and, therefore, are difficult to purify in native conformation. Here, we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other costimulatory proteins. The plasmid is coated onto 1 µm gold particles that are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA-coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy-chain antibodies. The VHH/Nb coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific Nbs are selected by phage display or by screening of Nb-based heavy-chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully

  11. Ramadan Fasting Decreases Body Fat but Not Protein Mass.

    Fahrial Syam, Ari; Suryani Sobur, Cecep; Abdullah, Murdani; Makmun, Dadang

    2016-01-01

    Many studies have shown various results regarding the effects of Ramadan fasting on weight and body composition in healthy individuals. This study aimed to evaluate the effect of Ramadan fasting on body composition in healthy Indonesian medical staff. In this study, we examined the influence of Ramadan fasting on body composition in healthy medical staff. The longitudinal study was performed during and after Ramadan fasting in 2013 (August to October). Fourty-three medical staff members (physicians, nurses and nutritionists) at the Internal Medicine Ward of the Dr. Cipto Mangunkusumo General Hospital were measured to compare their calorie intake, weight, body mass index, waist-to-hip ratio (WHR), and body composition, including body fat, protein, minerals and water, on the first and 28(th) days of Ramadan and also 4-5 weeks after Ramadan fasting. Measurements were obtained for all 43 subjects on the 28(th) day of Ramadan, but they were obtained for only 25 subjects 4 - 5 weeks after Ramadan. By the 28(th) day of Ramadan, it was found that the body weight, BMI, body fat, water and mineral measures had decreased significantly (-0.874 ± 0.859 kg, P Ramadan, body weight and composition had returned to the same levels as on the first day of Ramadan. Ramadan fasting resulted in weight loss even it was only a temporary effect, as the weight was quickly regained within one month after fasting. The catabolism catabolic state, which is related to protein loss, was not triggered during Ramadan fasting. Further research is needed to evaluate the effects of weight loss during Ramadan fasting in healthy individuals.

  12. TRIP13 is a protein-remodeling AAA+ ATPase that catalyzes MAD2 conformation switching.

    Ye, Qiaozhen; Rosenberg, Scott C; Moeller, Arne; Speir, Jeffrey A; Su, Tiffany Y; Corbett, Kevin D

    2015-04-28

    The AAA+ family ATPase TRIP13 is a key regulator of meiotic recombination and the spindle assembly checkpoint, acting on signaling proteins of the conserved HORMA domain family. Here we present the structure of the Caenorhabditis elegans TRIP13 ortholog PCH-2, revealing a new family of AAA+ ATPase protein remodelers. PCH-2 possesses a substrate-recognition domain related to those of the protein remodelers NSF and p97, while its overall hexameric architecture and likely structural mechanism bear close similarities to the bacterial protein unfoldase ClpX. We find that TRIP13, aided by the adapter protein p31(comet), converts the HORMA-family spindle checkpoint protein MAD2 from a signaling-active 'closed' conformer to an inactive 'open' conformer. We propose that TRIP13 and p31(comet) collaborate to inactivate the spindle assembly checkpoint through MAD2 conformational conversion and disassembly of mitotic checkpoint complexes. A parallel HORMA protein disassembly activity likely underlies TRIP13's critical regulatory functions in meiotic chromosome structure and recombination.

  13. TRIP13 is a protein-remodeling AAA+ ATPase that catalyzes MAD2 conformation switching

    Ye, Qiaozhen [Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, United States; Rosenberg, Scott C. [Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, United States; Moeller, Arne [National Resource for Automated Molecular Microscopy, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, United States; Speir, Jeffrey A. [National Resource for Automated Molecular Microscopy, Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, United States; Su, Tiffany Y. [Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, United States; Corbett, Kevin D. [Ludwig Institute for Cancer Research, San Diego Branch, La Jolla, United States; Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, United States

    2015-04-28

    The AAA+ family ATPase TRIP13 is a key regulator of meiotic recombination and the spindle assembly checkpoint, acting on signaling proteins of the conserved HORMA domain family. Here we present the structure of the Caenorhabditis elegans TRIP13 ortholog PCH-2, revealing a new family of AAA+ ATPase protein remodelers. PCH-2 possesses a substrate-recognition domain related to those of the protein remodelers NSF and p97, while its overall hexameric architecture and likely structural mechanism bear close similarities to the bacterial protein unfoldase ClpX. We find that TRIP13, aided by the adapter protein p31(comet), converts the HORMA-family spindle checkpoint protein MAD2 from a signaling-active ‘closed’ conformer to an inactive ‘open’ conformer. We propose that TRIP13 and p31(comet) collaborate to inactivate the spindle assembly checkpoint through MAD2 conformational conversion and disassembly of mitotic checkpoint complexes. A parallel HORMA protein disassembly activity likely underlies TRIP13's critical regulatory functions in meiotic chromosome structure and recombination.

  14. Experimental conformational energy maps of proteins and peptides.

    Balaji, Govardhan A; Nagendra, H G; Balaji, Vitukudi N; Rao, Shashidhar N

    2017-06-01

    We have presented an extensive analysis of the peptide backbone dihedral angles in the PDB structures and computed experimental Ramachandran plots for their distributions seen under a various constraints on X-ray resolution, representativeness at different sequence identity percentages, and hydrogen bonding distances. These experimental distributions have been converted into isoenergy contour plots using the approach employed previously by F. M. Pohl. This has led to the identification of energetically favored minima in the Ramachandran (ϕ, ψ) plots in which global minima are predominantly observed either in the right-handed α-helical or the polyproline II regions. Further, we have identified low energy pathways for transitions between various minima in the (ϕ,ψ) plots. We have compared and presented the experimental plots with published theoretical plots obtained from both molecular mechanics and quantum mechanical approaches. In addition, we have developed and employed a root mean square deviation (RMSD) metric for isoenergy contours in various ranges, as a measure (in kcal.mol -1 ) to compare any two plots and determine the extent of correlation and similarity between their isoenergy contours. In general, we observe a greater degree of compatibility with experimental plots for energy maps obtained from molecular mechanics methods compared to most quantum mechanical methods. The experimental energy plots we have investigated could be helpful in refining protein structures obtained from X-ray, NMR, and electron microscopy and in refining force field parameters to enable simulations of peptide and protein structures that have higher degree of consistency with experiments. Proteins 2017; 85:979-1001. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Adjustments of Protein Metabolism in Fasting Arctic Charr, Salvelinus alpinus.

    Alicia A Cassidy

    Full Text Available Protein metabolism, including the interrelated processes of synthesis and degradation, mediates the growth of an animal. In ectothermic animals, protein metabolism is responsive to changes in both biotic and abiotic conditions. This study aimed to characterise responses of protein metabolism to food deprivation that occur in the coldwater salmonid, Arctic charr, Salvelinus alpinus. We compared two groups of Arctic charr: one fed continuously and the other deprived of food for 36 days. We measured the fractional rate of protein synthesis (KS in individuals from the fed and fasted groups using a flooding dose technique modified for the use of deuterium-labelled phenylalanine. The enzyme activities of the three major protein degradation pathways (ubiquitin proteasome, lysosomal cathepsins and the calpain systems were measured in the same fish. This study is the first to measure both KS and the enzymatic activity of protein degradation in the same fish, allowing us to examine the apparent contribution of different protein degradation pathways to protein turnover in various tissues (red and white muscle, liver, heart and gills. KS was lower in the white muscle and in liver of the fasted fish compared to the fed fish. There were no observable effects of food deprivation on the protease activities in any of the tissues with the exception of liver, where the ubiquitin proteasome pathway seemed to be activated during fasting conditions. Lysosomal proteolysis appears to be the primary degradation pathway for muscle protein, while the ubiquitin proteasome pathway seems to predominate in the liver. We speculate that Arctic charr regulate protein metabolism during food deprivation to conserve proteins.

  16. Characterizing protein conformations by correlation analysis of coarse-grained contact matrices

    Lindsay, Richard J.; Siess, Jan; Lohry, David P.; McGee, Trevor S.; Ritchie, Jordan S.; Johnson, Quentin R.; Shen, Tongye

    2018-01-01

    We have developed a method to capture the essential conformational dynamics of folded biopolymers using statistical analysis of coarse-grained segment-segment contacts. Previously, the residue-residue contact analysis of simulation trajectories was successfully applied to the detection of conformational switching motions in biomolecular complexes. However, the application to large protein systems (larger than 1000 amino acid residues) is challenging using the description of residue contacts. Also, the residue-based method cannot be used to compare proteins with different sequences. To expand the scope of the method, we have tested several coarse-graining schemes that group a collection of consecutive residues into a segment. The definition of these segments may be derived from structural and sequence information, while the interaction strength of the coarse-grained segment-segment contacts is a function of the residue-residue contacts. We then perform covariance calculations on these coarse-grained contact matrices. We monitored how well the principal components of the contact matrices is preserved using various rendering functions. The new method was demonstrated to assist the reduction of the degrees of freedom for describing the conformation space, and it potentially allows for the analysis of a system that is approximately tenfold larger compared with the corresponding residue contact-based method. This method can also render a family of similar proteins into the same conformational space, and thus can be used to compare the structures of proteins with different sequences.

  17. Conformational analysis by quantitative NOE measurements of the β-proton pairs across individual disulfide bonds in proteins

    Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune

    2012-01-01

    NOEs between the β-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U- 13 C, 15 N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal β-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[β- 13 C; α,β- 2 H 2 ] Cys and (2R, 3R)-[β- 13 C; α,β- 2 H 2 ] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely β2 (proS) or β3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the β-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the β2 and β3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ 2 and χ 3 , can be determined based on the precise proton distances of the four β-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.

  18. Heteronuclear Adiabatic Relaxation Dispersion (HARD) for quantitative analysis of conformational dynamics in proteins.

    Traaseth, Nathaniel J; Chao, Fa-An; Masterson, Larry R; Mangia, Silvia; Garwood, Michael; Michaeli, Shalom; Seelig, Burckhard; Veglia, Gianluigi

    2012-06-01

    NMR relaxation methods probe biomolecular motions over a wide range of timescales. In particular, the rotating frame spin-lock R(1ρ) and Carr-Purcell-Meiboom-Gill (CPMG) R(2) experiments are commonly used to characterize μs to ms dynamics, which play a critical role in enzyme folding and catalysis. In an effort to complement these approaches, we introduced the Heteronuclear Adiabatic Relaxation Dispersion (HARD) method, where dispersion in rotating frame relaxation rate constants (longitudinal R(1ρ) and transverse R(2ρ)) is created by modulating the shape and duration of adiabatic full passage (AFP) pulses. Previously, we showed the ability of the HARD method to detect chemical exchange dynamics in the fast exchange regime (k(ex)∼10(4)-10(5) s(-1)). In this article, we show the sensitivity of the HARD method to slower exchange processes by measuring R(1ρ) and R(2ρ) relaxation rates for two soluble proteins (ubiquitin and 10C RNA ligase). One advantage of the HARD method is its nominal dependence on the applied radio frequency field, which can be leveraged to modulate the dispersion in the relaxation rate constants. In addition, we also include product operator simulations to define the dynamic range of adiabatic R(1ρ) and R(2ρ) that is valid under all exchange regimes. We conclude from both experimental observations and simulations that this method is complementary to CPMG-based and rotating frame spin-lock R(1ρ) experiments to probe conformational exchange dynamics for biomolecules. Finally, this approach is germane to several NMR-active nuclei, where relaxation rates are frequency-offset independent. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Correlated motion of protein subdomains and large-scale conformational flexibility of RecA protein filament

    Yu, Garmay; A, Shvetsov; D, Karelov; D, Lebedev; A, Radulescu; M, Petukhov; V, Isaev-Ivanov

    2012-02-01

    Based on X-ray crystallographic data available at Protein Data Bank, we have built molecular dynamics (MD) models of homologous recombinases RecA from E. coli and D. radiodurans. Functional form of RecA enzyme, which is known to be a long helical filament, was approximated by a trimer, simulated in periodic water box. The MD trajectories were analyzed in terms of large-scale conformational motions that could be detectable by neutron and X-ray scattering techniques. The analysis revealed that large-scale RecA monomer dynamics can be described in terms of relative motions of 7 subdomains. Motion of C-terminal domain was the major contributor to the overall dynamics of protein. Principal component analysis (PCA) of the MD trajectories in the atom coordinate space showed that rotation of C-domain is correlated with the conformational changes in the central domain and N-terminal domain, that forms the monomer-monomer interface. Thus, even though C-terminal domain is relatively far from the interface, its orientation is correlated with large-scale filament conformation. PCA of the trajectories in the main chain dihedral angle coordinate space implicates a co-existence of a several different large-scale conformations of the modeled trimer. In order to clarify the relationship of independent domain orientation with large-scale filament conformation, we have performed analysis of independent domain motion and its implications on the filament geometry.

  20. Detergent-associated solution conformations of helical and beta-barrel membrane proteins.

    Mo, Yiming; Lee, Byung-Kwon; Ankner, John F; Becker, Jeffrey M; Heller, William T

    2008-10-23

    Membrane proteins present major challenges for structural biology. In particular, the production of suitable crystals for high-resolution structural determination continues to be a significant roadblock for developing an atomic-level understanding of these vital cellular systems. The use of detergents for extracting membrane proteins from the native membrane for either crystallization or reconstitution into model lipid membranes for further study is assumed to leave the protein with the proper fold with a belt of detergent encompassing the membrane-spanning segments of the structure. Small-angle X-ray scattering was used to probe the detergent-associated solution conformations of three membrane proteins, namely bacteriorhodopsin (BR), the Ste2p G-protein coupled receptor from Saccharomyces cerevisiae, and the Escherichia coli porin OmpF. The results demonstrate that, contrary to the traditional model of a detergent-associated membrane protein, the helical proteins BR and Ste2p are not in the expected, compact conformation and associated with detergent micelles, while the beta-barrel OmpF is indeed embedded in a disk-like micelle in a properly folded state. The comparison provided by the BR and Ste2p, both members of the 7TM family of helical membrane proteins, further suggests that the interhelical interactions between the transmembrane helices of the two proteins differ, such that BR, like other rhodopsins, can properly refold to crystallize, while Ste2p continues to prove resistant to crystallization from an initially detergent-associated state.

  1. Using simulation to interpret experimental data in terms of protein conformational ensembles.

    Allison, Jane R

    2017-04-01

    In their biological environment, proteins are dynamic molecules, necessitating an ensemble structural description. Molecular dynamics simulations and solution-state experiments provide complimentary information in the form of atomically detailed coordinates and averaged or distributions of structural properties or related quantities. Recently, increases in the temporal and spatial scale of conformational sampling and comparison of the more diverse conformational ensembles thus generated have revealed the importance of sampling rare events. Excitingly, new methods based on maximum entropy and Bayesian inference are promising to provide a statistically sound mechanism for combining experimental data with molecular dynamics simulations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Conformational sampling in template-free protein loop structure modeling: an overview.

    Li, Yaohang

    2013-01-01

    Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a "mini protein folding problem" under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized.

  3. CONFORMATIONAL SAMPLING IN TEMPLATE-FREE PROTEIN LOOP STRUCTURE MODELING: AN OVERVIEW

    Yaohang Li

    2013-02-01

    Full Text Available Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increasing number of known structures available in PDB. This mini review provides an overview on the recent computational approaches for loop structure modeling. In particular, we focus on the approaches of sampling loop conformation space, which is a critical step to obtain high resolution models in template-free methods. We review the potential energy functions for loop modeling, loop buildup mechanisms to satisfy geometric constraints, and loop conformation sampling algorithms. The recent loop modeling results are also summarized.

  4. Relation between Protein Intrinsic Normal Mode Weights and Pre-Existing Conformer Populations.

    Ozgur, Beytullah; Ozdemir, E Sila; Gursoy, Attila; Keskin, Ozlem

    2017-04-20

    Intrinsic fluctuations of a protein enable it to sample a large repertoire of conformers including the open and closed forms. These distinct forms of the protein called conformational substates pre-exist together in equilibrium as an ensemble independent from its ligands. The role of ligand might be simply to alter the equilibrium toward the most appropriate form for binding. Normal mode analysis is proved to be useful in identifying the directions of conformational changes between substates. In this study, we demonstrate that the ratios of normalized weights of a few normal modes driving the protein between its substates can give insights about the ratios of kinetic conversion rates of the substates, although a direct relation between the eigenvalues and kinetic conversion rates or populations of each substate could not be observed. The correlation between the normalized mode weight ratios and the kinetic rate ratios is around 83% on a set of 11 non-enzyme proteins and around 59% on a set of 17 enzymes. The results are suggestive that mode motions carry intrinsic relations with thermodynamics and kinetics of the proteins.

  5. Structural investigation of ribonuclease A conformational preferences using high pressure protein crystallography

    Kurpiewska, Katarzyna, E-mail: kurpiews@chemia.uj.edu.pl [Jagiellonian University, Faculty of Chemistry, Department of Crystal Chemistry and Crystal Physics, Protein Crystallography Group, Ingardena 3, 30-060 Kraków (Poland); Dziubek, Kamil; Katrusiak, Andrzej [Adam Mickiewicz University, Faculty of Chemistry, Department of Materials Chemistry, Umultowska 89b, 61-61 Poznań (Poland); Font, Josep [School of Medical Science, University of Sydney, NSW 2006 (Australia); Ribò, Marc; Vilanova, Maria [Universitat de Girona, Laboratorid’Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Campus de Montilivi, 17071 Girona (Spain); Lewiński, Krzysztof [Jagiellonian University, Faculty of Chemistry, Department of Crystal Chemistry and Crystal Physics, Protein Crystallography Group, Ingardena 3, 30-060 Kraków (Poland)

    2016-04-01

    Highlights: • A unique crystallographic studies of wild-type and mutated form of the same protein under high pressure. • Compressibility of RNase A molecule is significantly affected by a single amino acid substitution. • High pressure protein crystallography helps understanding protein flexibility and identify conformational substrates. - Abstract: Hydrostatic pressure in range 0.1–1.5 GPa is used to modify biological system behaviour mostly in biophysical studies of proteins in solution. Due to specific influence on the system equilibrium high pressure can act as a filter that enables to identify and investigate higher energy protein conformers. The idea of the presented experiments is to examine the behaviour of RNase A molecule under high pressure before and after introduction of destabilizing mutation. For the first time crystal structures of wild-type bovine pancreatic ribonuclease A and its markedly less stable variant modified at position Ile106 were determined at different pressures. X-ray diffraction experiments at high pressure showed that the secondary structure of RNase A is well preserved even beyond 0.67 GPa at room temperature. Detailed structural analysis of ribonuclease A conformation observed under high pressure revealed that pressure influences hydrogen bonds pattern, cavity size and packing of molecule.

  6. Conformational Analysis of Misfolded Protein Aggregation by FRET and Live-Cell Imaging Techniques

    Akira Kitamura

    2015-03-01

    Full Text Available Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS and Huntington’s disease (HD. A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET, fluorescence correlation spectroscopy (FCS, and bimolecular fluorescence complementation (BiFC have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes, and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.

  7. Structural investigation of ribonuclease A conformational preferences using high pressure protein crystallography

    Kurpiewska, Katarzyna; Dziubek, Kamil; Katrusiak, Andrzej; Font, Josep; Ribò, Marc; Vilanova, Maria; Lewiński, Krzysztof

    2016-01-01

    Highlights: • A unique crystallographic studies of wild-type and mutated form of the same protein under high pressure. • Compressibility of RNase A molecule is significantly affected by a single amino acid substitution. • High pressure protein crystallography helps understanding protein flexibility and identify conformational substrates. - Abstract: Hydrostatic pressure in range 0.1–1.5 GPa is used to modify biological system behaviour mostly in biophysical studies of proteins in solution. Due to specific influence on the system equilibrium high pressure can act as a filter that enables to identify and investigate higher energy protein conformers. The idea of the presented experiments is to examine the behaviour of RNase A molecule under high pressure before and after introduction of destabilizing mutation. For the first time crystal structures of wild-type bovine pancreatic ribonuclease A and its markedly less stable variant modified at position Ile106 were determined at different pressures. X-ray diffraction experiments at high pressure showed that the secondary structure of RNase A is well preserved even beyond 0.67 GPa at room temperature. Detailed structural analysis of ribonuclease A conformation observed under high pressure revealed that pressure influences hydrogen bonds pattern, cavity size and packing of molecule.

  8. A correspondence between solution-state dynamics of an individual protein and the sequence and conformational diversity of its family.

    Gregory D Friedland

    2009-05-01

    Full Text Available Conformational ensembles are increasingly recognized as a useful representation to describe fundamental relationships between protein structure, dynamics and function. Here we present an ensemble of ubiquitin in solution that is created by sampling conformational space without experimental information using "Backrub" motions inspired by alternative conformations observed in sub-Angstrom resolution crystal structures. Backrub-generated structures are then selected to produce an ensemble that optimizes agreement with nuclear magnetic resonance (NMR Residual Dipolar Couplings (RDCs. Using this ensemble, we probe two proposed relationships between properties of protein ensembles: (i a link between native-state dynamics and the conformational heterogeneity observed in crystal structures, and (ii a relation between dynamics of an individual protein and the conformational variability explored by its natural family. We show that the Backrub motional mechanism can simultaneously explore protein native-state dynamics measured by RDCs, encompass the conformational variability present in ubiquitin complex structures and facilitate sampling of conformational and sequence variability matching those occurring in the ubiquitin protein family. Our results thus support an overall relation between protein dynamics and conformational changes enabling sequence changes in evolution. More practically, the presented method can be applied to improve protein design predictions by accounting for intrinsic native-state dynamics.

  9. Structure of Human Pancreatic Lipase-Related Protein 2 with the Lid in an Open Conformation

    Eydoux, Cecilia; Spinelli, Silvia; Davis, Tara L.; Walker, John R.; Seitova, Alma; Dhe-Paganon, Sirano; De Caro, Alain; Cambillau, Christian; Carriere, Frederic (CNRS-UMR); (Toronto)

    2008-10-02

    Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.

  10. The role of PEG conformation in mixed layers: from protein corona substrate to steric stabilization avoiding protein adsorption

    Joan Comenge

    2015-03-01

    Full Text Available Although nanoparticles (NPs have been traditionally modified with a single ligand layer, mixture of ligands might help to combine different functionalities and to further engineer the NP surface. A detailed study of the competition between an alkanethiol (11-mercaptoundecanoic acid and SH-PEG for the surface of AuNPs and the resultant behaviors of this model nanoconjugate is presented here. As a result, the physicochemical properties of these conjugates can be progressively tuned by controlling the composition and especially the conformation of the mixed monolayer. This has implications in the physiological stability. The controlled changes on the SH-PEG conformation rather than its concentration induce a change in the stabilization mechanism from electrostatic repulsion to steric hindrance, which changes the biological fate of NPs. Importantly, the adsorption of proteins on the conjugates can be tailored by tuning the composition and conformation of the mixed layer.

  11. Influence of multiple well defined conformations on small-angle scattering of proteins in solution.

    Heller, William T

    2005-01-01

    A common structural motif for many proteins comprises rigid domains connected by a flexible hinge or linker. The flexibility afforded by these domains is important for proper function and such proteins may be able to adopt more than one conformation in solution under equilibrium conditions. Small-angle scattering of proteins in solution samples all conformations that exist in the sampled volume during the time of the measurement, providing an ensemble-averaged intensity. In this paper, the influence of sampling an ensemble of well defined protein structures on the small-angle solution scattering intensity profile is examined through common analysis methods. Two tests were performed using simulated data: one with the extended and collapsed states of the bilobal calcium-binding protein calmodulin and the second with the catalytic subunit of protein kinase A, which has two globular domains connected by a glycine hinge. In addition to analyzing the simulated data for the radii of gyration Rg, distance distribution function P(r) and particle volume, shape restoration was applied to the simulated data. Rg and P(r) of the ensemble profiles could be easily mistaken for a single intermediate state. The particle volumes and models of the ensemble intensity profiles show that some indication of multiple conformations exists in the case of calmodulin, which manifests an enlarged volume and shapes that are clear superpositions of the conformations used. The effect on the structural parameters and models is much more subtle in the case of the catalytic subunit of protein kinase A. Examples of how noise influences the data and analyses are also presented. These examples demonstrate the loss of the indications of multiple conformations in cases where even broad distributions of structures exist. While the tests using calmodulin show that the ensemble states remain discernible from the other ensembles tested or a single partially collapsed state, the tests performed using the

  12. Accessing a hidden conformation of the maltose binding protein using accelerated molecular dynamics.

    Denis Bucher

    2011-04-01

    Full Text Available Periplasmic binding proteins (PBPs are a large family of molecular transporters that play a key role in nutrient uptake and chemotaxis in Gram-negative bacteria. All PBPs have characteristic two-domain architecture with a central interdomain ligand-binding cleft. Upon binding to their respective ligands, PBPs undergo a large conformational change that effectively closes the binding cleft. This conformational change is traditionally viewed as a ligand induced-fit process; however, the intrinsic dynamics of the protein may also be crucial for ligand recognition. Recent NMR paramagnetic relaxation enhancement (PRE experiments have shown that the maltose binding protein (MBP - a prototypical member of the PBP superfamily - exists in a rapidly exchanging (ns to µs regime mixture comprising an open state (approx 95%, and a minor partially closed state (approx 5%. Here we describe accelerated MD simulations that provide a detailed picture of the transition between the open and partially closed states, and confirm the existence of a dynamical equilibrium between these two states in apo MBP. We find that a flexible part of the protein called the balancing interface motif (residues 175-184 is displaced during the transformation. Continuum electrostatic calculations indicate that the repacking of non-polar residues near the hinge region plays an important role in driving the conformational change. Oscillations between open and partially closed states create variations in the shape and size of the binding site. The study provides a detailed description of the conformational space available to ligand-free MBP, and has implications for understanding ligand recognition and allostery in related proteins.

  13. Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor*

    Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K.; Prosser, R. Scott

    2012-01-01

    The G protein-coupled β2-adrenoreceptor (β2AR) signals through the heterotrimeric G proteins Gs and Gi and β-arrestin. As such, the energy landscape of β2AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β2AR with a trifluoromethyl probe, 19F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β2AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β2AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor. PMID:22893704

  14. Conformational variation of proteins at room temperature is not dominated by radiation damage

    Russi, Silvia; González, Ana; Kenner, Lillian R.; Keedy, Daniel A.; Fraser, James S.; Bedem, Henry van den

    2017-01-01

    Protein crystallography data collection at synchrotrons is routinely carried out at cryogenic temperatures to mitigate radiation damage. Although damage still takes place at 100 K and below, the immobilization of free radicals increases the lifetime of the crystals by approximately 100-fold. Recent studies have shown that flash-cooling decreases the heterogeneity of the conformational ensemble and can hide important functional mechanisms from observation. These discoveries have motivated increasing numbers of experiments to be carried out at room temperature. However, the trade-offs between increased risk of radiation damage and increased observation of alternative conformations at room temperature relative to cryogenic temperature have not been examined. A considerable amount of effort has previously been spent studying radiation damage at cryo-temperatures, but the relevance of these studies to room temperature diffraction is not well understood. Here, the effects of radiation damage on the conformational landscapes of three different proteins (T. danielli thaumatin, hen egg-white lysozyme and human cyclophilin A) at room (278 K) and cryogenic (100 K) temperatures are investigated. Increasingly damaged datasets were collected at each temperature, up to a maximum dose of the order of 10 7 Gy at 100 K and 10 5 Gy at 278 K. Although it was not possible to discern a clear trend between damage and multiple conformations at either temperature, it was observed that disorder, monitored by B-factor-dependent crystallographic order parameters, increased with higher absorbed dose for the three proteins at 100 K. At 278 K, however, the total increase in this disorder was only statistically significant for thaumatin. A correlation between specific radiation damage affecting side chains and the amount of disorder was not observed. Lastly, this analysis suggests that elevated conformational heterogeneity in crystal structures at room temperature is observed despite radiation

  15. Role of detergents in conformational exchange of a G protein-coupled receptor.

    Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K; Prosser, R Scott

    2012-10-19

    The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

  16. Conformational changes in acetylcholine binding protein investigated by temperature accelerated molecular dynamics.

    Zeynab Mohammad Hosseini Naveh

    Full Text Available Despite the large number of studies available on nicotinic acetylcholine receptors, a complete account of the mechanistic aspects of their gating transition in response to ligand binding still remains elusive. As a first step toward dissecting the transition mechanism by accelerated sampling techniques, we study the ligand-induced conformational changes of the acetylcholine binding protein (AChBP, a widely accepted model for the full receptor extracellular domain. Using unbiased Molecular Dynamics (MD and Temperature Accelerated Molecular Dynamics (TAMD simulations we investigate the AChBP transition between the apo and the agonist-bound state. In long standard MD simulations, both conformations of the native protein are stable, while the agonist-bound structure evolves toward the apo one if the orientation of few key sidechains in the orthosteric cavity is modified. Conversely, TAMD simulations initiated from the native conformations are able to produce the spontaneous transition. With respect to the modified conformations, TAMD accelerates the transition by at least a factor 10. The analysis of some specific residue-residue interactions points out that the transition mechanism is based on the disruption/formation of few key hydrogen bonds. Finally, while early events of ligand dissociation are observed already in standard MD, TAMD accelerates the ligand detachment and, at the highest TAMD effective temperature, it is able to produce a complete dissociation path in one AChBP subunit.

  17. Lipid Regulated Intramolecular Conformational Dynamics of SNARE-Protein Ykt6

    Dai, Yawei; Seeger, Markus; Weng, Jingwei; Song, Song; Wang, Wenning; Tan, Yan-Wen

    2016-08-01

    Cellular informational and metabolic processes are propagated with specific membrane fusions governed by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNARE). SNARE protein Ykt6 is highly expressed in brain neurons and plays a critical role in the membrane-trafficking process. Studies suggested that Ykt6 undergoes a conformational change at the interface between its longin domain and the SNARE core. In this work, we study the conformational state distributions and dynamics of rat Ykt6 by means of single-molecule Förster Resonance Energy Transfer (smFRET) and Fluorescence Cross-Correlation Spectroscopy (FCCS). We observed that intramolecular conformational dynamics between longin domain and SNARE core occurred at the timescale ~200 μs. Furthermore, this dynamics can be regulated and even eliminated by the presence of lipid dodecylphoshpocholine (DPC). Our molecular dynamic (MD) simulations have shown that, the SNARE core exhibits a flexible structure while the longin domain retains relatively stable in apo state. Combining single molecule experiments and theoretical MD simulations, we are the first to provide a quantitative dynamics of Ykt6 and explain the functional conformational change from a qualitative point of view.

  18. A network model to correlate conformational change and the impedance spectrum of single proteins

    Alfinito, Eleonora; Pennetta, Cecilia; Reggiani, Lino [Dipartimento di Ingegneria dell' Innovazione, Universita del Salento, Via Arnesano, Lecce (Italy); Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM) (Italy)

    2008-02-13

    Integrated nanodevices based on proteins or biomolecules are attracting increasing interest in today's research. In fact, it has been shown that proteins such as azurin and bacteriorhodopsin manifest some electrical properties that are promising for the development of active components of molecular electronic devices. Here we focus on two relevant kinds of protein: bovine rhodopsin, prototype of G-protein-coupled-receptor (GPCR) proteins, and the enzyme acetylcholinesterase (AChE), whose inhibition is one of the most qualified treatments of Alzheimer's disease. Both these proteins exert their function starting with a conformational change of their native structure. Our guess is that such a change should be accompanied with a detectable variation of their electrical properties. To investigate this conjecture, we present an impedance network model of proteins, able to estimate the different impedance spectra associated with the different configurations. The distinct types of conformational change of rhodopsin and AChE agree with their dissimilar electrical responses. In particular, for rhodopsin the model predicts variations of the impedance spectra up to about 30%, while for AChE the same variations are limited to about 10%, which supports the existence of a dynamical equilibrium between its native and complexed states.

  19. Conformational dynamics of a protein in the folded and the unfolded state

    Fitter, Joerg

    2003-08-01

    In a quasielastic neutron scattering experiment, the picosecond dynamics of {alpha}-amylase was investigated for the folded and the unfolded state of the protein. In order to ensure a reasonable interpretation of the internal protein dynamics, the protein was measured in D{sub 2}O-buffer solution. The much higher structural flexibility of the pH induced unfolded state as compared to the native folded state was quantified using a simple analytical model, describing a local diffusion inside a sphere. In terms of this model the conformational volume, which is explored mainly by confined protein side-chain movements, is parameterized by the radius of a sphere (folded state, r=1.2 A; unfolded state, 1.8 A). Differences in conformational dynamics between the folded and the unfolded state of a protein are of fundamental interest in the field of protein science, because they are assumed to play an important role for the thermodynamics of folding/unfolding transition and for protein stability.

  20. Optimization of the GBMV2 implicit solvent force field for accurate simulation of protein conformational equilibria.

    Lee, Kuo Hao; Chen, Jianhan

    2017-06-15

    Accurate treatment of solvent environment is critical for reliable simulations of protein conformational equilibria. Implicit treatment of solvation, such as using the generalized Born (GB) class of models arguably provides an optimal balance between computational efficiency and physical accuracy. Yet, GB models are frequently plagued by a tendency to generate overly compact structures. The physical origins of this drawback are relatively well understood, and the key to a balanced implicit solvent protein force field is careful optimization of physical parameters to achieve a sufficient level of cancellation of errors. The latter has been hampered by the difficulty of generating converged conformational ensembles of non-trivial model proteins using the popular replica exchange sampling technique. Here, we leverage improved sampling efficiency of a newly developed multi-scale enhanced sampling technique to re-optimize the generalized-Born with molecular volume (GBMV2) implicit solvent model with the CHARMM36 protein force field. Recursive optimization of key GBMV2 parameters (such as input radii) and protein torsion profiles (via the CMAP torsion cross terms) has led to a more balanced GBMV2 protein force field that recapitulates the structures and stabilities of both helical and β-hairpin model peptides. Importantly, this force field appears to be free of the over-compaction bias, and can generate structural ensembles of several intrinsically disordered proteins of various lengths that seem highly consistent with available experimental data. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Lipid-protein interaction induced domains: Kinetics and conformational changes in multicomponent vesicles

    Sreeja, K. K.; Sunil Kumar, P. B.

    2018-04-01

    The spatio-temporal organization of proteins and the associated morphological changes in membranes are of importance in cell signaling. Several mechanisms that promote the aggregation of proteins at low cell surface concentrations have been investigated in the past. We show, using Monte Carlo simulations, that the affinity of proteins for specific lipids can hasten their aggregation kinetics. The lipid membrane is modeled as a dynamically triangulated surface with the proteins defined as in-plane fields at the vertices. We show that, even at low protein concentrations, strong lipid-protein interactions can result in large protein clusters indicating a route to lipid mediated signal amplification. At high protein concentrations, the domains form buds similar to that seen in lipid-lipid interaction induced phase separation. Protein interaction induced domain budding is suppressed when proteins act as anisotropic inclusions and exhibit nematic orientational order. The kinetics of protein clustering and resulting conformational changes are shown to be significantly different for the isotropic and anisotropic curvature inducing proteins.

  2. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian, E-mail: jianzhou@scut.edu.cn

    2016-07-30

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  3. Molecular dynamics simulations of conformation changes of HIV-1 regulatory protein on graphene

    Zhao, Daohui; Li, Libo; He, Daohang; Zhou, Jian

    2016-01-01

    Graphical abstract: Preferential adsorption of Vpr13-33 on graphene accompanied by early conformational change from α-helix to β-sheet structures was observed by molecular simulations. This work presents the molecular mechanism of graphene-induced peptide conformational alteration and sheds light on developing graphene-based materials to inhibit HIV. - Highlights: • Graphene induced early structural transition of Vpr13-33 is studied by MD simulations. • Both π-π stacking and hydrophobic interactions orchestrate the peptide adsorption. • Vpr has an increased propensity of β-sheet content on graphene surface. • To develop graphene-based materials to inhibit HIV is possible. - Abstract: The fragment of viral protein R (Vpr), Vpr13-33, plays an important role in regulating nuclear importing of HIV genes through channel formation in which it adopts a leucine-zipper-like alpha-helical conformation. A recent experimental study reported that helical Vpr13-33 would transform to β-sheet or random coil structures and aggregate on the surface of graphene or graphene oxide through hydrophobic interactions. Due to experimental limitations, however, there is still a considerable lack of understanding on the adsorption dynamics at the early stage of the conformational transition at water-graphene interface and the underlying driving force at molecular level. In this study, atomistic molecular dynamics simulations were used to explore the conformation transition phenomena. Vpr13-33 kept α-helical structure in solution, but changed to β-sheet structure when strongly adsorbed onto graphene. Preferential adsorption of Vpr13-33 on graphene is dominated by hydrophobic interactions. The cluster analysis identified the most significant populated conformation and the early stage of structure conversion from α-helical to β-sheet was found, but the full β-sheet propagation was not observed. Free energy landscape analysis further complemented the transformation analysis of

  4. MOLECULAR MODELING INDICATES THAT HOMOCYSTEINE INDUCES CONFORMATIONAL CHANGES IN THE STRUCTURE OF PUTATIVE TARGET PROTEINS

    Yumnam Silla

    2015-09-01

    Full Text Available An elevated level of homocysteine, a reactive thiol containing amino acid is associated with a multitude of complex diseases. A majority (>80% of homocysteine in circulation is bound to protein cysteine residues. Although, till date only 21 proteins have been experimentally shown to bind with homocysteine, using an insilico approach we had earlier identified several potential target proteins that could bind with homocysteine. Shomocysteinylation of proteins could potentially alter the structure and/or function of the protein. Earlier studies have shown that binding of homocysteine to protein alters its function. However, the effect of homocysteine on the target protein structure has not yet been documented. In the present work, we assess conformational or structural changes if any due to protein homocysteinylation using two proteins, granzyme B (GRAB and junctional adhesion molecule 1 (JAM1, which could potentially bind to homocysteine. We, for the first time, constructed computational models of homocysteine bound to target proteins and monitored their structural changes using explicit solvent molecular dynamic (MD simulation. Analysis of homocysteine bound trajectories revealed higher flexibility of the active site residues and local structural perturbations compared to the unbound native structure’s simulation, which could affect the stability of the protein. In addition, secondary structure analysis of homocysteine bound trajectories also revealed disappearance of â-helix within the G-helix and linker region that connects between the domain regions (as defined in the crystal structure. Our study thus captures the conformational transitions induced by homocysteine and we suggest these structural alterations might have implications for hyperhomocysteinemia induced pathologies.

  5. On the effect of hydrostatic pressure on the conformational stability of globular proteins.

    Graziano, Giuseppe

    2015-12-01

    The model developed for cold denaturation (Graziano, PCCP 2010, 12, 14245-14252) is extended to rationalize the dependence of protein conformational stability upon hydrostatic pressure, at room temperature. A pressure- volume work is associated with the process of cavity creation for the need to enlarge the liquid volume against hydrostatic pressure. This contribution destabilizes the native state that has a molecular volume slightly larger than the denatured state due to voids existing in the protein core. Therefore, there is a hydrostatic pressure value at which the pressure-volume contribution plus the conformational entropy loss of the polypeptide chain are able to overwhelm the stabilizing gain in translational entropy of water molecules, due to the decrease in water accessible surface area upon folding, causing denaturation. © 2015 Wiley Periodicals, Inc.

  6. Characterization of conformational properties of protein/trehalose/water system by neutron scattering

    Brandt, A. [Hahn-Meitner-Institut, BENSC (NI), Glienicker Strasse, 14109 Berlin (Germany); Magazu' , S.; Mangione, A.; Migliardo, F. [Dipartimento di Fisica and INFM, Universita' di Messina, PO Box 55, 98166 Messina (Italy); Vertessy, B.G. [Institute of Enzymology, Hungarian Academy of Sciences, P. O. Box 7, 1518 Budapest (Hungary)

    2002-07-01

    In this contribution we report results of a small-angle neutron scattering (SANS) investigation of dUTPase/D{sub 2}O solutions. Data were collected by the V4 spectrometer at the BENSC facility (Berlin, Germany). The results allow us to characterize the conformational properties of the protein in solution as a function of temperature and in the presence of trehalose, a disaccharide with a noticeable bioprotective action. (orig.)

  7. Characterization of conformational properties of protein/trehalose/water system by neutron scattering

    Brandt, A; Mangione, A; Migliardo, F; Vertessy, B G

    2002-01-01

    In this contribution we report results of a small-angle neutron scattering (SANS) investigation of dUTPase/D sub 2 O solutions. Data were collected by the V4 spectrometer at the BENSC facility (Berlin, Germany). The results allow us to characterize the conformational properties of the protein in solution as a function of temperature and in the presence of trehalose, a disaccharide with a noticeable bioprotective action. (orig.)

  8. Conformationally Preorganized Diastereomeric Norbornane-Based Maltosides for Membrane Protein Study

    Das, Manabendra; Du, Yang; Ribeiro, Orquidea

    2017-01-01

    were generally better at stabilizing membrane proteins than short alkyl chain agents. Furthermore, use of one well-behaving NBM enabled us to attain a marked stabilization and clear visualization of a challenging membrane protein complex using electron microscopy. Thus, this study not only describes......Detergents are essential tools for functional and structural studies of membrane proteins. However, conventional detergents are limited in their scope and utility, particularly for eukaryotic membrane proteins. Thus, there are major efforts to develop new amphipathic agents with enhanced properties....... Here, a novel class of diastereomeric agents with a preorganized conformation, designated norbornane-based maltosides (NBMs), were prepared and evaluated for their ability to solubilize and stabilize membrane proteins. Representative NBMs displayed enhanced behaviors compared to n...

  9. Conformational and functional variants of CD44-targeted protein nanoparticles bio-produced in bacteria

    Pesarrodona, Mireia; Conchillo-Solé, Oscar; Unzueta, Ugutz; Xu, Zhikun; Ferrer-Miralles, Neus; Daura, Xavier; Vázquez, Esther; Villaverde, Antonio; Fernández, Yolanda; Foradada, Laia; Schwartz, Simó Jr; Abasolo, Ibane; Sánchez-Chardi, Alejandro; Roldán, Mónica; Villegas, Sandra; Rinas, Ursula

    2016-01-01

    Biofabrication is attracting interest as a means to produce nanostructured functional materials because of its operational versatility and full scalability. Materials based on proteins are especially appealing, as the structure and functionality of proteins can be adapted by genetic engineering. Furthermore, strategies and tools for protein production have been developed and refined steadily for more than 30 years. However, protein conformation and therefore activity might be sensitive to production conditions. Here, we have explored whether the downstream strategy influences the structure and biological activities, in vitro and in vivo, of a self-assembling, CD44-targeted protein-only nanoparticle produced in Escherichia coli. This has been performed through the comparative analysis of particles built from soluble protein species or protein versions obtained by in vitro protein extraction from inclusion bodies, through mild, non-denaturing procedures. These methods have been developed recently as a convenient alternative to the use of toxic chaotropic agents for protein resolubilization from protein aggregates. The results indicate that the resulting material shows substantial differences in its physicochemical properties and its biological performance at the systems level, and that its building blocks are sensitive to the particular protein source. (paper)

  10. Nuclear magnetic resonance provides a quantitative description of protein conformational flexibility on physiologically important time scales.

    Salmon, Loïc; Bouvignies, Guillaume; Markwick, Phineus; Blackledge, Martin

    2011-04-12

    A complete description of biomolecular activity requires an understanding of the nature and the role of protein conformational dynamics. In recent years, novel nuclear magnetic resonance-based techniques that provide hitherto inaccessible detail concerning biomolecular motions occurring on physiologically important time scales have emerged. Residual dipolar couplings (RDCs) provide precise information about time- and ensemble-averaged structural and dynamic processes with correlation times up to the millisecond and thereby encode key information for understanding biological activity. In this review, we present the application of two very different approaches to the quantitative description of protein motion using RDCs. The first is purely analytical, describing backbone dynamics in terms of diffusive motions of each peptide plane, using extensive statistical analysis to validate the proposed dynamic modes. The second is based on restraint-free accelerated molecular dynamics simulation, providing statistically sampled free energy-weighted ensembles that describe conformational fluctuations occurring on time scales from pico- to milliseconds, at atomic resolution. Remarkably, the results from these two approaches converge closely in terms of distribution and absolute amplitude of motions, suggesting that this kind of combination of analytical and numerical models is now capable of providing a unified description of protein conformational dynamics in solution.

  11. Navigating the conformational landscape of G protein-coupled receptor kinases during allosteric activation.

    Yao, Xin-Qiu; Cato, M Claire; Labudde, Emily; Beyett, Tyler S; Tesmer, John J G; Grant, Barry J

    2017-09-29

    G protein-coupled receptors (GPCRs) are essential for transferring extracellular signals into carefully choreographed intracellular responses controlling diverse aspects of cell physiology. The duration of GPCR-mediated signaling is primarily regulated via GPCR kinase (GRK)-mediated phosphorylation of activated receptors. Although many GRK structures have been reported, the mechanisms underlying GRK activation are not well-understood, in part because it is unknown how these structures map to the conformational landscape available to this enzyme family. Unlike most other AGC kinases, GRKs rely on their interaction with GPCRs for activation and not phosphorylation. Here, we used principal component analysis of available GRK and protein kinase A crystal structures to identify their dominant domain motions and to provide a framework that helps evaluate how close each GRK structure is to being a catalytically competent state. Our results indicated that disruption of an interface formed between the large lobe of the kinase domain and the regulator of G protein signaling homology domain (RHD) is highly correlated with establishment of the active conformation. By introducing point mutations in the GRK5 RHD-kinase domain interface, we show with both in silico and in vitro experiments that perturbation of this interface leads to higher phosphorylation activity. Navigation of the conformational landscape defined by this bioinformatics-based study is likely common to all GPCR-activated GRKs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Conformational responses to changes in the state of ionization of titrable groups in proteins

    Richman, Daniel Eric

    Electrostatic energy links the structural properties of proteins with some of their important biological functions, including catalysis, energy transduction, and binding and recognition. Accurate calculation of electrostatic energy is essential for predicting and for analyzing function from structure. All proteins have many ionizable residues at the protein-water interface. These groups tend to have ionization equilibria (pK a values) shifted slightly relative to their values in water. In contrast, groups buried in the hydrophobic interior usually have highly anomalous p Ka values. These shifts are what structure-based calculations have to reproduce to allow examination of contributions from electrostatics to stability, solubility and interactions of proteins. Electrostatic energies are challenging to calculate accurately because proteins are heterogeneous dielectric materials. Any individual ionizable group can experience very different local environments with different dielectric properties. The studies in this thesis examine the hypothesis that proteins reorganize concomitant with changes in their state of ionization. It appears that the pKa value measured experimentally reflects the average of pKa values experienced in the different electrostatic environments corresponding to different conformational microstates. Current computational models fail to sample conformational reorganization of the backbone correctly. Staphyloccocal nuclease (SNase) was used as a model protein in nuclear magnetic resonance (NMR) spectroscopy studies to characterize the conformational rearrangements of the protein coupled to changes in the ionization state of titrable groups. One set of experiments tests the hypothesis that proton binding to surface Asp and Glu side chains drives local unfolding by stabilizing less-native, more water-solvated conformations in which the side chains have normalized pKa values. Increased backbone flexibility in the ps-ns timescale, hydrogen bond (H

  13. Biomimetic conformation-specific assembly of proteins at artificial binding sites nano-patterned on silicon

    de la Rica, Roberto; Matsui, Hiroshi

    2009-01-01

    Biomolecules such as enzymes and antibodies possess binding sites where the molecular architecture and the physicochemical properties are optimum for their interaction with a particular target, in some cases even differentiating between stereoisomers. Here, we mimic this exquisite specificity via the creation of a suitable chemical environment by fabricating artificial binding sites for the protein calmodulin (CaM). By downscaling well-known surface chemical modification methodologies to the nanometer scale via silicon nanopatterning, the Ca2+-CaM conformer was found to selectively bind the biomimetic binding sites. The methodology could be adapted to mimic other protein-receptor interactions for sensing and catalysis. PMID:19757782

  14. Methodology for fast evaluation of Bacillus thuringiensis crystal protein content

    Alves Lúcia M. Carareto

    2000-01-01

    Full Text Available The development of the production and use of Bacillus thuringiensis in Brazil at a commercial scale faces certain difficulties, among them the establishment of efficient methodologies for the quantitation of toxic products to be commercialized. Presently, the amount of toxin is given in percentage by analyzing the samples total protein content. Such methodology however, does not measure the actual amount of active protein present in the product, since most strains express different endotoxin genes and might even produce b-toxin. Since the various types of toxins exhibit different antigenic characteristics, this work has as objective the utilization of fast immunological techniques to quantify the level of crystal protein. Crystal protein produced by a subspecies of Bacillus thuringiensis var. israelensis was purified by ultracentrifugation and utilized to immunize rabbits and to produce hiperimmune sera. Such sera were latter used to evaluate the level of proteins on commercial bioinsecticide and on laboratory cultures of B. thuringiensis through the immunodot technique. The results were obtained by comparison of data obtained from reactions with known concentrations of crystal protein permitting to evaluate the level of such protein on various materials.

  15. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics

    Dai, Jin; He, Jianfeng; Niemi, Antti J.

    2016-01-01

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  16. Conformational landscape of an amyloid intra-cellular domain and Landau-Ginzburg-Wilson paradigm in protein dynamics

    Dai, Jin; He, Jianfeng, E-mail: Antti.Niemi@physics.uu.se, E-mail: hjf@bit.edu.cn [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Niemi, Antti J., E-mail: Antti.Niemi@physics.uu.se, E-mail: hjf@bit.edu.cn [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Department of Physics and Astronomy, Uppsala University, P.O. Box 803, S-75108 Uppsala (Sweden); Laboratoire de Mathematiques et Physique Theorique CNRS UMR 6083, Fédération Denis Poisson, Université de Tours, Parc de Grandmont, F37200 Tours (France)

    2016-07-28

    The Landau-Ginzburg-Wilson paradigm is proposed as a framework, to investigate the conformational landscape of intrinsically unstructured proteins. A universal Cα-trace Landau free energy is deduced from general symmetry considerations, with the ensuing all-atom structure modeled using publicly available reconstruction programs Pulchra and Scwrl. As an example, the conformational stability of an amyloid precursor protein intra-cellular domain (AICD) is inspected; the reference conformation is the crystallographic structure with code 3DXC in Protein Data Bank (PDB) that describes a heterodimer of AICD and a nuclear multi-domain adaptor protein Fe65. Those conformations of AICD that correspond to local or near-local minima of the Landau free energy are identified. For this, the response of the original 3DXC conformation to variations in the ambient temperature is investigated, using the Glauber algorithm. The conclusion is that in isolation the AICD conformation in 3DXC must be unstable. A family of degenerate conformations that minimise the Landau free energy is identified, and it is proposed that the native state of an isolated AICD is a superposition of these conformations. The results are fully in line with the presumed intrinsically unstructured character of isolated AICD and should provide a basis for a systematic analysis of AICD structure in future NMR experiments.

  17. Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6

    Chen, Hui; Mustafi, Sourajit M. [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); LeMaster, David M. [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); University at Albany – SUNY, Empire State Plaza, Albany, NY 12201 (United States); Li, Zhong [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); Héroux, Annie [Brookhaven National Laboratory, Upton, NY 11973 (United States); Li, Hongmin; Hernández, Griselda, E-mail: griselda@wadsworth.org [New York State Department of Health, Empire State Plaza, Albany, NY 12201 (United States); University at Albany – SUNY, Empire State Plaza, Albany, NY 12201 (United States)

    2014-03-01

    Two crystal forms of unligated FKBP12.6 exhibit multiple conformations in the active site and in the 80s loop, the primary site for known protein-recognition interactions. The previously unreported NMR backbone assignment of FKBP12.6 revealed extensive doubling of amide resonances, which reflects a slow conformational transition centered in the 80s loop. The primary known physiological function of FKBP12.6 involves its role in regulating the RyR2 isoform of ryanodine receptor Ca{sup 2+} channels in cardiac muscle, pancreatic β islets and the central nervous system. With only a single previously reported X-ray structure of FKBP12.6, bound to the immunosuppressant rapamycin, structural inferences for this protein have been drawn from the more extensive studies of the homologous FKBP12. X-ray structures at 1.70 and 1.90 Å resolution from P2{sub 1} and P3{sub 1}21 crystal forms are reported for an unligated cysteine-free variant of FKBP12.6 which exhibit a notable diversity of conformations. In one monomer from the P3{sub 1}21 crystal form, the aromatic ring of Phe59 at the base of the active site is rotated perpendicular to its typical orientation, generating a steric conflict for the immunosuppressant-binding mode. The peptide unit linking Gly89 and Val90 at the tip of the protein-recognition ‘80s loop’ is flipped in the P2{sub 1} crystal form. Unlike the >30 reported FKBP12 structures, the backbone conformation of this loop closely follows that of the first FKBP domain of FKBP51. The NMR resonances for 21 backbone amides of FKBP12.6 are doubled, corresponding to a slow conformational transition centered near the tip of the 80s loop, as recently reported for 31 amides of FKBP12. The comparative absence of doubling for residues along the opposite face of the active-site pocket in FKBP12.6 may in part reflect attenuated structural coupling owing to increased conformational plasticity around the Phe59 ring.

  18. A microscopic insight from conformational thermodynamics to functional ligand binding in proteins.

    Sikdar, Samapan; Chakrabarti, J; Ghosh, Mahua

    2014-12-01

    We show that the thermodynamics of metal ion-induced conformational changes aid to understand the functions of protein complexes. This is illustrated in the case of a metalloprotein, alpha-lactalbumin (aLA), a divalent metal ion binding protein. We use the histograms of dihedral angles of the protein, generated from all-atom molecular dynamics simulations, to calculate conformational thermodynamics. The thermodynamically destabilized and disordered residues in different conformational states of a protein are proposed to serve as binding sites for ligands. This is tested for β-1,4-galactosyltransferase (β4GalT) binding to the Ca(2+)-aLA complex, in which the binding residues are known. Among the binding residues, the C-terminal residues like aspartate (D) 116, glutamine (Q) 117, tryptophan (W) 118 and leucine (L) 119 are destabilized and disordered and can dock β4GalT onto Ca(2+)-aLA. No such thermodynamically favourable binding residues can be identified in the case of the Mg(2+)-aLA complex. We apply similar analysis to oleic acid binding and predict that the Ca(2+)-aLA complex can bind to oleic acid through the basic histidine (H) 32 of the A2 helix and the hydrophobic residues, namely, isoleucine (I) 59, W60 and I95, of the interfacial cleft. However, the number of destabilized and disordered residues in Mg(2+)-aLA are few, and hence, the oleic acid binding to Mg(2+)-bound aLA is less stable than that to the Ca(2+)-aLA complex. Our analysis can be generalized to understand the functionality of other ligand bound proteins.

  19. Protein adsorption on tailored substrates: long-range forces and conformational changes

    Bellion, M; Santen, L [Department of Theoretical Physics, Saarland University, 66041 Saarbruecken (Germany); Mantz, H; Haehl, H; Quinn, A; Nagel, A; Gilow, C; Weitenberg, C; Schmitt, Y; Jacobs, K [Department of Experimental Physics, Saarland University, 66041 Saarbruecken (Germany)], E-mail: k.jacobs@physik.uni-saarland.de

    2008-10-08

    Adsorption of proteins onto solid surfaces is an everyday phenomenon that is not yet fully understood. To further the current understanding, we have performed in situ ellipsometry studies to reveal the adsorption kinetics of three different proteins, lysozyme, {alpha}-amylase and bovine serum albumin. As substrates we offer Si wafers with a controlled Si oxide layer thickness and a hydrophilic or hydrophobic surface functionalization, allowing the tailoring of the influence of short- and long-range interactions. Our studies show that not only the surface chemistry determines the properties of an adsorbed protein layer but also the van der Waals contributions of a composite substrate. We compare the experimental findings to results of a colloidal Monte Carlo approach that includes conformational changes of the adsorbed proteins induced by density fluctuations.

  20. Developing Fast Fluorescent Protein Voltage Sensors by Optimizing FRET Interactions.

    Uhna Sung

    Full Text Available FRET (Förster Resonance Energy Transfer-based protein voltage sensors can be useful for monitoring neuronal activity in vivo because the ratio of signals between the donor and acceptor pair reduces common sources of noise such as heart beat artifacts. We improved the performance of FRET based genetically encoded Fluorescent Protein (FP voltage sensors by optimizing the location of donor and acceptor FPs flanking the voltage sensitive domain of the Ciona intestinalis voltage sensitive phosphatase. First, we created 39 different "Nabi1" constructs by positioning the donor FP, UKG, at 8 different locations downstream of the voltage-sensing domain and the acceptor FP, mKO, at 6 positions upstream. Several of these combinations resulted in large voltage dependent signals and relatively fast kinetics. Nabi1 probes responded with signal size up to 11% ΔF/F for a 100 mV depolarization and fast response time constants both for signal activation (~2 ms and signal decay (~3 ms. We improved expression in neuronal cells by replacing the mKO and UKG FRET pair with Clover (donor FP and mRuby2 (acceptor FP to create Nabi2 probes. Nabi2 probes also had large signals and relatively fast time constants in HEK293 cells. In primary neuronal culture, a Nabi2 probe was able to differentiate individual action potentials at 45 Hz.

  1. Detection of small conformational changes of proteins by small-angle scattering

    Durchschlag, H.; Purr, G.; Zipper, P.; Wilfing, R.

    1991-01-01

    In the past the technique of small-angle scattering has been a powerful tool for studying conformational changes of protein which occur, for example, upon binding with ligands. Results obtained by different authors from X-ray and neutron experiments on a variety of proteins and under various conditions have been compiled. This offers the possibility of comparing the extent of changes in the molecular parameters investigated (e.g. change of the radius of gyration). Problems encountered with the detection of small changes are discussed. As an example, conformational changes of the enzyme citrate synthase upon substrate binding (oxaloacetate) are presented. X-ray crystallography had already found distinct changes between open and closed forms of the enzyme. Small-angle X-ray scattering studies registered slight changes of some parameters in solution. These changes could be paralleled with the results of other solution techniques (UV absorption, fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation). The results found for citrate synthase are also compared with previous findings for malate synthase, an enzyme of similar enzymatic function. Above all, this study shows that care has to be taken when studying small conformational changes. It is absolutely necessary to use different methods and conditions and to study the problem from different points of view to avoid pitfalls. (orig.)

  2. Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery.

    Choy, Meng S; Li, Yang; Machado, Luciana E S F; Kunze, Micha B A; Connors, Christopher R; Wei, Xingyu; Lindorff-Larsen, Kresten; Page, Rebecca; Peti, Wolfgang

    2017-02-16

    Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Compositional and conformational analysis of yam proteins by near infrared fourier transform Raman spectroscopy.

    Liao, Yu-Hsiu; Wang, Chih-Hsien; Tseng, Chin-Yin; Chen, Hsiu-Ling; Lin, Long-Liu; Chen, Wenlung

    2004-12-29

    Fourier transform (FT)-Raman spectroscopy was employed to study the molecular structure of yam proteins isolated from three commonly consumed yam species including Dioscorea alata L., D. alata L. var. purpurea, and Dioscorea japonica. Although D. alata L. and D. alata L. var. purpurea consisted of similar amino acid residues, they still exhibited significant differences in conformational arrangement. The secondary structure of D. alata L. was mainly an alpha-helix, while D. alata L. var. purpurea was mostly in antiparallel beta-sheets. In contrast, D. japonica, which belongs to a different species, exhibited explicit differences in amino acid compositions and molecular structures of which the conformation was a mixed form of alpha-helices and antiparallel beta-sheets. FT-Raman directly proved the existence of S-S in yam proteins, implying that oligomer formation in yam proteins might be due to disulfide linking of dioscorin (32 kDa). The microenvironment of aromatic amino acids and the state of S-S in yam proteins were also discussed.

  4. Probing Conformational Changes of Human DNA Polymerase λ Using Mass Spectrometry-Based Protein Footprinting

    Fowler, Jason D.; Brown, Jessica A.; Kvaratskhelia, Mamuka; Suo, Zucai

    2009-01-01

    SUMMARY Crystallographic studies of the C-terminal, DNA polymerase β-like domain of human DNA polymerase lambda (fPolλ) suggested that the catalytic cycle might not involve a large protein domain rearrangement as observed with several replicative DNA polymerases and DNA polymerase β. To examine solution-phase protein conformation changes in fPolλ, which also contains a breast cancer susceptibility gene 1 C-terminal domain and a Proline-rich domain at its N-terminus, we used a mass spectrometry - based protein footprinting approach. In parallel experiments, surface accessibility maps for Arg residues were compared for the free fPolλ versus the binary complex of enzyme•gapped DNA and the ternary complex of enzyme•gapped DNA•dNTP. These experiments suggested that fPolλ does not undergo major conformational changes during the catalysis in the solution phase. Furthermore, the mass spectrometry-based protein footprinting experiments revealed that active site residue R386 was shielded from the surface only in the presence of both a gapped DNA substrate and an incoming nucleotide dNTP. Site-directed mutagenesis and pre-steady state kinetic studies confirmed the importance of R386 for the enzyme activity, and indicated the key role for its guanidino group in stabilizing the negative charges of an incoming nucleotide and the leaving pyrophosphate product. We suggest that such interactions could be shared by and important for catalytic functions of other DNA polymerases. PMID:19467241

  5. Influence of Tableting on the Conformation and Thermal Stability of Trypsin as a Model Protein

    Klukkert, Marten; Van De Weert, Marco; Fanø, Mathias

    2015-01-01

    was performed to determine the Tm as well as the folding reversibility after thermal denaturation of the reconstituted samples. It was found that compacted samples showed reduced activity accompanied by an altered secondary structure. Conformational changes that occur in the solid state were partially...... reversible upon tablet reconstitution. Aqueous-state IR spectroscopy combined with partial least squares was shown to be a powerful tool to follow irreversible structural changes and evaluate sample bioactivity. Besides its conformation, the thermal stability of trypsin was altered as a result of the applied...... compaction pressure, indicated by a reduced folding reversibility. In conclusion, this study reveals that tableting can have a negative impact on the biological quality of protein APIs....

  6. Ultra-thin, conformal, and hydratable color-absorbers using silk protein hydrogel

    Umar, Muhammad; Min, Kyungtaek; Jo, Minsik; Kim, Sunghwan

    2018-06-01

    Planar and multilayered photonic devices offer unprecedented opportunities in biological and chemical sensing due to strong light-matter interactions. However, uses of rigid substances such as semiconductors and dielectrics confront photonic devices with issues of biocompatibility and a mechanical mismatch for their application on humid, uneven, and soft biological surfaces. Here, we report that favorable material traits of natural silk protein led to the fabrication of an ultra-thin, conformal, and water-permeable (hydratable) metal-insulator-metal (MIM) color absorber that was mapped on soft, curved, and hydrated biological interfaces. Strong absorption was induced in the MIM structure and could be tuned by hydration and tilting of the sample. The transferred MIM color absorbers reached the exhibition of a very strong resonant absorption in the visible and near infra-red ranges. In addition, we demonstrated that the conformal resonator could function as a refractometric glucose sensor applied on a contact lens.

  7. Molecular dynamics analysis of conformational change of paramyxovirus F protein during the initial steps of membrane fusion

    Martín-García, Fernando; Mendieta-Moreno, Jesús Ignacio; Mendieta, Jesús; Gómez-Puertas, Paulino

    2012-01-01

    Highlights: ► Initial conformational change of paramyxovirus F protein is caused only by mechanical forces. ► HRA region undergoes a structural change from a beta + alpha conformation to an extended coil and then to an all-alpha conformation. ► HRS domains of F protein form three single α-helices prior to generation of the coiled coil. -- Abstract: The fusion of paramyxovirus to the cell membrane is mediated by fusion protein (F protein) present in the virus envelope, which undergoes a dramatic conformational change during the process. Unlike hemagglutinin in orthomyxovirus, this change is not mediated by an alteration of environmental pH, and its cause remains unknown. Steered molecular dynamics analysis leads us to suggest that the conformational modification is mediated only by stretching mechanical forces once the transmembrane fusion peptide of the protein is anchored to the cell membrane. Such elongating forces will generate major secondary structure rearrangement in the heptad repeat A region of the F protein; from β-sheet conformation to an elongated coil and then spontaneously to an α-helix. In addition, it is proposed that the heptad repeat A region adopts a final three-helix coiled coil and that this structure appears after the formation of individual helices in each monomer.

  8. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  9. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

    2010-01-01

    Research highlights: → Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. → The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. → Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  10. Enhanced conformational sampling to visualize a free-energy landscape of protein complex formation.

    Iida, Shinji; Nakamura, Haruki; Higo, Junichi

    2016-06-15

    We introduce various, recently developed, generalized ensemble methods, which are useful to sample various molecular configurations emerging in the process of protein-protein or protein-ligand binding. The methods introduced here are those that have been or will be applied to biomolecular binding, where the biomolecules are treated as flexible molecules expressed by an all-atom model in an explicit solvent. Sampling produces an ensemble of conformations (snapshots) that are thermodynamically probable at room temperature. Then, projection of those conformations to an abstract low-dimensional space generates a free-energy landscape. As an example, we show a landscape of homo-dimer formation of an endothelin-1-like molecule computed using a generalized ensemble method. The lowest free-energy cluster at room temperature coincided precisely with the experimentally determined complex structure. Two minor clusters were also found in the landscape, which were largely different from the native complex form. Although those clusters were isolated at room temperature, with rising temperature a pathway emerged linking the lowest and second-lowest free-energy clusters, and a further temperature increment connected all the clusters. This exemplifies that the generalized ensemble method is a powerful tool for computing the free-energy landscape, by which one can discuss the thermodynamic stability of clusters and the temperature dependence of the cluster networks. © 2016 The Author(s).

  11. DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction*

    Algasaier, Sana I.; Exell, Jack C.; Bennet, Ian A.; Thompson, Mark J.; Gotham, Victoria J. B.; Shaw, Steven J.; Craggs, Timothy D.; Finger, L. David; Grasby, Jane A.

    2016-01-01

    Human flap endonuclease-1 (hFEN1) catalyzes the essential removal of single-stranded flaps arising at DNA junctions during replication and repair processes. hFEN1 biological function must be precisely controlled, and consequently, the protein relies on a combination of protein and substrate conformational changes as a prerequisite for reaction. These include substrate bending at the duplex-duplex junction and transfer of unpaired reacting duplex end into the active site. When present, 5′-flaps are thought to thread under the helical cap, limiting reaction to flaps with free 5′-termini in vivo. Here we monitored DNA bending by FRET and DNA unpairing using 2-aminopurine exciton pair CD to determine the DNA and protein requirements for these substrate conformational changes. Binding of DNA to hFEN1 in a bent conformation occurred independently of 5′-flap accommodation and did not require active site metal ions or the presence of conserved active site residues. More stringent requirements exist for transfer of the substrate to the active site. Placement of the scissile phosphate diester in the active site required the presence of divalent metal ions, a free 5′-flap (if present), a Watson-Crick base pair at the terminus of the reacting duplex, and the intact secondary structure of the enzyme helical cap. Optimal positioning of the scissile phosphate additionally required active site conserved residues Tyr40, Asp181, and Arg100 and a reacting duplex 5′-phosphate. These studies suggest a FEN1 reaction mechanism where junctions are bound and 5′-flaps are threaded (when present), and finally the substrate is transferred onto active site metals initiating cleavage. PMID:26884332

  12. On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

    Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian; Roitberg, Adrian E.

    2015-01-01

    The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data

  13. On the analysis and comparison of conformer-specific essential dynamics upon ligand binding to a protein

    Grosso, Marcos; Kalstein, Adrian; Parisi, Gustavo; Fernandez-Alberti, Sebastian, E-mail: sfalberti@gmail.com [Universidad Nacional de Quilmes, Roque Saenz Peña 352, B1876BXD Bernal (Argentina); Roitberg, Adrian E. [Departments of Physics and Chemistry, University of Florida, Gainesville, Florida 32611 (United States)

    2015-06-28

    The native state of a protein consists of an equilibrium of conformational states on an energy landscape rather than existing as a single static state. The co-existence of conformers with different ligand-affinities in a dynamical equilibrium is the basis for the conformational selection model for ligand binding. In this context, the development of theoretical methods that allow us to analyze not only the structural changes but also changes in the fluctuation patterns between conformers will contribute to elucidate the differential properties acquired upon ligand binding. Molecular dynamics simulations can provide the required information to explore these features. Its use in combination with subsequent essential dynamics analysis allows separating large concerted conformational rearrangements from irrelevant fluctuations. We present a novel procedure to define the size and composition of essential dynamics subspaces associated with ligand-bound and ligand-free conformations. These definitions allow us to compare essential dynamics subspaces between different conformers. Our procedure attempts to emphasize the main similarities and differences between the different essential dynamics in an unbiased way. Essential dynamics subspaces associated to conformational transitions can also be analyzed. As a test case, we study the glutaminase interacting protein (GIP), composed of a single PDZ domain. Both GIP ligand-free state and glutaminase L peptide-bound states are analyzed. Our findings concerning the relative changes in the flexibility pattern upon binding are in good agreement with experimental Nuclear Magnetic Resonance data.

  14. Steric exclusion and protein conformation determine the localization of plasma membrane transporters.

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

    2018-02-05

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

  15. The Role of Conformational Flexibility on Protein Supercharging in Native Electrospray Ionization

    Sterling, Harry J.; Cassou, Catherine A.; Trnka, Michael J.; Burlingame, A. L.; Krantz, Bryan A.; Williams, Evan R.

    2012-01-01

    Effects of covalent intramolecular bonds, either native disulfide bridges or chemical crosslinks, on ESI supercharging of proteins from aqueous solutions were investigated. Chemically modifying cytochrome c with up to seven crosslinks or ubiquitin with up to two crosslinks did not affect the average or maximum charge states of these proteins in the absence of m-nitrobenzyl alcohol (m-NBA), but the extent of supercharging induced by m-NBA increased with decreasing numbers of crosslinks. For the model random coil polypeptide reduced/alkylated RNase A, a decrease in charging with increasing m-NBA concentration attributable to reduced surface tension of the ESI droplet was observed, whereas native RNase A electrosprayed from these same solutions exhibited enhanced charging. The inverse relationship between the extent of supercharging and the number of intramolecular crosslinks for folded proteins, as well as the absence of supercharging for proteins that are random coils in aqueous solution, indicate that conformational restrictions induced by the crosslinks reduce the extent of supercharging. These results provide additional evidence that protein and protein complex supercharging from aqueous solution is primarily due to partial or significant unfolding that occurs as a result of chemical and/or thermal denaturation induced by the supercharging reagent late in the ESI droplet lifetime. PMID:21399817

  16. How and why do toxic conformers of aberrant proteins accumulate during ageing?

    Josefson, Rebecca; Andersson, Rebecca; Nyström, Thomas

    2017-07-15

    Ageing can be defined as a gradual decline in cellular and physical functions accompanied by an increased sensitivity to the environment and risk of death. The increased risk of mortality is causally connected to a gradual, intracellular accumulation of so-called ageing factors, of which damaged and aggregated proteins are believed to be one. Such aggregated proteins also contribute to several age-related neurodegenerative disorders e.g. Alzheimer's, Parkinson's, and Huntington's diseases, highlighting the importance of protein quality control (PQC) in ageing and its associated diseases. PQC consists of two interrelated systems: the temporal control system aimed at refolding, repairing, and/or removing aberrant proteins and their aggregates and the spatial control system aimed at harnessing the potential toxicity of aberrant proteins by sequestering them at specific cellular locations. The accumulation of toxic conformers of aberrant proteins during ageing is often declared to be a consequence of an incapacitated temporal PQC system-i.e. a gradual decline in the activity of chaperones and proteases. Here, we review the current knowledge on PQC in relation to ageing and highlight that the breakdown of both temporal and spatial PQC may contribute to ageing and thus comprise potential targets for therapeutic interventions of the ageing process. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  17. Ultra-fast evaluation of protein energies directly from sequence.

    Gevorg Grigoryan

    2006-06-01

    Full Text Available The structure, function, stability, and many other properties of a protein in a fixed environment are fully specified by its sequence, but in a manner that is difficult to discern. We present a general approach for rapidly mapping sequences directly to their energies on a pre-specified rigid backbone, an important sub-problem in computational protein design and in some methods for protein structure prediction. The cluster expansion (CE method that we employ can, in principle, be extended to model any computable or measurable protein property directly as a function of sequence. Here we show how CE can be applied to the problem of computational protein design, and use it to derive excellent approximations of physical potentials. The approach provides several attractive advantages. First, following a one-time derivation of a CE expansion, the amount of time necessary to evaluate the energy of a sequence adopting a specified backbone conformation is reduced by a factor of 10(7 compared to standard full-atom methods for the same task. Second, the agreement between two full-atom methods that we tested and their CE sequence-based expressions is very high (root mean square deviation 1.1-4.7 kcal/mol, R2 = 0.7-1.0. Third, the functional form of the CE energy expression is such that individual terms of the expansion have clear physical interpretations. We derived expressions for the energies of three classic protein design targets-a coiled coil, a zinc finger, and a WW domain-as functions of sequence, and examined the most significant terms. Single-residue and residue-pair interactions are sufficient to accurately capture the energetics of the dimeric coiled coil, whereas higher-order contributions are important for the two more globular folds. For the task of designing novel zinc-finger sequences, a CE-derived energy function provides significantly better solutions than a standard design protocol, in comparable computation time. Given these advantages

  18. Conformation transitions of blood proteins under influence of physical factors on microwave dielectric method

    Gorobchenko, O.A.; Nikolov, O.T.; Gatash, S.V.

    2006-01-01

    In this article, the influence of γ-irradiation and temperature on albumin and fibrinogen conformation and dielectric properties of protein solutions have been studied by the microwave dielectric method. Both the values of the real part ε' (dielectric permittivity) and the imaginary part ε'' (dielectric losses) of the complex dielectric permittivity of the aqueous solution of bovine serum albumin and human fibrinogen as functions of temperature and γ-irradiation dose have been obtained. The time of dielectric relaxation of water molecules in the protein solutions was calculated. The hydration of the albumin and fibrinogen molecules was determined. The temperature dependencies of hydration are non-monotonous and have a number of characteristic features at the temperatures 30-34 and 44-47 deg. C for serum albumin, and 24 and 32 deg. C for fibrinogen

  19. Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor.

    Weiss, Dahlia R; Ahn, SeungKirl; Sassano, Maria F; Kleist, Andrew; Zhu, Xiao; Strachan, Ryan; Roth, Bryan L; Lefkowitz, Robert J; Shoichet, Brian K

    2013-05-17

    A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

  20. The role of side chain conformational flexibility in surface recognition by Tenebrio molitor antifreeze protein

    Daley, Margaret E.; Sykes, Brian D.

    2003-01-01

    Two-dimensional nuclear magnetic resonance spectroscopy was used to investigate the flexibility of the threonine side chains in the β-helical Tenebrio molitor antifreeze protein (TmAFP) at low temperatures. From measurement of the 3Jαβ 1H-1H scalar coupling constants, the χ1 angles and preferred rotamer populations can be calculated. It was determined that the threonines on the ice-binding face of the protein adopt a preferred rotameric conformation at near freezing temperatures, whereas the threonines not on the ice-binding face sample many rotameric states. This suggests that TmAFP maintains a preformed ice-binding conformation in solution, wherein the rigid array of threonines that form the AFP-ice interface matches the ice crystal lattice. A key factor in binding to the ice surface and inhibition of ice crystal growth appears to be the close surface-to-surface complementarity between the AFP and crystalline ice, and the lack of an entropic penalty associated with freezing out motions in a flexible ligand. PMID:12824479

  1. Prion protein β2–α2 loop conformational landscape

    Caldarulo, Enrico; Wüthrich, Kurt; Parrinello, Michele

    2017-01-01

    In transmissible spongiform encephalopathies (TSEs), which are lethal neurodegenerative diseases that affect humans and a wide range of other mammalian species, the normal “cellular” prion protein (PrPC) is transformed into amyloid aggregates representing the “scrapie form” of the protein (PrPSc). Continued research on this system is of keen interest, since new information on the physiological function of PrPC in healthy organisms is emerging, as well as new data on the mechanism of the transformation of PrPC to PrPSc. In this paper we used two different approaches: a combination of the well-tempered ensemble (WTE) and parallel tempering (PT) schemes and metadynamics (MetaD) to characterize the conformational free-energy surface of PrPC. The focus of the data analysis was on an 11-residue polypeptide segment in mouse PrPC(121–231) that includes the β2–α2 loop of residues 167–170, for which a correlation between structure and susceptibility to prion disease has previously been described. This study includes wild-type mouse PrPC and a variant with the single-residue replacement Y169A. The resulting detailed conformational landscapes complement in an integrative manner the available experimental data on PrPC, providing quantitative insights into the nature of the structural transition-related function of the β2–α2 loop. PMID:28827331

  2. Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

    Coleman, Bradley M.; Nisbet, Rebecca M.; Han, Sen; Cappai, Roberto; Hatters, Danny M.; Hill, Andrew F.

    2009-01-01

    Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP C ) into a disease associated form (PrP Sc ). Recombinant PrP can be refolded into either an α-helical rich conformation (α-PrP) resembling PrP C or a β-sheet rich, protease resistant form similar to PrP Sc . Here, we generated tetracysteine tagged recombinant PrP, folded this into α- or β-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished β-PrP from α-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the α-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the β-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP Sc from PrP C . This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer's, Huntington's and Parkinson's diseases.

  3. Probing Conformational Dynamics of Tau Protein by Hydrogen/Deuterium Exchange Mass Spectrometry

    Huang, Richard Y.-C.; Iacob, Roxana E.; Sankaranarayanan, Sethu; Yang, Ling; Ahlijanian, Michael; Tao, Li; Tymiak, Adrienne A.; Chen, Guodong

    2018-01-01

    Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. [Figure not available: see fulltext.

  4. Conformational co-dependence between Plasmodium berghei LCCL proteins promotes complex formation and stability.

    Saeed, Sadia; Tremp, Annie Z; Dessens, Johannes T

    2012-10-01

    Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Introduction to the conformational investigation of peptides and proteins by using two-dimensional proton NMR experiments

    Neumann, J.M.; Macquaire, F.

    1991-01-01

    This report presents the elementary bases for an initiation to the conformational study of peptides and proteins by using two-dimensional proton NMR experiments. First, some general features of protein structures are summarized. A second chapter is devoted to the basic NMR experiments and to the spectral parameters which provide a structural information. This description is illustrated by NMR spectra of peptides. The third chapter concerns the most standard two-dimensional proton NMR experiments and their use for a conformational study of peptides and proteins. Lastly, an example of NMR structural investigation of a peptide is reported [fr

  6. Conformational aspects of proteins at the air/water interface studied by infrared reflection-adsorption spectroscopy

    Martin, A.H.; Meinders, M.B.J.; Bos, M.A.; Cohen Stuart, M.A.; Vliet, van T.

    2003-01-01

    From absorption spectra obtained with infrared reflection-absorption spectroscopy (IRRAS), it is possible to obtain information on conformational changes at a secondary folding level of proteins adsorbed at the air/water interface. In addition, information on protein concentration at the interface

  7. Conformational aspects of proteins at the air/water interface studied by infrared reflection-absorption spectroscopy

    Martin, A.H.; Meinders, M.B.J.; Bos, M.A.; Cohen Stuart, M.A.; Vliet, T. van

    2003-01-01

    From absorption spectra obtained with infrared reflection - absorption spectroscopy (IRRAS), it is possible to obtain information on conformational changes at a secondary folding level of proteins adsorbed at the air/water interface. In addition, information on protein concentration at the interface

  8. Determining In Situ Protein Conformation and Orientation from the Amide-I Sum-Frequency Generation Spectrum: Theory and Experiment

    Roeters, S.J.; van Dijk, C.N.; Torres Knoop, A.; Backus, E.H.G.; Campen, R.K.; Bonn, M.; Woutersen, S.

    2013-01-01

    Vibrational sum-frequency generation (VSFG) spectra of the amide-I band of proteins can give detailed insight into biomolecular processes near membranes. However, interpreting these spectra in terms of the conformation and orientation of a protein can be difficult, especially in the case of complex

  9. Refinement of protein termini in template-based modeling using conformational space annealing.

    Park, Hahnbeom; Ko, Junsu; Joo, Keehyoung; Lee, Julian; Seok, Chaok; Lee, Jooyoung

    2011-09-01

    The rapid increase in the number of experimentally determined protein structures in recent years enables us to obtain more reliable protein tertiary structure models than ever by template-based modeling. However, refinement of template-based models beyond the limit available from the best templates is still needed for understanding protein function in atomic detail. In this work, we develop a new method for protein terminus modeling that can be applied to refinement of models with unreliable terminus structures. The energy function for terminus modeling consists of both physics-based and knowledge-based potential terms with carefully optimized relative weights. Effective sampling of both the framework and terminus is performed using the conformational space annealing technique. This method has been tested on a set of termini derived from a nonredundant structure database and two sets of termini from the CASP8 targets. The performance of the terminus modeling method is significantly improved over our previous method that does not employ terminus refinement. It is also comparable or superior to the best server methods tested in CASP8. The success of the current approach suggests that similar strategy may be applied to other types of refinement problems such as loop modeling or secondary structure rearrangement. Copyright © 2011 Wiley-Liss, Inc.

  10. Conformational energy calculations on polypeptides and proteins: use of a statistical mechanical procedure for evaluating structure and properties.

    Scheraga, H A; Paine, G H

    1986-01-01

    We are using a variety of theoretical and computational techniques to study protein structure, protein folding, and higher-order structures. Our earlier work involved treatments of liquid water and aqueous solutions of nonpolar and polar solutes, computations of the stabilities of the fundamental structures of proteins and their packing arrangements, conformations of small cyclic and open-chain peptides, structures of fibrous proteins (collagen), structures of homologous globular proteins, introduction of special procedures as constraints during energy minimization of globular proteins, and structures of enzyme-substrate complexes. Recently, we presented a new methodology for predicting polypeptide structure (described here); the method is based on the calculation of the probable and average conformation of a polypeptide chain by the application of equilibrium statistical mechanics in conjunction with an adaptive, importance sampling Monte Carlo algorithm. As a test, it was applied to Met-enkephalin.

  11. Interconversion of two GDP-bound conformations and their selection in an Arf-family small G protein.

    Okamura, Hideyasu; Nishikiori, Masaki; Xiang, Hongyu; Ishikawa, Masayuki; Katoh, Etsuko

    2011-07-13

    ADP-ribosylation factor (Arf) and other Arf-family small G proteins participate in many cellular functions via their characteristic GTP/GDP conformational cycles, during which a nucleotide(∗)Mg(2+)-binding site communicates with a remote N-terminal helix. However, the conformational interplay between the nucleotides, the helix, the protein core, and Mg(2+) has not been fully delineated. Herein, we report a study of the dynamics of an Arf-family protein, Arl8, under various conditions by means of NMR relaxation spectroscopy. The data indicated that, when GDP is bound, the protein core, which does not include the N-terminal helix, reversibly transition between an Arf-family GDP form and another conformation that resembles the Arf-family GTP form. Additionally, we found that the N-terminal helix and Mg(2+), respectively, stabilize the aforementioned former and latter conformations in a population-shift manner. Given the dynamics of the conformational changes, we can describe the Arl8 GTP/GDP cycle in terms of an energy diagram. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. A multiscale approach to simulating the conformational properties of unbound multi-C₂H₂ zinc finger proteins.

    Liu, Lei; Wade, Rebecca C; Heermann, Dieter W

    2015-09-01

    The conformational properties of unbound multi-Cys2 His2 (mC2H2) zinc finger proteins, in which zinc finger domains are connected by flexible linkers, are studied by a multiscale approach. Three methods on different length scales are utilized. First, atomic detail molecular dynamics simulations of one zinc finger and its adjacent flexible linker confirmed that the zinc finger is more rigid than the flexible linker. Second, the end-to-end distance distributions of mC2H2 zinc finger proteins are computed using an efficient atomistic pivoting algorithm, which only takes excluded volume interactions into consideration. The end-to-end distance distribution gradually changes its profile, from left-tailed to right-tailed, as the number of zinc fingers increases. This is explained by using a worm-like chain model. For proteins of a few zinc fingers, an effective bending constraint favors an extended conformation. Only for proteins containing more than nine zinc fingers, is a somewhat compacted conformation preferred. Third, a mesoscale model is modified to study both the local and the global conformational properties of multi-C2H2 zinc finger proteins. Simulations of the CCCTC-binding factor (CTCF), an important mC2H2 zinc finger protein for genome spatial organization, are presented. © 2015 Wiley Periodicals, Inc.

  13. An Improved Methodology for Multidimensional High-Throughput Preformulation Characterization of Protein Conformational Stability

    Maddux, Nathaniel R.; Rosen, Ilan T.; Hu, Lei; Olsen, Christopher M.; Volkin, David B.; Middaugh, C. Russell

    2013-01-01

    The Empirical Phase Diagram (EPD) technique is a vector-based multidimensional analysis method for summarizing large data sets from a variety of biophysical techniques. It can be used to provide comprehensive preformulation characterization of a macromolecule’s higher-order structural integrity and conformational stability. In its most common mode, it represents a type of stimulus-response diagram using environmental variables such as temperature, pH, and ionic strength as the stimulus, with alterations in macromolecular structure being the response. Until now EPD analysis has not been available in a high throughput mode because of the large number of experimental techniques and environmental stressor/stabilizer variables typically employed. A new instrument has been developed that combines circular dichroism, UV-absorbance, fluorescence spectroscopy and light scattering in a single unit with a 6-position temperature controlled cuvette turret. Using this multifunctional instrument and a new software system we have generated EPDs for four model proteins. Results confirm the reproducibility of the apparent phase boundaries and protein behavior within the boundaries. This new approach permits two EPDs to be generated per day using only 0.5 mg of protein per EPD. Thus, the new methodology generates reproducible EPDs in high-throughput mode, and represents the next step in making such determinations more routine. PMID:22447621

  14. Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

    Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

    2017-09-01

    Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Handheld highly selective plasmonic chem/biosensor using engineered binding proteins for extreme conformational changes

    Kosciolek, Derek J.; Sonar, Ajay; Lepak, Lori A.; Schnatz, Peter; Bendoym, Igor; Brown, Mia C.; Koder, Ronald L.; Crouse, David T.

    2017-08-01

    In this project we develop a handheld, portable, highly selective and sensitive chem/biosensor that has potential applications in both airborne and water-based environmental sensing. The device relies on a plasmonic chip of subwavelength-scale periodic gold rods engineered to resonate in the near infrared. The chip is functionalized with a novel class of proteins that exhibit large conformational changes upon binding to a specific target analyte. The subsequent change in local refractive index near the surface of the gold is one to two orders of magnitude greater than current conventional methods, which produces a readily measurable 5 to 10 percent difference in light transmission. This allows us to forgo traditional, bulky tabletop setups in favor of a compact form factor. Using commercially available optics to construct a transmission-based optical train, measured changes in bulk refractive index are presented here. While synthesis of binding protein efforts are focused on heme as analyte for proof of concept validation, the functionalized protein can be engineered to pair with a wide variety of analytes with minimal alterations to the plasmonic chip or device design. Such flexibility allows for this device to potentially meet the needs of first responders and health care professionals in a multitude of scenarios.

  16. The Staphylococcus aureus extracellular adherence protein (Eap) adopts an elongated but structured conformation in solution.

    Hammel, Michal; Nemecek, Daniel; Keightley, J Andrew; Thomas, George J; Geisbrecht, Brian V

    2007-12-01

    The extracellular adherence protein (Eap) of Staphylococcus aureus participates in a wide range of protein-protein interactions that facilitate the initiation and dissemination of Staphylococcal disease. In this report, we describe the use of a multidisciplinary approach to characterize the solution structure of full-length Eap. In contrast to previous reports suggesting that a six-domain isoform of Eap undergoes multimerization, sedimentation equilibrium analytical ultracentrifugation data revealed that a four-domain isoform of Eap is a monomer in solution. In vitro proteolysis and solution small angle X-ray scattering studies both indicate that Eap adopts an extended conformation in solution, where the linkers connecting sequential EAP modules are solvent exposed. Construction of a low-resolution model of full-length Eap using a combination of ab initio deconvolution of the SAXS data and rigid body modeling of the EAP domain crystal structure suggests that full-length Eap may present several unique concave surfaces capable of participating in ligand binding. These results also raise the possibility that such surfaces may be held together by additional interactions between adjacent EAP modules. This hypothesis is supported by a comparative Raman spectroscopic analysis of full-length Eap and a stoichiometric solution of the individual EAP modules, which indicates the presence of additional secondary structure and a greater extent of hydrogen/deuterium exchange protection in full-length Eap. Our results provide the first insight into the solution structure of full-length Eap and an experimental basis for interpreting the EAP domain crystal structures within the context of the full-length molecule. They also lay a foundation for future studies into the structural and molecular bases of Eap-mediated protein-protein interactions with its many ligands.

  17. Biospectroscopy of Nanodiamond-Induced Alterations in Conformation of Intra- and Extracellular Proteins: A Nanoscale IR Study.

    Khanal, Dipesh; Kondyurin, Alexey; Hau, Herman; Knowles, Jonathan C; Levinson, Olga; Ramzan, Iqbal; Fu, Dong; Marcott, Curtis; Chrzanowski, Wojciech

    2016-08-02

    The toxicity of nanomaterials raises major concerns because of the impact that nanomaterials may have on health, which remains poorly understood. We need to explore the fate of individual nanoparticles in cells at nano and molecular levels to establish their safety. Conformational changes in secondary protein structures are one of the main indicators of impaired biological function, and hence, the ability to identify these changes at a nanoscale level offers unique insights into the nanotoxicity of materials. Here, we used nanoscale infrared spectroscopy and demonstrated for the first time that nanodiamond-induced alterations in both extra- and intracellular secondary protein structures lead to the formation of antiparallel β-sheet, β-turns, intermolecular β-sheet, and aggregation of proteins. These conformational changes of the protein structure may result in the loss of functionality of proteins and in turn lead to adverse effects.

  18. Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

    Eddy, Matthew T.; Su, Yongchao; Silvers, Robert; Andreas, Loren; Clark, Lindsay; Wagner, Gerhard; Pintacuda, Guido; Emsley, Lyndon; Griffin, Robert G.

    2015-01-01

    The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for 13 C line widths and <0.5 ppm 15 N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported

  19. Fasting Lipoprotein Lipase Protein Levels Can Predict a Postmeal Increment of Triglyceride Levels in Fasting Normohypertriglyceridemic Subjects.

    Tsuzaki, Kokoro; Kotani, Kazuhiko; Yamada, Kazunori; Sakane, Naoki

    2016-09-01

    Although a postprandial increment in triglyceride (TG) levels is considered to be a risk factor for atherogenesis, tests (e.g., fat load) to assess postprandial changes in TG levels cannot be easily applied to clinical practice. Therefore, fasting markers that predict postprandial TG states are needed to be developed. One current candidate is lipoprotein lipase (LPL) protein, a molecule that hydrides TGs. This study investigated whether fasting LPL levels could predict postprandial TG levels. A total of 17 subjects (11 men, 6 women, mean age 52 ± 11 years) with normotriglyceridemia during fasting underwent the meal test. Several fasting parameters, including LPL, were measured for the area under the curve of postprandial TGs (AUC-TG). The subjects' mean fasting TG level was 1.30 mmol/l, and their mean LPL level was 41.6 ng/ml. The subjects' TG levels increased after loading (they peaked after two postprandial hours). Stepwise multiple regression analysis demonstrated that fasting TG levels were a predictor of the AUC-TG. In addition, fasting LPL mass levels were found to be a predictor of the AUC-TG (β = 0.65, P fasting TG levels. Fasting LPL levels may be useful to predict postprandial TG increment in this population. © 2015 Wiley Periodicals, Inc.

  20. Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites

    Serrano, Pedro; Pedrini, Bill; Geralt, Michael; Jaudzems, Kristaps; Mohanty, Biswaranjan; Horst, Reto; Herrmann, Torsten; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

    2010-01-01

    Tools for systematic comparisons of NMR and crystal structures developed by the JCSG were applied to two proteins with known functions: the T. maritima anti-σ factor antagonist TM1081 and the mouse γ-glutamylamine cyclotransferase A2LD1 (gi:13879369). In an attempt to exploit the complementarity of crystal and NMR data, the combined use of the two structure-determination techniques was explored for the initial steps in the challenge of searching proteins of unknown functions for putative active sites. The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-σ factor antagonist, and A2LD1 (gi:13879369), a mouse γ-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298 K, respectively, using the current JCSG protocol based on the software package UNIO for extensive automation. The corresponding crystal structures were solved by the JCSG at 100 K and 1.6 Å resolution and at 100 K and 1.9 Å resolution, respectively. The NMR and crystal structures of the two proteins share the same overall molecular architectures. However, the precision of the structure determination along the amino-acid sequence varies over a significantly wider range in the NMR structures than in the crystal structures. Thereby, in each of the two NMR structures about 65% of the residues have displacements below the average and in both proteins the less well ordered residues include large parts of the active sites, in addition to some highly solvent-exposed surface areas. Whereas the latter show increased disorder in the crystal and in solution, the active-site regions display increased displacements only in the NMR structures, where they undergo local conformational exchange on the

  1. Conformational intermediate of the amyloidogenic protein beta 2-microglobulin at neutral pH

    Heegaard, N H; Sen, J W; Kaarsholm, N C

    2001-01-01

    electrophoresis that two conformers spontaneously exist in aqueous buffers at neutral pH. Upon treatment of wild-type beta(2)-microglobulin with acetonitrile or trifluoroethanol, two conformations were also observed. These conformations were in equilibrium dependent on the sample temperature and the percentage...

  2. Assessing protein conformational sampling methods based on bivariate lag-distributions of backbone angles

    Maadooliat, Mehdi; Gao, Xin; Huang, Jianhua Z.

    2012-01-01

    Despite considerable progress in the past decades, protein structure prediction remains one of the major unsolved problems in computational biology. Angular-sampling-based methods have been extensively studied recently due to their ability to capture the continuous conformational space of protein structures. The literature has focused on using a variety of parametric models of the sequential dependencies between angle pairs along the protein chains. In this article, we present a thorough review of angular-sampling-based methods by assessing three main questions: What is the best distribution type to model the protein angles? What is a reasonable number of components in a mixture model that should be considered to accurately parameterize the joint distribution of the angles? and What is the order of the local sequence-structure dependency that should be considered by a prediction method? We assess the model fits for different methods using bivariate lag-distributions of the dihedral/planar angles. Moreover, the main information across the lags can be extracted using a technique called Lag singular value decomposition (LagSVD), which considers the joint distribution of the dihedral/planar angles over different lags using a nonparametric approach and monitors the behavior of the lag-distribution of the angles using singular value decomposition. As a result, we developed graphical tools and numerical measurements to compare and evaluate the performance of different model fits. Furthermore, we developed a web-tool (http://www.stat.tamu. edu/~madoliat/LagSVD) that can be used to produce informative animations. © The Author 2012. Published by Oxford University Press.

  3. Assessing protein conformational sampling methods based on bivariate lag-distributions of backbone angles

    Maadooliat, Mehdi

    2012-08-27

    Despite considerable progress in the past decades, protein structure prediction remains one of the major unsolved problems in computational biology. Angular-sampling-based methods have been extensively studied recently due to their ability to capture the continuous conformational space of protein structures. The literature has focused on using a variety of parametric models of the sequential dependencies between angle pairs along the protein chains. In this article, we present a thorough review of angular-sampling-based methods by assessing three main questions: What is the best distribution type to model the protein angles? What is a reasonable number of components in a mixture model that should be considered to accurately parameterize the joint distribution of the angles? and What is the order of the local sequence-structure dependency that should be considered by a prediction method? We assess the model fits for different methods using bivariate lag-distributions of the dihedral/planar angles. Moreover, the main information across the lags can be extracted using a technique called Lag singular value decomposition (LagSVD), which considers the joint distribution of the dihedral/planar angles over different lags using a nonparametric approach and monitors the behavior of the lag-distribution of the angles using singular value decomposition. As a result, we developed graphical tools and numerical measurements to compare and evaluate the performance of different model fits. Furthermore, we developed a web-tool (http://www.stat.tamu. edu/~madoliat/LagSVD) that can be used to produce informative animations. © The Author 2012. Published by Oxford University Press.

  4. Dynamic fluctuations provide the basis of a conformational switch mechanism in apo cyclic AMP receptor protein.

    Burcu Aykaç Fas

    Full Text Available Escherichia coli cyclic AMP Receptor Protein (CRP undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD simulations and Gaussian Network Model (GNM. The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

  5. Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

    Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

    2014-02-01

    In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  6. Functional and conformational properties of phaseolin (Phaseolus vulgris L.) and kidney bean protein isolate: a comparative study.

    Yin, Shou-Wei; Tang, Chuan-He; Wen, Qi-Biao; Yang, Xiao-Quan

    2010-03-15

    Kidney bean (Phaseolus vulgris L.) seed is an underutilised plant protein source with good potential to be applied in the food industry. Phaseolin (also named G1 globulin) represents about 50 g kg(-1) of total storage protein in the seed. The aim of the present study was to characterise physicochemical, functional and conformational properties of phaseolin, and to compare these properties with those of kidney bean protein isolate (KPI). Compared with kidney bean protein isolate (KPI), the acid-extracted phaseolin-rich protein product (PRP) had much lower protein recovery of 320 g kg(-1) (dry weight basis) but higher phaseolin purity (over 950 g kg(-1)). PRP contained much lower sulfhydryl (SH) and disulfide bond contents than KPI. Differential scanning calorimetry analyses showed that the phaseolin in PRP was less denatured than in KPI. Thermal analyses in the presence or absence of dithiothreitol, in combination with SH and SS content analyses showed the contributions of SS to the thermal stability of KPI. The analyses of near-UV circular dichroism and intrinsic fluorescence spectra indicated more compacted tertiary conformation of the proteins in PRP than in KPI. PRP exhibited much better protein solubility, emulsifying activity index, and gel-forming ability than KPI. The relatively poor functional properties of KPI may be associated with protein denaturation/unfolding, with subsequent protein aggregation. The results presented here suggest the potential for acid-extracted PRP to be applied in food formulations, in view of its functional properties.

  7. Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

    Furebring Christina

    2009-03-01

    Full Text Available Abstract Background The Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS blocks the Complement fragment C5a receptor (C5aR and formylated peptide receptor (FPR and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function in vitro. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity. Results In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS31–121 protein. Mapped epitopes were verified by in vitro mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding. Conclusion Conformational epitopes recognized by human antibodies

  8. The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane

    Juan Fontana

    2017-08-01

    Full Text Available All enveloped viruses, including herpesviruses, must fuse their envelope with the host membrane to deliver their genomes into target cells, making this essential step subject to interference by antibodies and drugs. Viral fusion is mediated by a viral surface protein that transits from an initial prefusion conformation to a final postfusion conformation. Strikingly, the prefusion conformation of the herpesvirus fusion protein, gB, is poorly understood. Herpes simplex virus (HSV, a model system for herpesviruses, causes diseases ranging from mild skin lesions to serious encephalitis and neonatal infections. Using cryo-electron tomography and subtomogram averaging, we have characterized the structure of the prefusion conformation and fusion intermediates of HSV-1 gB. To this end, we have set up a system that generates microvesicles displaying full-length gB on their envelope. We confirmed proper folding of gB by nondenaturing electrophoresis-Western blotting with a panel of monoclonal antibodies (MAbs covering all gB domains. To elucidate the arrangement of gB domains, we labeled them by using (i mutagenesis to insert fluorescent proteins at specific positions, (ii coexpression of gB with Fabs for a neutralizing MAb with known binding sites, and (iii incubation of gB with an antibody directed against the fusion loops. Our results show that gB starts in a compact prefusion conformation with the fusion loops pointing toward the viral membrane and suggest, for the first time, a model for gB’s conformational rearrangements during fusion. These experiments further illustrate how neutralizing antibodies can interfere with the essential gB structural transitions that mediate viral entry and therefore infectivity.

  9. A Novel Method Using Abstract Convex Underestimation in Ab-Initio Protein Structure Prediction for Guiding Search in Conformational Feature Space.

    Hao, Xiao-Hu; Zhang, Gui-Jun; Zhou, Xiao-Gen; Yu, Xu-Feng

    2016-01-01

    To address the searching problem of protein conformational space in ab-initio protein structure prediction, a novel method using abstract convex underestimation (ACUE) based on the framework of evolutionary algorithm was proposed. Computing such conformations, essential to associate structural and functional information with gene sequences, is challenging due to the high-dimensionality and rugged energy surface of the protein conformational space. As a consequence, the dimension of protein conformational space should be reduced to a proper level. In this paper, the high-dimensionality original conformational space was converted into feature space whose dimension is considerably reduced by feature extraction technique. And, the underestimate space could be constructed according to abstract convex theory. Thus, the entropy effect caused by searching in the high-dimensionality conformational space could be avoided through such conversion. The tight lower bound estimate information was obtained to guide the searching direction, and the invalid searching area in which the global optimal solution is not located could be eliminated in advance. Moreover, instead of expensively calculating the energy of conformations in the original conformational space, the estimate value is employed to judge if the conformation is worth exploring to reduce the evaluation time, thereby making computational cost lower and the searching process more efficient. Additionally, fragment assembly and the Monte Carlo method are combined to generate a series of metastable conformations by sampling in the conformational space. The proposed method provides a novel technique to solve the searching problem of protein conformational space. Twenty small-to-medium structurally diverse proteins were tested, and the proposed ACUE method was compared with It Fix, HEA, Rosetta and the developed method LEDE without underestimate information. Test results show that the ACUE method can more rapidly and more

  10. Increasing the sampling efficiency of protein conformational transition using velocity-scaling optimized hybrid explicit/implicit solvent REMD simulation

    Yu, Yuqi; Wang, Jinan; Shao, Qiang, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn; Zhu, Weiliang, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn [ACS Key Laboratory of Receptor Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203 (China); Shi, Jiye, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn [UCB Pharma, 216 Bath Road, Slough SL1 4EN (United Kingdom)

    2015-03-28

    The application of temperature replica exchange molecular dynamics (REMD) simulation on protein motion is limited by its huge requirement of computational resource, particularly when explicit solvent model is implemented. In the previous study, we developed a velocity-scaling optimized hybrid explicit/implicit solvent REMD method with the hope to reduce the temperature (replica) number on the premise of maintaining high sampling efficiency. In this study, we utilized this method to characterize and energetically identify the conformational transition pathway of a protein model, the N-terminal domain of calmodulin. In comparison to the standard explicit solvent REMD simulation, the hybrid REMD is much less computationally expensive but, meanwhile, gives accurate evaluation of the structural and thermodynamic properties of the conformational transition which are in well agreement with the standard REMD simulation. Therefore, the hybrid REMD could highly increase the computational efficiency and thus expand the application of REMD simulation to larger-size protein systems.

  11. Alternative Conformations of the Tau Repeat Domain in Complex with an Engineered Binding Protein*

    Grüning, Clara S. R.; Mirecka, Ewa A.; Klein, Antonia N.; Mandelkow, Eckhard; Willbold, Dieter; Marino, Stephen F.; Stoldt, Matthias; Hoyer, Wolfgang

    2014-01-01

    The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337–342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau. PMID:24966331

  12. Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases

    Hickman, D.T.; Nand, D.; Baldus, M.; Muhs, A.

    2011-01-01

    Synthetic peptide immunogens which mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters

  13. Spectroscopic and theoretical investigation of conformational changes of proteins by synthesized pyrimidine derivative and its sensitivity towards FRET application

    Ghosh, Swadesh; Singharoy, Dipti; Bhattacharya, Subhash Chandra

    2018-04-01

    Interest in synthesizing and characterizing (IR, NMR and HRMS spectroscopic methods) a pyrimidine based Schiff-base ligand, 2-(2-(Anthracen-9-ylmethylene) hydrazinyl)-4,6-dimethyl pyrimidine (ANHP) has been developed for its application to ascertain the conformational change of protein and sensitivity towards fluorescence resonance energy transfer (FRET) process. Location of ANHP in bovine serum albumin (BSA) and human serum albumin (HSA) proteins environment has been determined using different spectroscopic techniques. Weakly fluorescent ANHP have shown greater protein induced fluorescence enhancement (PIFE) in case of HSA than BSA, though in both cases energy transfer efficiency are almost same but difference in binding constant values encourages us to find the location of ANHP within the complex protein environment. From the FRET parameter and α-helicity change, it has been found that ANHP bound with Trp-214 of HSA and surface Trp-134 of BSA. Conformational changes of proteins have been observed more for HSA than BSA in presence of ANHP, which has confirmed the location of ANHP in both the protein environments. Coupled with experimental studies, molecular docking analysis has also been done to explain the locations and distance dependent FRET process of ANHP in both proteins.

  14. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

    Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

    2016-03-01

    DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

  15. Conformational Changes in the Hepatitis B Virus Core Protein Are Consistent with a Role for Allostery in Virus Assembly

    Packianathan, Charles; Katen, Sarah P.; Dann, III, Charles E.; Zlotnick, Adam (Indiana)

    2010-01-12

    In infected cells, virus components must be organized at the right place and time to ensure assembly of infectious virions. From a different perspective, assembly must be prevented until all components are available. Hypothetically, this can be achieved by allosterically controlling assembly. Consistent with this hypothesis, here we show that the structure of the hepatitis B virus (HBV) core protein dimer, which can spontaneously self-assemble, is incompatible with capsid assembly. Systematic differences between core protein dimer and capsid conformations demonstrate linkage between the intradimer interface and interdimer contact surface. These structures also provide explanations for the capsid-dimer selectivity of some antibodies and the activities of assembly effectors. Solution studies suggest that the assembly-inactive state is more accurately an ensemble of conformations. Simulations show that allostery supports controlled assembly and results in capsids that are resistant to dissociation. We propose that allostery, as demonstrated in HBV, is common to most self-assembling viruses.

  16. Effect of dietary protein level and length of fattening period on dressing percentage and carcass conformation in broiler chickens

    Dosković, Vladimir; Bogosavljević-Bošković, Snežana; Škrbić, Zdenka; Đoković, Radojica; Rakonjac, Simeon; Petričević, Veselin

    2017-01-01

    This study analyses the effect of different protein levels in broiler feeds (supplemented with protease) and different lengths of fattening period on some parameters related to dressed carcass quality. Medium-growing Master Gris broiler chickens were used in a fattening trial lasting 63 days. At slaughter, dressing percentages and abdominal fat percentages were determined based on traditionally dressed carcass weights and abdominal fat weights of broilers at 49 and 63 days, and conformation i...

  17. The making of the minibody: an engineered beta-protein for the display of conformationally constrained peptides.

    Tramontano, A; Bianchi, E; Venturini, S; Martin, F; Pessi, A; Sollazzo, M

    1994-03-01

    Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost the drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue beta-protein aimed at retaining some desirable features of immunoglobulin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of the main chain conformation of the same regions, based on the 'canonical structures' model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical synthesis and expression in E. coli and characterized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low solubility. We were able to overcome this problem both by further mutagenesis of the framework and by addition of a solubilizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.

  18. Protein energetic conformational analysis from NMR chemical shifts (PECAN) and its use in determining secondary structural elements

    Eghbalnia, Hamid R.; Wang Liya; Bahrami, Arash [National Magnetic Resonance Facility at Madison, Biochemistry Department (United States); Assadi, Amir [University of Wisconsin-Madison, Mathematics Department (United States); Markley, John L. [National Magnetic Resonance Facility at Madison, Biochemistry Department (United States)], E-mail: eghbalni@nmrfam.wisc.edu

    2005-05-15

    We present an energy model that combines information from the amino acid sequence of a protein and available NMR chemical shifts for the purposes of identifying low energy conformations and determining elements of secondary structure. The model ('PECAN', Protein Energetic Conformational Analysis from NMR chemical shifts) optimizes a combination of sequence information and residue-specific statistical energy function to yield energetic descriptions most favorable to predicting secondary structure. Compared to prior methods for secondary structure determination, PECAN provides increased accuracy and range, particularly in regions of extended structure. Moreover, PECAN uses the energetics to identify residues located at the boundaries between regions of predicted secondary structure that may not fit the stringent secondary structure class definitions. The energy model offers insights into the local energetic patterns that underlie conformational preferences. For example, it shows that the information content for defining secondary structure is localized about a residue and reaches a maximum when two residues on either side are considered. The current release of the PECAN software determines the well-defined regions of secondary structure in novel proteins with assigned chemical shifts with an overall accuracy of 90%, which is close to the practical limit of achievable accuracy in classifying the states.

  19. Protein energetic conformational analysis from NMR chemical shifts (PECAN) and its use in determining secondary structural elements

    Eghbalnia, Hamid R.; Wang Liya; Bahrami, Arash; Assadi, Amir; Markley, John L.

    2005-01-01

    We present an energy model that combines information from the amino acid sequence of a protein and available NMR chemical shifts for the purposes of identifying low energy conformations and determining elements of secondary structure. The model ('PECAN', Protein Energetic Conformational Analysis from NMR chemical shifts) optimizes a combination of sequence information and residue-specific statistical energy function to yield energetic descriptions most favorable to predicting secondary structure. Compared to prior methods for secondary structure determination, PECAN provides increased accuracy and range, particularly in regions of extended structure. Moreover, PECAN uses the energetics to identify residues located at the boundaries between regions of predicted secondary structure that may not fit the stringent secondary structure class definitions. The energy model offers insights into the local energetic patterns that underlie conformational preferences. For example, it shows that the information content for defining secondary structure is localized about a residue and reaches a maximum when two residues on either side are considered. The current release of the PECAN software determines the well-defined regions of secondary structure in novel proteins with assigned chemical shifts with an overall accuracy of 90%, which is close to the practical limit of achievable accuracy in classifying the states

  20. Conformational Flexibility of Proteins Involved in Ribosome Biogenesis: Investigations via Small Angle X-ray Scattering (SAXS

    Dritan Siliqi

    2018-02-01

    Full Text Available The dynamism of proteins is central to their function, and several proteins have been described as flexible, as consisting of multiple domains joined by flexible linkers, and even as intrinsically disordered. Several techniques exist to study protein structures, but small angle X-ray scattering (SAXS has proven to be particularly powerful for the quantitative analysis of such flexible systems. In the present report, we have used SAXS in combination with X-ray crystallography to highlight their usefulness at characterizing flexible proteins, using as examples two proteins involved in different steps of ribosome biogenesis. The yeast BRCA2 and CDKN1A-interactig protein, Bcp1, is a chaperone for Rpl23 of unknown structure. We showed that it consists of a rigid, slightly elongated protein, with a secondary structure comprising a mixture of alpha helices and beta sheets. As an example of a flexible molecule, we studied the SBDS (Shwachman-Bodian-Diamond Syndrome protein that is involved in the cytoplasmic maturation of the 60S subunit and constitutes the mutated target in the Shwachman-Diamond Syndrome. In solution, this protein coexists in an ensemble of three main conformations, with the N- and C-terminal ends adopting different orientations with respect to the central domain. The structure observed in the protein crystal corresponds to an average of those predicted by the SAXS flexibility analysis.

  1. Synthesis of 19-substituted geldanamycins with altered conformations and their binding to heat shock protein Hsp90

    Kitson, Russell R. A.; Chang, Chuan-Hsin; Xiong, Rui; Williams, Huw E. L.; Davis, Adrienne L.; Lewis, William; Dehn, Donna L.; Siegel, David; Roe, S. Mark; Prodromou, Chrisostomos; Ross, David; Moody, Christopher J.

    2013-01-01

    The benzoquinone ansamycin geldanamycin and its derivatives are inhibitors of heat shock protein Hsp90, an emerging target for novel therapeutic agents both in cancer and in neurodegeneration. However, toxicity of these compounds to normal cells has been ascribed to reaction with thiol nucleophiles at the quinone 19-position. We reasoned that blocking this position would ameliorate toxicity, and that it might also enforce a favourable conformational switch of the trans-amide group into the cis-form required for protein binding. We report here an efficient synthesis of such 19-substituted compounds and realization of our hypotheses. Protein crystallography established that the new compounds bind to Hsp90 with, as expected, a cis-amide conformation. Studies on Hsp90 inhibition in cells demonstrated the molecular signature of Hsp90 inhibitors: decreases in client proteins with compensatory increases in other heat shock proteins in both human breast cancer and dopaminergic neural cells, demonstrating their potential for use in the therapy of cancer or neurodegenerative diseases. PMID:23511419

  2. Measurement of conformational constraints in an elastin-mimetic protein by residue-pair selected solid-state NMR

    Hong, Mei [Iowa State University, Department of Chemistry (United States)], E-mail: mhong@iastate.edu; McMillan, R. Andrew; Conticello, Vincent P. [Emory University, Department of Chemistry (United States)

    2002-02-15

    We introduce a solid-state NMR technique for selective detection of a residue pair in multiply labeled proteins to obtain site-specific structural constraints. The method exploits the frequency-offset dependence of cross polarization to achieve {sup 13}CO{sub i} {sup {yields}} {sup 15}N{sub i} {sup {yields}} {sup 13}C{alpha}{sub i} transfer between two residues. A {sup 13}C, {sup 15}N-labeled elastin mimetic protein (VPGVG){sub n} is used to demonstrate the method. The technique selected the Gly3 C{alpha} signal while suppressing the Gly5 C{alpha} signal, and allowed the measurement of the Gly3 C{alpha} chemical shift anisotropy to derive information on the protein conformation. This residue-pair selection technique should simplify the study of protein structure at specific residues.

  3. Measurement of conformational constraints in an elastin-mimetic protein by residue-pair selected solid-state NMR

    Hong, Mei; McMillan, R. Andrew; Conticello, Vincent P.

    2002-01-01

    We introduce a solid-state NMR technique for selective detection of a residue pair in multiply labeled proteins to obtain site-specific structural constraints. The method exploits the frequency-offset dependence of cross polarization to achieve 13 CO i → 15 N i → 13 Cα i transfer between two residues. A 13 C, 15 N-labeled elastin mimetic protein (VPGVG) n is used to demonstrate the method. The technique selected the Gly3 Cα signal while suppressing the Gly5 Cα signal, and allowed the measurement of the Gly3 Cα chemical shift anisotropy to derive information on the protein conformation. This residue-pair selection technique should simplify the study of protein structure at specific residues

  4. Ligand binding turns moth pheromone-binding protein into a pH sensor: effect on the Antheraea polyphemus PBP1 conformation.

    Katre, Uma V; Mazumder, Suman; Prusti, Rabi K; Mohanty, Smita

    2009-11-13

    In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of approximately 1:1. Mutation of His(70) and His(95) to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.

  5. Fasting-induced adipose factor/angiopoietin-like protein 4: a potential target for dyslipidemia?

    Zandbergen, F.J.; Dijk, van S.; Müller, M.R.; Kersten, A.H.

    2006-01-01

    Recently, several proteins with homology to angiopoietins have been discovered. Three members of this new group, designated angiopoietin-like proteins (ANGPTLs), have been linked to regulation of energy metabolism. This review will focus on the fasting-induced adipose factor (FIAF)/ANGPTL4 as an

  6. Protein determination in soya bean by fast neutron activation analysis

    Szegedi, S.; Mosbah, D.S.; Varadi, M.; Szaloki, I.

    1988-01-01

    For a non-destructive determination of the protein content in soya bean samples, 14-MeV neutron activation analysis was applied. To check the method, the results obtained by X-ray fluorescence analysis and the Kjeldahl procedure were compared. For pressed pellet samples of about 1 g with 15 min irradiation and 10 min measuring times the accuracy of the protein determination was found to be 15%. (author) 7 refs.; 4 figs.; 3 tabs

  7. Fast Proton Titration Scheme for Multiscale Modeling of Protein Solutions.

    Teixeira, Andre Azevedo Reis; Lund, Mikael; da Silva, Fernando Luís Barroso

    2010-10-12

    Proton exchange between titratable amino acid residues and the surrounding solution gives rise to exciting electric processes in proteins. We present a proton titration scheme for studying acid-base equilibria in Metropolis Monte Carlo simulations where salt is treated at the Debye-Hückel level. The method, rooted in the Kirkwood model of impenetrable spheres, is applied on the three milk proteins α-lactalbumin, β-lactoglobulin, and lactoferrin, for which we investigate the net-charge, molecular dipole moment, and charge capacitance. Over a wide range of pH and salt conditions, excellent agreement is found with more elaborate simulations where salt is explicitly included. The implicit salt scheme is orders of magnitude faster than the explicit analog and allows for transparent interpretation of physical mechanisms. It is shown how the method can be expanded to multiscale modeling of aqueous salt solutions of many biomolecules with nonstatic charge distributions. Important examples are protein-protein aggregation, protein-polyelectrolyte complexation, and protein-membrane association.

  8. Cholesterol Corrects Altered Conformation of MHC-II Protein in Leishmania donovani Infected Macrophages: Implication in Therapy

    Chakrabarti, Saikat; Roy, Syamal

    2016-01-01

    Background Previously we reported that Kala-azar patients show progressive decrease in serum cholesterol as a function of splenic parasite burden. Splenic macrophages (MΦ) of Leishmania donovani (LD) infected mice show decrease in membrane cholesterol, while LD infected macrophages (I-MΦ) show defective T cell stimulating ability that could be corrected by liposomal delivery of cholesterol. T helper cells recognize peptide antigen in the context of class II MHC molecule. It is known that the conformation of a large number of membrane proteins is dependent on membrane cholesterol. In this investigation we tried to understand the influence of decreased membrane cholesterol in I-MΦ on the conformation of MHC-II protein and peptide-MHC-II stability, and its bearing on the antigen specific T-cell activation. Methodology/Principal Findings MΦ of CBA/j mice were infected with Leishmania donovani (I-MΦ). Two different anti-Aκ mAbs were used to monitor the status of MHC-II protein under parasitized condition. One of them (11.5–2) was conformation specific, whereas the other one (10.2.16) was not. Under parasitized condition, the binding of 11.5–2 decreased significantly with respect to the normal counterpart, whereas that of 10.2.16 remained unaltered. The binding of 11.5–2 was restored to normal upon liposomal delivery of cholesterol in I-MΦ. By molecular dynamics (MD) simulation studies we found that there was considerable conformational fluctuation in the transmembrane domain of the MHC-II protein in the presence of membrane cholesterol than in its absence, which possibly influenced the distal peptide binding groove. This was evident from the faster dissociation of the cognate peptide from peptide-MHC complex under parasitized condition, which could be corrected by liposomal delivery of cholesterol in I-MΦ. Conclusion The decrease in membrane cholesterol in I-MΦ may lead to altered conformation of MHC II, and this may contribute to a faster dissociation of

  9. A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP

    Rozentur-Shkop, Eva; Goobes, Gil; Chill, Jordan H., E-mail: Jordan.Chill@biu.ac.il [Bar Ilan University, Department of Chemistry (Israel)

    2016-12-15

    Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J({sup 15}N,{sup 13}Cα) couplings. This concept was realized using a unidirectional (H)NCO {sup 13}C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J({sup 15}N,{sup 13}Cα) couplings were acquired for WIP residues 2–65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16–23 and 45–60, and a helical tendency at residues 28–42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP{sup 2–65} conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein–protein interactions that stabilize the unfolded

  10. The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA.

    Aparicio, Frederic; Vilar, Marçal; Perez-Payá, Enrique; Pallás, Vicente

    2003-08-15

    Binding of coat protein (CP) to the 3' nontranslated region (3'-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo- and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3'-NTR of its genomic RNA using purified E. coli- expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3'-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3'-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg(2+), lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3'-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3'-NTR level in Alfamo- and Ilarvirus genera.

  11. The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA

    Aparicio, Frederic; Vilar, Marcal; Perez-Paya, Enrique; Pallas, Vicente

    2003-01-01

    Binding of coat protein (CP) to the 3' nontranslated region (3'-NTR) of viral RNAs is a crucial requirement to establish the infection of Alfamo- and Ilarviruses. In vitro binding properties of the Prunus necrotic ringspot ilarvirus (PNRSV) CP to the 3'-NTR of its genomic RNA using purified E. coli- expressed CP and different synthetic peptides corresponding to a 26-residue sequence near the N-terminus were investigated by electrophoretic mobility shift assays. PNRSV CP bound to, at least, three different sites existing on the 3'-NTR. Moreover, the N-terminal region between amino acid residues 25 to 50 of the protein could function as an independent RNA-binding domain. Single exchange of some arginine residues by alanine eliminated the RNA-interaction capacity of the synthetic peptides, consistent with a crucial role for Arg residues common to many RNA-binding proteins possessing Arg-rich domains. Circular dichroism spectroscopy revealed that the RNA conformation is altered when amino-terminal CP peptides bind to the viral RNA. Finally, mutational analysis of the 3'-NTR suggested the presence of a pseudoknotted structure at this region on the PNRSV RNA that, when stabilized by the presence of Mg 2+ , lost its capability to bind the coat protein. The existence of two mutually exclusive conformations for the 3'-NTR of PNRSV strongly suggests a similar regulatory mechanism at the 3'-NTR level in Alfamo- and Ilarvirus genera

  12. Rational design of a conformation-switchable Ca2+- and Tb3+-binding protein without the use of multiple coupled metal-binding sites.

    Li, Shunyi; Yang, Wei; Maniccia, Anna W; Barrow, Doyle; Tjong, Harianto; Zhou, Huan-Xiang; Yang, Jenny J

    2008-10-01

    Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+-induced conformational changes. Investigation into the determinants for Ca2+-induced conformational change is often impeded by cooperativity between multiple metal-binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+-dependent conformational changes, we report the design of a single-site Ca2+-binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2).CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 +/- 0.1 and 90 +/- 25 microM, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native-like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+-dependent proteins and coupled metal-binding sites, represents an important step in the design of trigger proteins.

  13. Practical considerations for investigation of protein conformational dynamics by {sup 15}N R{sub 1ρ} relaxation dispersion

    Walinda, Erik [Kyoto University, Department of Molecular and Cellular Physiology, Graduate School of Medicine (Japan); Morimoto, Daichi; Shirakawa, Masahiro; Sugase, Kenji, E-mail: sugase@moleng.kyoto-u.ac.jp [Kyoto University, Department of Molecular Engineering, Graduate School of Engineering (Japan)

    2017-03-15

    It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure (“excited states”) has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by {sup 15}N R{sub 1ρ} relaxation dispersion. The schemes use selective Hartmann–Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713–721, 2005; Hansen et al. in J Am Chem Soc 131:3818–3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R{sub 1ρ} relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.

  14. Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.

    Devost, Dominic; Sleno, Rory; Pétrin, Darlaine; Zhang, Alice; Shinjo, Yuji; Okde, Rakan; Aoki, Junken; Inoue, Asuka; Hébert, Terence E

    2017-03-31

    Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gα q , Gα 11 , Gα 12 , and Gα 13 or β-arrestins, we showed that Gα q and Gα 11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Conformationally rigid histone deacetylase inhibitors correct DF508-CFTR protein function

    Vickers, Chris J.; Olsen, Christian Adam; Hutt, Darren M.

    2011-01-01

    and bacterial infection, therapy using HDAC inhibitors has the potential to treat and correct the underlying etiology associated with the disorder. Subsequently, we have synthesized conformationally well-defined cyclic tetrapeptide derivatives based on the natural product HDAC inhibitor Apicidin, in order...

  16. Differential Mobility Spectrometry-Hydrogen Deuterium Exchange (DMS-HDX) as a Probe of Protein Conformation in Solution.

    Zhu, Shaolong; Campbell, J Larry; Chernushevich, Igor; Le Blanc, J C Yves; Wilson, Derek J

    2016-06-01

    Differential mobility spectrometry (DMS) is an ion mobility technique that has been adopted chiefly as a pre-filter for small- to medium-sized analytes (DMS-field asymmetric waveform ion mobility spectroscopy (FAIMS)-the application of DMS to intact biomacromolecules remains largely unexplored. In this work, we employ DMS combined with gas-phase hydrogen deuterium exchange (DMS-HDX) to probe the gas-phase conformations generated from proteins that were initially folded, partially-folded, and unfolded in solution. Our findings indicate that proteins with distinct structural features in solution exhibit unique deuterium uptake profiles as function of their optimal transmission through the DMS. Ultimately we propose that DMS-HDX can, if properly implemented, provide rapid measurements of liquid-phase protein structural stability that could be of use in biopharmaceuticals development. Graphical Abstract ᅟ.

  17. Conformational Stability of the NH2-Terminal Propeptide of the Precursor of Pulmonary Surfactant Protein SP-B.

    Ángeles Bañares-Hidalgo

    Full Text Available Assembly of pulmonary surfactant lipid-protein complexes depends on conformational changes coupled with proteolytic maturation of proSP-B, the precursor of pulmonary surfactant protein B (SP-B, along the surfactant biogenesis pathway in pneumocytes. Conformational destabilization of the N-terminal propeptide of proSP-B (SP-BN triggers exposure of the mature SP-B domain for insertion into surfactant lipids. We have studied the conformational stability during GdmCl- or urea-promoted unfolding of SP-BN with trp fluorescence and circular dichroism spectroscopies. Binding of the intermediate states to bis-ANS suggests their molten globule-like character. ΔG0H2O was ~ 12.7 kJ·mol-1 either with urea or GdmCl. None of the thermal transitions of SP-BN detected by CD correspond to protein unfolding. Differential scanning calorimetry of SP-BN evidenced two endothermic peaks involved in oligomer dissociation as confirmed with 2 M urea. Ionic strength was relevant since at 150 mM NaCl, the process originating the endotherm at the highest temperature was irreversible (Tm2 = 108.5°C with an activation energy of 703.8 kJ·mol-1. At 500 mM NaCl the process became reversible (Tm2 = 114.4°C and data were fitted to the Non-two States model with two subpeaks. No free thiols in the propeptide could be titrated by DTNB with or without 5.7 M GdmCl, indicating disulfide bonds establishment.

  18. Calculation of relative free energies for ligand-protein binding, solvation, and conformational transitions using the GROMOS software.

    Riniker, Sereina; Christ, Clara D; Hansen, Halvor S; Hünenberger, Philippe H; Oostenbrink, Chris; Steiner, Denise; van Gunsteren, Wilfred F

    2011-11-24

    The calculation of the relative free energies of ligand-protein binding, of solvation for different compounds, and of different conformational states of a polypeptide is of considerable interest in the design or selection of potential enzyme inhibitors. Since such processes in aqueous solution generally comprise energetic and entropic contributions from many molecular configurations, adequate sampling of the relevant parts of configurational space is required and can be achieved through molecular dynamics simulations. Various techniques to obtain converged ensemble averages and their implementation in the GROMOS software for biomolecular simulation are discussed, and examples of their application to biomolecules in aqueous solution are given. © 2011 American Chemical Society

  19. Effective Application of Bicelles for Conformational Analysis of G Protein-Coupled Receptors by Hydrogen/Deuterium Exchange Mass Spectrometry

    Duc, Nguyen Minh; Du, Yang; Thorsen, Thor S.; Lee, Su Youn; Zhang, Cheng; Kato, Hideaki; Kobilka, Brian K.; Chung, Ka Young

    2015-05-01

    G protein-coupled receptors (GPCRs) have important roles in physiology and pathology, and 40% of drugs currently on the market target GPCRs for the treatment of various diseases. Because of their therapeutic importance, the structural mechanism of GPCR signaling is of great interest in the field of drug discovery. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for analyzing ligand binding sites, the protein-protein interaction interface, and conformational changes of proteins. However, its application to GPCRs has been limited for various reasons, including the hydrophobic nature of GPCRs and the use of detergents in their preparation. In the present study, we tested the application of bicelles as a means of solubilizing GPCRs for HDX-MS studies. GPCRs (e.g., β2-adrenergic receptor [β2AR], μ-opioid receptor, and protease-activated receptor 1) solubilized in bicelles produced better sequence coverage (greater than 90%) than GPCRs solubilized in n-dodecyl-β-D-maltopyranoside (DDM), suggesting that bicelles are a more effective method of solubilization for HDX-MS studies. The HDX-MS profile of β2AR in bicelles showed that transmembrane domains (TMs) undergo lower deuterium uptake than intracellular or extracellular regions, which is consistent with the fact that the TMs are highly ordered and embedded in bicelles. The overall HDX-MS profiles of β2AR solubilized in bicelles and in DDM were similar except for intracellular loop 3. Interestingly, we detected EX1 kinetics, an important phenomenon in protein dynamics, at the C-terminus of TM6 in β2AR. In conclusion, we suggest the application of bicelles as a useful method for solubilizing GPCRs for conformational analysis by HDX-MS.

  20. FAST

    Zuidmeer-Jongejan, Laurian; Fernandez-Rivas, Montserrat; Poulsen, Lars K.

    2012-01-01

    ABSTRACT: The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqu...

  1. Multiple conformational states of DnaA protein regulate its interaction with DnaA boxes in the initiation of DNA replication.

    Patel, Meera J; Bhatia, Lavesh; Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

    2017-09-01

    DnaA protein is the initiator of genomic DNA replication in prokaryotes. It binds to specific DNA sequences in the origin of DNA replication and unwinds small AT-rich sequences downstream for the assembly of the replisome. The mechanism of activation of DnaA that enables it to bind and organize the origin DNA and leads to replication initiation remains unclear. In this study, we have developed double-labeled fluorescent DnaA probes to analyze conformational states of DnaA protein upon binding DNA, nucleotide, and Soj sporulation protein using Fluorescence Resonance Energy Transfer (FRET). Our studies demonstrate that DnaA protein undergoes large conformational changes upon binding to substrates and there are multiple distinct conformational states that enable it to initiate DNA replication. DnaA protein adopted a relaxed conformation by expanding ~15Å upon binding ATP and DNA to form the ATP·DnaA·DNA complex. Hydrolysis of bound ATP to ADP led to a contraction of DnaA within the complex. The relaxed conformation of DnaA is likely required for the formation of the multi-protein ATP·DnaA·DNA complex. In the initiation of sporulation, Soj binding to DnaA prevented relaxation of its conformation. Soj·ADP appeared to block the activation of DnaA, suggesting a mechanism for Soj·ADP in switching initiation of DNA replication to sporulation. Our studies demonstrate that multiple conformational states of DnaA protein regulate its binding to DNA in the initiation of DNA replication. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Visceral organ mass and hepatic protein synthetic capacity in fed and fasted rats

    Burrin, D.G.; Britton, R.A.; Ferrell, C.L.

    1986-01-01

    Forty-two male rats (avg wt. = 320 g) were used to assess the effect of severe nutrient restriction (72 h fast) on visceral organ mass and hepatic protein synthetic capacity as measured by in vitro incorporation of U- 14 -C-VALINE ( 14 C-VAL) into isolated hepatocytes. Organ weights expressed as a percent of empty body weight for fed vs. fasted rats were; liver (5.21 +/- .54 vs 3.82 +/- .46), kidney (.87 +/- 0.6 vs .89 +/- .05), stomach (.60 +/- .06 vs .61 +/- .06), intestines (3.70 +/- .44 vs 3.41 +/- .37). No differences were observed in in vitro oxygen consumption (15.7 +/- 3.1 vs 16.1 +/- 3.3, umole min -1 g -1 dry tissue) or 14 -C VAL incorporation (4.93 +/- 1.28 vs 4.31 +/- 1.48, dpm min -1 mg -1 dry tissue) for hepatocytes from fed vs. fasted rats. Analysis of perfused liver tissue indicated fed rats had higher protein (152.1 +/- 16.3 vs 136.6 +/- 29.6, mg/g tissue) and RNA (8.81 +/- 1.66 vs 5.97 +/- 1.87, mg/g tissue) with lower DNA (2.19 +/- .31 vs 3.19 +/- .54, mg/g tissue) compared to fasted rats. Protein-nucleic acid ratios suggest liver tissue from fed rats had a greater capacity for protein synthesis compared to fasted rats, however, this was not evident from in vitro hepatocyte 14 -C VAL incorporation estimates. These data indicate that severe nutrient restriction (72 h fast) affects visceral organ mass largely by reduced liver and gut size as well as decreased hepatic protein synthetic capacity

  3. pH-induced conformational changes of AcrA, the membrane fusion protein of Escherichia coli multidrug efflux system.

    Ip, Hermia; Stratton, Kelly; Zgurskaya, Helen; Liu, Jun

    2003-12-12

    The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.

  4. Loss of conformational entropy in protein folding calculated using realistic ensembles and its implications for NMR-based calculations

    Baxa, Michael C.; Haddadian, Esmael J.; Jumper, John M.; Freed, Karl F.; Sosnick, Tobin R.

    2014-01-01

    The loss of conformational entropy is a major contribution in the thermodynamics of protein folding. However, accurate determination of the quantity has proven challenging. We calculate this loss using molecular dynamic simulations of both the native protein and a realistic denatured state ensemble. For ubiquitin, the total change in entropy is TΔSTotal = 1.4 kcal⋅mol−1 per residue at 300 K with only 20% from the loss of side-chain entropy. Our analysis exhibits mixed agreement with prior studies because of the use of more accurate ensembles and contributions from correlated motions. Buried side chains lose only a factor of 1.4 in the number of conformations available per rotamer upon folding (ΩU/ΩN). The entropy loss for helical and sheet residues differs due to the smaller motions of helical residues (TΔShelix−sheet = 0.5 kcal⋅mol−1), a property not fully reflected in the amide N-H and carbonyl C=O bond NMR order parameters. The results have implications for the thermodynamics of folding and binding, including estimates of solvent ordering and microscopic entropies obtained from NMR. PMID:25313044

  5. Conformational effects of a common codon 751 polymorphism on the C-terminal domain of the xeroderma pigmentosum D protein

    Monaco Regina

    2009-01-01

    Full Text Available Aim: The xeroderma pigmentosum D (XPD protein is a DNA helicase involved in the repair of DNA damage, including nucleotide excision repair (NER and transcription-coupled repair (TCR. The C-terminal domain of XPD has been implicated in interactions with other components of the TFIIH complex, and it is also the site of a common genetic polymorphism in XPD at amino acid residue 751 (Lys->Gln. Some evidence suggests that this polymorphism may alter DNA repair capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XPD induced by the polymorphism. Materials and Methods: Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the C-terminal domain of XPD and differences in structure produced by the polymorphic substitution were determined. Results: The results indicate that, although the general configuration of both proteins is similar, the substitution produces a significant conformational change immediately N-terminal to the site of the polymorphism. Conclusion: These results provide support for the hypothesis that this polymorphism in XPD could affect DNA repair capability, and hence cancer risk, by altering the structure of the C-terminal domain.

  6. Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein

    Langowski, J.; Benight, A.S.; Fujimoto, B.S.; Schurr, J.M.; Schomburg, U.

    1985-01-01

    The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D 0 ) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (D/sub app/) obtained from dynamic light scattering display the well-known increase with K 2 (K = scattering vector), leveling off toward a plateau value (D/sub plat/) at high K 2 . For both DNAs, the difference D/sub plat/ - D 0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed

  7. Fast conformational exchange between the sulfur-free and persulfide-bound rhodanese domain of E. coli YgaP

    Wang, Wei [Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026 (China); Zhou, Peng [High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); He, Yao; Yu, Lu; Xiong, Ying [Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026 (China); Tian, Changlin, E-mail: cltian@ustc.edu.cn [Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026 (China); High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui 230031 (China); Wu, Fangming, E-mail: fmwu@hmfl.ac.cn [High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, Anhui 230031 (China)

    2014-09-26

    Highlights: • Three dimensional solution NMR structure of YgaP rhodanese domain. • Function validation of YgaP rhodanese domain to substrate Na{sub 2}S{sub 2}O{sub 3}. • Fast exchange between the intact and persulfide-intermediate rhodanese domain. - Abstract: Rhodanese domains are abundant structural modules that catalyze the transfer of a sulfur atom from thiolsulfates to cyanide via formation of a covalent persulfide intermediate that is bound to an essential conserved cysteine residue. In this study, the three-dimensional structure of the rhodanese domain of YgaP from Escherichia coli was determined using solution NMR. A typical rhodanese domain fold was observed, as expected from the high homology with the catalytic domain of other sulfur transferases. The initial sulfur-transfer step and formation of the rhodanese persulfide intermediate were monitored by addition of sodium thiosulfate using two-dimensional {sup 1}H–{sup 15}N correlation spectroscopy. Discrete sharp signals were observed upon substrate addition, indicting fast exchange between sulfur-free and persulfide-intermediate forms. Residues exhibiting pronounced chemical shift changes were mapped to the structure, and included both substrate binding and surrounding residues.

  8. Conformational Sampling in Template-Free Protein Loop Structure Modeling: An Overview

    Li, Yaohang

    2013-01-01

    Accurately modeling protein loops is an important step to predict three-dimensional structures as well as to understand functions of many proteins. Because of their high flexibility, modeling the three-dimensional structures of loops is difficult and is usually treated as a “mini protein folding problem” under geometric constraints. In the past decade, there has been remarkable progress in template-free loop structure modeling due to advances of computational methods as well as stably increas...

  9. Dynamic conformational change regulates the protein-DNA recognition: an investigation on binding of a Y-family polymerase to its target DNA.

    Xiakun Chu

    2014-09-01

    Full Text Available Protein-DNA recognition is a central biological process that governs the life of cells. A protein will often undergo a conformational transition to form the functional complex with its target DNA. The protein conformational dynamics are expected to contribute to the stability and specificity of DNA recognition and therefore may control the functional activity of the protein-DNA complex. Understanding how the conformational dynamics influences the protein-DNA recognition is still challenging. Here, we developed a two-basin structure-based model to explore functional dynamics in Sulfolobus solfataricus DNA Y-family polymerase IV (DPO4 during its binding to DNA. With explicit consideration of non-specific and specific interactions between DPO4 and DNA, we found that DPO4-DNA recognition is comprised of first 3D diffusion, then a short-range adjustment sliding on DNA and finally specific binding. Interestingly, we found that DPO4 is under a conformational equilibrium between multiple states during the binding process and the distributions of the conformations vary at different binding stages. By modulating the strength of the electrostatic interactions, the flexibility of the linker, and the conformational dynamics in DPO4, we drew a clear picture on how DPO4 dynamically regulates the DNA recognition. We argue that the unique features of flexibility and conformational dynamics in DPO4-DNA recognition have direct implications for low-fidelity translesion DNA synthesis, most of which is found to be accomplished by the Y-family DNA polymerases. Our results help complete the description of the DNA synthesis process for the Y-family polymerases. Furthermore, the methods developed here can be widely applied for future investigations on how various proteins recognize and bind specific DNA substrates.

  10. Effects of N-glycosylation on protein conformation and dynamics: Protein Data Bank analysis and molecular dynamics simulation study.

    Lee, Hui Sun; Qi, Yifei; Im, Wonpil

    2015-03-09

    N-linked glycosylation is one of the most important, chemically complex, and ubiquitous post-translational modifications in all eukaryotes. The N-glycans that are covalently linked to proteins are involved in numerous biological processes. There is considerable interest in developments of general approaches to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in protein design with advantageous properties. In this study, the impacts of N-glycans on protein structure and dynamics are systematically investigated using an integrated computational approach of the Protein Data Bank structure analysis and atomistic molecular dynamics simulations of glycosylated and deglycosylated proteins. Our study reveals that N-glycosylation does not induce significant changes in protein structure, but decreases protein dynamics, likely leading to an increase in protein stability. Overall, these results suggest not only a common role of glycosylation in proteins, but also a need for certain proteins to be properly glycosylated to gain their intrinsic dynamic properties.

  11. Selecting for Fast Protein-Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP.

    Cohen-Khait, Ruth; Schreiber, Gideon

    2018-04-27

    Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-β-lactamase library against its natural nanomolar affinity binder β-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.

  12. Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis

    Schlegel Brigitte

    2004-03-01

    Full Text Available Abstract Background High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination. Results 88 different E. coli expression constructs for 17 human protein domains were analysed using high-throughput cloning, purification and folding analysis to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, protein domains were directly analysed using 1D 1H-NMR spectroscopy. In addition, analytical hydrophobic interaction chromatography (HIC was used to detect natively folded protein. With these two analytical methods, six constructs (representing two domains were quickly identified as being well folded and suitable for structural analysis. Conclusion The described approach facilitates high-throughput structural analysis. Clones expressing natively folded proteins suitable for NMR structure determination were quickly identified upon small scale expression screening using 1D 1H-NMR and/or analytical HIC. This procedure is especially effective as a fast and inexpensive screen for the 'low hanging fruits' in structural genomics.

  13. A Unified Conformational Selection and Induced Fit Approach to Protein-Peptide Docking

    Trellet, M.; Melquiond, A.S.J.; Bonvin, A.M.J.J.

    2013-01-01

    Protein-peptide interactions are vital for the cell. They mediate, inhibit or serve as structural components in nearly 40% of all macromolecular interactions, and are often associated with diseases, making them interesting leads for protein drug design. In recent years, large-scale technologies have

  14. Conformational and functional analysis of the C-terminal globular head of the reovirus cell attachment protein.

    Duncan, R; Horne, D; Strong, J E; Leone, G; Pon, R T; Yeung, M C; Lee, P W

    1991-06-01

    We have been investigating structure-function relationships in the reovirus cell attachment protein sigma 1 using various deletion mutants and protease analysis. In the present study, a series of deletion mutants were constructed which lacked 90, 44, 30, 12, or 4 amino acids from the C-terminus of the 455-amino acid-long reovirus type 3 (T3) sigma 1 protein. The full-length and truncated sigma 1 proteins were expressed in an in vitro transcription/translation system and assayed for L cell binding activity. It was found that the removal of as few as four amino acids from the C-terminus drastically affected the cell binding function of the sigma 1 protein. The C-terminal-truncated proteins were further characterized using trypsin, chymotrypsin, and monoclonal and polyclonal antibodies. Our results indicated that the C-terminal portions of the mutant proteins were misfolded, leading to a loss in cell binding function. The N-terminal fibrous tail of the proteins was unaffected by the deletions as was sigma 1 oligomerization, further illustrating the discrete structural and functional roles of the N- and C-terminal domains of sigma 1. In an attempt to identify smaller, functional peptides, full-length sigma 1 expressed in vitro was digested with trypsin and subsequently with chymotrypsin under various conditions. The results clearly demonstrated the highly stable nature of the C-terminal globular head of sigma 1, even when separated from the N-terminal fibrous tail. We concluded that: (1) the C-terminal globular head of sigma 1 exists as a compact, protease-resistant oligomeric structure; (2) an intact C-terminus is required for proper head folding and generation of the conformationally dependent cell binding domain.

  15. Constrained Maximum Likelihood Estimation of Relative Abundances of Protein Conformation in a Heterogeneous Mixture from Small Angle X-Ray Scattering Intensity Measurements

    Onuk, A. Emre; Akcakaya, Murat; Bardhan, Jaydeep P.; Erdogmus, Deniz; Brooks, Dana H.; Makowski, Lee

    2015-01-01

    In this paper, we describe a model for maximum likelihood estimation (MLE) of the relative abundances of different conformations of a protein in a heterogeneous mixture from small angle X-ray scattering (SAXS) intensities. To consider cases where the solution includes intermediate or unknown conformations, we develop a subset selection method based on k-means clustering and the Cramér-Rao bound on the mixture coefficient estimation error to find a sparse basis set that represents the space spanned by the measured SAXS intensities of the known conformations of a protein. Then, using the selected basis set and the assumptions on the model for the intensity measurements, we show that the MLE model can be expressed as a constrained convex optimization problem. Employing the adenylate kinase (ADK) protein and its known conformations as an example, and using Monte Carlo simulations, we demonstrate the performance of the proposed estimation scheme. Here, although we use 45 crystallographically determined experimental structures and we could generate many more using, for instance, molecular dynamics calculations, the clustering technique indicates that the data cannot support the determination of relative abundances for more than 5 conformations. The estimation of this maximum number of conformations is intrinsic to the methodology we have used here. PMID:26924916

  16. Conformational study of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) by tryptophan fluorescence and differential scanning calorimetry.

    Yin, Shou-Wei; Tang, Chuan-He; Yang, Xiao-Quan; Wen, Qi-Biao

    2011-01-12

    Fluorescence and differential scanning calorimetry (DSC) were used to study changes in the conformation of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) under various environmental conditions. The possible relationship between fluorescence data and DSC characteristics was also discussed. Tryptophan fluorescence and fluorescence quenching analyses indicated that the tryptophan residues in KPI, exhibiting multiple fluorophores with different accessibilities to acrylamide, are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains close to at least some of the tryptophan residues. GdnHCl was more effective than urea and SDS in denaturing KPI. SDS and urea caused variable red shifts, 2-5 nm, in the emission λ(max), suggesting the conformational compactness of KPI. The result was further supported by DSC characteristics that a discernible endothermic peak was still detected up to 8 M urea or 30 mM SDS, also evidenced by the absence of any shift in emission maximum (λ(max)) at different pH conditions. Marked decreases in T(d) and enthalpy (ΔH) were observed at extreme alkaline and/or acidic pH, whereas the presence of NaCl resulted in higher T(d) and ΔH, along with greater cooperativity of the transition. Decreases in T(d) and ΔH were observed in the presence of protein perturbants, for example, SDS and urea, indicating partial denaturation and decrease in thermal stability. Dithiothreitol and N-ethylmaleimide have a slight effect on the thermal properties of KPI. Interestingly, a close linear relationship between the T(d) (or ΔH) and the λ(max) was observed for KPI in the presence of 0-6 M urea.

  17. Protein conformational transitions at the liquid-gas interface as studied by dilational surface rheology.

    Noskov, Boris A

    2014-04-01

    Experimental results on the dynamic dilational surface elasticity of protein solutions are analyzed and compared. Short reviews of the protein behavior at the liquid-gas interface and the dilational surface rheology precede the main sections of this work. The kinetic dependencies of the surface elasticity differ strongly for the solutions of globular and non-globular proteins. In the latter case these dependencies are similar to those for solutions of non-ionic amphiphilic polymers and have local maxima corresponding to the formation of the distal region of the surface layer (type I). In the former case the dynamic surface elasticity is much higher (>60 mN/m) and the kinetic dependencies are monotonical and similar to the data for aqueous dispersions of solid nanoparticles (type II). The addition of strong denaturants to solutions of bovine serum albumin and β-lactoglobulin results in an abrupt transition from the type II to type I dependencies if the denaturant concentration exceeds a certain critical value. These results give a strong argument in favor of the preservation of the protein globular structure in the course of adsorption without any denaturants. The addition of cationic surfactants also can lead to the non-monotonical kinetic dependencies of the dynamic surface elasticity indicating destruction of the protein tertiary and secondary structures. The addition of anionic surfactants gives similar results only for the protein solutions of high ionic strength. The influence of cationic surfactants on the local maxima of the kinetic dependencies of the dynamic surface elasticity for solutions of a non-globular protein (β-casein) differs from the influence of anionic surfactants due to the heterogeneity of the charge distribution along the protein chain. In this case one can use small admixtures of ionic surfactants as probes of the adsorption mechanism. The effect of polyelectrolytes on the kinetic dependencies of the dynamic surface elasticity of protein

  18. Determination of conformation and orientation of immobilized peptides and proteins at buried interfaces

    Shen, Lei; Ulrich, Nathan W.; Mello, Charlene M.; Chen, Zhan

    2015-01-01

    Surface immobilized peptides/proteins have important applications such as antimicrobial coating and biosensing. We report a study of such peptides/proteins using sum frequency generation vibrational spectroscopy and ATR-FTIR. Immobilization on surfaces via physical adsorption and chemical coupling revealed that structures of chemically immobilized peptides are determined by immobilization sites, chemical environments, and substrate surfaces. In addition, controlling enzyme orientation by engineering the surface immobilization site demonstrated that structures can be well-correlated to measured chemical activity. This research facilitates the development of immobilized peptides/proteins with improved activities by optimizing their surface orientation and structure.

  19. Rates and equilibrium constants of the ligand-induced conformational transition of an HCN ion channel protein domain determined by DEER spectroscopy.

    Collauto, Alberto; DeBerg, Hannah A; Kaufmann, Royi; Zagotta, William N; Stoll, Stefan; Goldfarb, Daniella

    2017-06-14

    Ligand binding can induce significant conformational changes in proteins. The mechanism of this process couples equilibria associated with the ligand binding event and the conformational change. Here we show that by combining the application of W-band double electron-electron resonance (DEER) spectroscopy with microfluidic rapid freeze quench (μRFQ) it is possible to resolve these processes and obtain both equilibrium constants and reaction rates. We studied the conformational transition of the nitroxide labeled, isolated carboxy-terminal cyclic-nucleotide binding domain (CNBD) of the HCN2 ion channel upon binding of the ligand 3',5'-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the μRFQ DEER experiments directly provide fractional populations of the open and closed conformations as a function of time. We modeled the ligand-induced conformational change in the protein using a four-state model: apo/open (AO), apo/closed (AC), bound/open (BO), bound/closed (BC). These species interconvert according to AC + L ⇌ AO + L ⇌ BO ⇌ BC. By analyzing the concentration dependence of the relative contributions of the closed and open conformations at equilibrium, we estimated the equilibrium constants for the two conformational equilibria and the open-state ligand dissociation constant. Analysis of the time-resolved μRFQ DEER data gave estimates for the intrinsic rates of ligand binding and unbinding as well as the rates of the conformational change. This demonstrates that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational change.

  20. Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins.

    Sherman, Eilon; Itkin, Anna; Kuttner, Yosef Yehuda; Rhoades, Elizabeth; Amir, Dan; Haas, Elisha; Haran, Gilad

    2008-06-01

    Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.

  1. The Staphylococcus aureus extracellular adherence protein (Eap) adopts an elongated but structured conformation in solution

    Hammel, Michal; Němeček, Daniel; Keightley, J. Andrew; Thomas, George J.; Geisbrecht, Brian V.

    2007-01-01

    The extracellular adherence protein (Eap) of Staphylococcus aureus participates in a wide range of protein–protein interactions that facilitate the initiation and dissemination of Staphylococcal disease. In this report, we describe the use of a multidisciplinary approach to characterize the solution structure of full-length Eap. In contrast to previous reports suggesting that a six-domain isoform of Eap undergoes multimerization, sedimentation equilibrium analytical ultracentrifugation data r...

  2. Open and closed conformations of two SpoIIAA-like proteins (YP-749275.1 and YP-001095227.1) provide insights into membrane association and ligand binding

    Kumar, Abhinav; Lomize, Andrei; Jin, Kevin K.; Carlton, Dennis; Miller, Mitchell D.; Jaroszewski, Lukasz; Abdubek, Polat; Astakhova, Tamara; Axelrod, Herbert L.; Chiu, Hsiu-Ju; Clayton, Thomas; Das, Debanu; Deller, Marc C.; Duan, Lian; Feuerhelm, Julie; Grant, Joanna C.; Grzechnik, Anna; Han, Gye Won; Klock, Heath E.; Knuth, Mark W.; Kozbial, Piotr; Krishna, S. Sri; Marciano, David; McMullan, Daniel; Morse, Andrew T.; Nigoghossian, Edward; Okach, Linda; Reyes, Ron; Rife, Christopher L.; Sefcovic, Natasha; Tien, Henry J.; Trame, Christine B.; Bedem, Henry van den; Weekes, Dana; Xu, Qingping; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2009-01-01

    The crystal structures of two orthologous proteins from different Shewanella species have uncovered a resemblance to CRAL-TRIO carrier proteins, which suggest that they function as transporters of small nonpolar molecules. One protein adopts an open conformation, while the other adopts a closed structure that may act as a conformational switch in the transport of ligands at the membrane surface. The crystal structures of the proteins encoded by the YP-749275.1 and YP-001095227.1 genes from Shewanella frigidimarina and S. loihica, respectively, have been determined at 1.8 and 2.25 Å resolution, respectively. These proteins are members of a novel family of bacterial proteins that adopt the α/β SpoIIAA-like fold found in STAS and CRAL-TRIO domains. Despite sharing 54% sequence identity, these two proteins adopt distinct conformations arising from different dispositions of their α2 and α3 helices. In the ‘open’ conformation (YP-001095227.1), these helices are 15 Å apart, leading to the creation of a deep nonpolar cavity. In the ‘closed’ structure (YP-749275.1), the helices partially unfold and rearrange, occluding the cavity and decreasing the solvent-exposed hydrophobic surface. These two complementary structures are reminiscent of the conformational switch in CRAL-TRIO carriers of hydrophobic compounds. It is suggested that both proteins may associate with the lipid bilayer in their ‘open’ monomeric state by inserting their amphiphilic helices, α2 and α3, into the lipid bilayer. These bacterial proteins may function as carriers of nonpolar substances or as interfacially activated enzymes

  3. Sequence and conformational preferences at termini of α-helices in membrane proteins: role of the helix environment.

    Shelar, Ashish; Bansal, Manju

    2014-12-01

    α-Helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α-helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C-termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α-helices in a high-resolution dataset of integral α-helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C-termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near-helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. © 2014 Wiley Periodicals, Inc.

  4. Fast protein tertiary structure retrieval based on global surface shape similarity.

    Sael, Lee; Li, Bin; La, David; Fang, Yi; Ramani, Karthik; Rustamov, Raif; Kihara, Daisuke

    2008-09-01

    Characterization and identification of similar tertiary structure of proteins provides rich information for investigating function and evolution. The importance of structure similarity searches is increasing as structure databases continue to expand, partly due to the structural genomics projects. A crucial drawback of conventional protein structure comparison methods, which compare structures by their main-chain orientation or the spatial arrangement of secondary structure, is that a database search is too slow to be done in real-time. Here we introduce a global surface shape representation by three-dimensional (3D) Zernike descriptors, which represent a protein structure compactly as a series expansion of 3D functions. With this simplified representation, the search speed against a few thousand structures takes less than a minute. To investigate the agreement between surface representation defined by 3D Zernike descriptor and conventional main-chain based representation, a benchmark was performed against a protein classification generated by the combinatorial extension algorithm. Despite the different representation, 3D Zernike descriptor retrieved proteins of the same conformation defined by combinatorial extension in 89.6% of the cases within the top five closest structures. The real-time protein structure search by 3D Zernike descriptor will open up new possibility of large-scale global and local protein surface shape comparison. 2008 Wiley-Liss, Inc.

  5. Protein Conformation Ensembles Monitored by HDX Reveal a Structural Rationale for Abscisic Acid Signaling Protein Affinities and Activities

    West, Graham M.; Pascal, Bruce D.; Ng, Ley-Moy; Soon, Fen-Fen; Melcher, Karsten; Xu, H. Eric; Chalmers, Michael J.; Griffin, Patrick R.

    2013-01-01

    Plants regulate growth and respond to environmental stress through abscisic acid (ABA) regulated pathways, and as such these pathways are of primary interest for biological and agricultural research. The ABA response is first perceived by the PYR/PYL/RCAR class of START protein receptors. These ABA activated receptors disrupt phosphatase inhibition of Snf1-related kinases (SnRKs) enabling kinase signaling. Here, insights into the structural mechanism of proteins in the ABA signaling pathway (...

  6. A novel mechanism for antiglycative action of limonene through stabilization of protein conformation.

    Joglekar, Madhav M; Panaskar, Shrimant N; Chougale, Ashok D; Kulkarni, Mahesh J; Arvindekar, Akalpita U

    2013-10-01

    Inhibition of protein glycation is known to ameliorate secondary complications in diabetes. In the present study antiglycative properties of limonene, a natural product, were evaluated using BSA as a model protein. AMG (aminoguanidine) was used as a positive control. Measurement of total AGEs (Advanced Glycation End-products) and specific AGEs revealed that limonene could inhibit protein glycation to the extent of 56.3% and 75.1% respectively at 50 μM concentration as against 54.4% and 82.2% by AMG at 1 mM. Congo red binding and CD (Circular Dichroism) analysis revealed inhibition of α-helix to β-sheet transition wherein 18.5% β-sheet structures were observed in glycated BSA (bovine serum albumin) as against 4.9% with limonene. Glycation of protein in the presence of urea was enhanced by 18%, while in the presence of limonene it was reduced by 23% revealing the stabilizing effect of limonene. Electrophoretic mobility was similar to the normal control and a zeta potential value of -12.1 mV as against -15.1 mV in diabetic control was observed. Inhibition of glycation in limonene treated samples was confirmed through LC-MS analysis wherein AGEs such as pentosidine, CML (N(ε)-(carboxymethyl)lysine), CEL (N(ε)-(carboxyethyl)lysine), MOLD (methylglyoxal-lysine dimer) and imidazolone observed in glycated samples were absent in limonene treated samples. PatchDock studies revealed that limonene could bind to the major glycation sites IB, IIA and IIB sub domains and AMG to the IIIA sub domain. Thus limonene is a potent protein glycation inhibitor that prevents protein glycation through a novel mechanism of stabilization of protein structure through hydrophobic interactions.

  7. Structural ordering of disordered ligand-binding loops of biotin protein ligase into active conformations as a consequence of dehydration.

    Vibha Gupta

    Full Text Available Mycobacterium tuberculosis (Mtb, a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC, an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA. The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of approximately 3.5 A in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In

  8. Conformational interconversions in peptide beta-turns: analysis of turns in proteins and computational estimates of barriers.

    Gunasekaran, K; Gomathi, L; Ramakrishnan, C; Chandrasekhar, J; Balaram, P

    1998-12-18

    The two most important beta-turn features in peptides and proteins are the type I and type II turns, which differ mainly in the orientation of the central peptide unit. Facile conformational interconversion is possible, in principle, by a flip of the central peptide unit. Homologous crystal structures afford an opportunity to structurally characterize both possible conformational states, thus allowing identification of sites that are potentially stereochemically mobile. A representative data set of 250 high-resolution (turns that are assigned different conformational types (type I/type II) in related structures. A total of 55 examples of beta-turns were identified as possible candidates for a stereochemically mobile site. Of the 55 examples, 45 could be classified as a potential site for interconversion between type I and type II beta-turns, while ten correspond to flips from type I' to type II' structures. As a further check, the temperature factors of the central peptide unit carbonyl oxygen atom of the 55 examples were examined. The analysis reveals that the turn assignments are indeed reliable. Examination of the secondary structures at the flanking positions of the flippable beta-turns reveals that seven examples occur in the loop region of beta-hairpins, indicating that the formation of ordered secondary structures on either side of the beta-turn does not preclude local conformational variations. In these beta-turns, Pro (11 examples), Lys (nine examples) and Ser (seven examples) were most often found at the i+1 position. Glycine was found to occur overwhelmingly at position i+2 (28 examples), while Ser (seven examples) and Asn (six examples) were amongst the most frequent residues. Activation energy barriers for the interconversion between type I and type II beta-turns were computed using the peptide models Ac-Pro-Aib-NHMe and Ac-Pro-Gly-NHMe within the framework of the AM1 semi-empirical molecular orbital procedure. In order to have a uniform basis for

  9. Fast axonal transport of labeled proteins in motoneurons of exercise-trained rats

    Jasmin, B.J.; Lavoie, P.A.; Gardiner, P.F.

    1988-01-01

    In this study, the fast orthograde axonal transport of radiolabeled proteins was measured to determine the effects of endurance-running training on transport velocity and amounts of transported proteins in rat sciatic motoneurons. Female rats were subjected to a progressive running-training program for 10-12 wk. Twenty-four hours after the last training session, rats underwent right L4-L5 dorsal root ganglionectomy. The next day, 20 microCi of [3H]leucine was injected bilaterally in the vicinity of the motoneuronal cell bodies supplying the sciatic nerve, to study axonal transport parameters. Results showed that peak and average transport velocities of labeled proteins were significantly (P less than 0.05) increased by 22 and 29%, respectively, in the deafferented nerves of the runners as compared with controls. Moreover, the amount of total transported protein-bound radioactivity was increased in both left (40%) and right (37%) sciatic nerves of the runners. An exhaustive exercise session reduced (P less than 0.05) peak displacement (8%) and total transported protein-bound radioactivity (36%) in the sciatic nerves of control rats, whereas no changes were noticed in trained animals. The data suggest that chronic endurance running induces significant adaptations in the fast axonal transport of labeled proteins

  10. Exploration of Protein Conformational Change with PELE and Meta-Dynamics.

    Cossins, Benjamin P; Hosseini, Ali; Guallar, Victor

    2012-03-13

    Atomistic molecular simulation methods are now able to explore complex protein or protein-ligand dynamical space in a tractable way with methods such as meta-dynamics or adaptive biasing force. However, many of these methods either require a careful selection of reaction coordinates or the knowledge of an initial pathway of some kind. Thus, it is important that effective methods are developed to produce this pathway data in an efficient fashion. PELE, a proven protein-ligand sampling code, has been developed to provide rapid protein sampling in highly flexible cases, using a reduced network model eigen problem approach. The resulting method is able to rapidly sample configuration space with very general driving information. When applied to ubiquitin, PELE was able to reproduce RMSD and average force data found in molecular dynamics simulations. PELE was also applied to explore the opening/closing transition of T4 lysozyme. A meta-dynamics exploration using a low energy pathway validated that the configurations explored by PELE represent the most populated regions of phase space. PELE and meta-dynamics explorations also discovered a low free energy region where a large cross-domain helix of T4 lysozyme is broken in two. There is previous NMR evidence for the validity of this unfolded helix region.

  11. Steric exclusion and protein conformation determine the localization of plasma membrane transporters

    Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

    2018-01-01

    The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to

  12. Conformationally Constrained Peptidomimetics as Inhibitors of the Protein Arginine Methyl Transferases

    Knuhtsen, Astrid; Legrand, Baptiste; Van der Poorten, Olivier

    2016-01-01

    Protein arginine N-methyl transferases (PRMTs) belong to a family of enzymes that modulate the epigenetic code through modifications of histones. In the present study, peptides emerging from a phage display screening were modified in the search for PRMT inhibitors through substitution with non-pr...

  13. Exploring the local conformational space of a membrane protein by site-directed spin labeling

    Stopar, D.; Strancar, J.; Spruijt, R.B.; Hemminga, M.A.

    2005-01-01

    Molecular modeling based on a hybrid evolutionary optimization and an information condensation algorithm, called GHOST, of spin label ESR spectra was applied to study the structure and dynamics of membrane proteins. The new method is capable of providing detailed molecular information about the

  14. Fragment growing induces conformational changes in acetylcholine-binding protein: A structural and thermodynamic analysis

    Edink, E.S.; Rucktooa, P.; Retra, K.; Akdemir, A.; Nahar, T.T.; Zuiderveld, O.P.; van Elk, R.; Janssen, E.; van Nierop, P.; van Muijlwijk-Koezen, J.E.; Smit, A.B.; Sixma, T.K.; Leurs, R.; de Esch, I.J.P.

    2011-01-01

    Optimization of fragment hits toward high-affinity lead compounds is a crucial aspect of fragment-based drug discovery (FBDD). In the current study, we have successfully optimized a fragment by growing into a ligand-inducible subpocket of the binding site of acetylcholine-binding protein (AChBP).

  15. Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline

    Roy, Santanu; Lessing, Joshua; Meisl, Georg; Ganim, Ziad; Tokmakoff, Andrei; Knoester, Jasper; Jansen, Thomas L. C.

    2011-01-01

    We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are

  16. Fish protein intake induces fast-muscle hypertrophy and reduces liver lipids and serum glucose levels in rats.

    Kawabata, Fuminori; Mizushige, Takafumi; Uozumi, Keisuke; Hayamizu, Kohsuke; Han, Li; Tsuji, Tomoko; Kishida, Taro

    2015-01-01

    In our previous study, fish protein was proven to reduce serum lipids and body fat accumulation by skeletal muscle hypertrophy and enhancing basal energy expenditure in rats. In the present study, we examined the precise effects of fish protein intake on different skeletal muscle fiber types and metabolic gene expression of the muscle. Fish protein increased fast-twitch muscle weight, reduced liver triglycerides and serum glucose levels, compared with the casein diet after 6 or 8 weeks of feeding. Furthermore, fish protein upregulated the gene expressions of a fast-twitch muscle-type marker and a glucose transporter in the muscle. These results suggest that fish protein induces fast-muscle hypertrophy, and the enhancement of basal energy expenditure by muscle hypertrophy and the increase in muscle glucose uptake reduced liver lipids and serum glucose levels. The present results also imply that fish protein intake causes a slow-to-fast shift in muscle fiber type.

  17. A miniaturized technique for assessing protein thermodynamics and function using fast determination of quantitative cysteine reactivity.

    Isom, Daniel G; Marguet, Philippe R; Oas, Terrence G; Hellinga, Homme W

    2011-04-01

    Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity, which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics. Copyright © 2010 Wiley-Liss, Inc.

  18. Reovirus FAST Protein Enhances Vesicular Stomatitis Virus Oncolytic Virotherapy in Primary and Metastatic Tumor Models

    Fabrice Le Boeuf

    2017-09-01

    Full Text Available The reovirus fusion-associated small transmembrane (FAST proteins are the smallest known viral fusogens (∼100–150 amino acids and efficiently induce cell-cell fusion and syncytium formation in multiple cell types. Syncytium formation enhances cell-cell virus transmission and may also induce immunogenic cell death, a form of apoptosis that stimulates immune recognition of tumor cells. These properties suggest that FAST proteins might serve to enhance oncolytic virotherapy. The oncolytic activity of recombinant VSVΔM51 (an interferon-sensitive vesicular stomatitis virus [VSV] mutant encoding the p14 FAST protein (VSV-p14 was compared with a similar construct encoding GFP (VSV-GFP in cell culture and syngeneic BALB/c tumor models. Compared with VSV-GFP, VSV-p14 exhibited increased oncolytic activity against MCF-7 and 4T1 breast cancer spheroids in culture and reduced primary 4T1 breast tumor growth in vivo. VSV-p14 prolonged survival in both primary and metastatic 4T1 breast cancer models, and in a CT26 metastatic colon cancer model. As with VSV-GFP, VSV-p14 preferentially replicated in vivo in tumors and was cleared rapidly from other sites. Furthermore, VSV-p14 increased the numbers of activated splenic CD4, CD8, natural killer (NK, and natural killer T (NKT cells, and increased the number of activated CD4 and CD8 cells in tumors. FAST proteins may therefore provide a multi-pronged approach to improving oncolytic virotherapy via syncytium formation and enhanced immune stimulation.

  19. Diet-induced obesity alters protein synthesis: Tissue-specific effects in fasted vs. fed mice

    Anderson, Stephanie R.; Gilge, Danielle A.; Steiber, Alison L.; Previs, Stephen F.

    2008-01-01

    The influence of obesity on protein dynamics is not clearly understood. We have designed experiments to test the hypothesis that obesity impairs the stimulation of tissue-specific protein synthesis following nutrient ingestion. C57BL/6J mice were randomized into two groups: group 1 (control, n = 16) were fed a low-fat, high-carbohydrate diet and group 2 (experimental, n = 16) were fed a high-fat, low-carbohydrate diet ad libitum for 9 weeks. On the experiment day, all mice were fasted for 6 h...

  20. Fast iodide-SAD phasing for high-throughput membrane protein structure determination.

    Melnikov, Igor; Polovinkin, Vitaly; Kovalev, Kirill; Gushchin, Ivan; Shevtsov, Mikhail; Shevchenko, Vitaly; Mishin, Alexey; Alekseev, Alexey; Rodriguez-Valera, Francisco; Borshchevskiy, Valentin; Cherezov, Vadim; Leonard, Gordon A; Gordeliy, Valentin; Popov, Alexander

    2017-05-01

    We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide-single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins-the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins, including a human G protein-coupled receptor (GPCR), and we show that I-SAD can be applied using data collection strategies based on either standard or serial x-ray crystallography techniques.

  1. Albumin-coated SPIONs: an experimental and theoretical evaluation of protein conformation, binding affinity and competition with serum proteins

    Yu, Siming; Perálvarez-Marín, Alex; Minelli, Caterina; Faraudo, Jordi; Roig, Anna; Laromaine, Anna

    2016-07-01

    The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the outside of the SPIONs and their binding strength to the SPIONs is about 3.5 × 10-4 M, ten times higher than the adsorption of fetal bovine serum (FBS) on the same SPIONs. We elucidate a strong electrostatic interaction between BSA and the SPIONs, although the secondary structure of the protein is not affected. We present data that supports the strong binding of the BSA monolayer on SPIONs and the properties of the BSA layer as a protein-resistant coating. We believe that a complete understanding of the behavior and morphology of BSA-SPIONs and how the protein interacts with SPIONs is crucial for improving NP surface design and expanding the potential applications of SPIONs in nanomedicine.The variety of nanoparticles (NPs) used in biological applications is increasing and the study of their interaction with biological media is becoming more important. Proteins are commonly the first biomolecules that NPs encounter when they interact with biological systems either in vitro or in vivo. Among NPs, super-paramagnetic iron oxide nanoparticles (SPIONs) show great promise for medicine. In this work, we study in detail the formation, composition, and structure of a monolayer of bovine serum albumin (BSA) on SPIONs. We determine, both by molecular simulations and experimentally, that ten molecules of BSA form a monolayer around the

  2. Novel Entropically Driven Conformation-specific Interactions with Tomm34 Protein Modulate Hsp70 Protein Folding and ATPase Activities

    Durech, M.; Trčka, F.; Man, Petr; Blackburn, E.A.; Hernychová, L.; Dvořáková, P.; Coufalová, D.; Kavan, Daniel; Vojtěšek, B.; Muller, P.

    2016-01-01

    Roč. 15, č. 5 (2016), s. 1710-1727 ISSN 1535-9476 R&D Projects: GA MŠk(CZ) LO1509 EU Projects: Wellcome Trust(CZ) 01527/Z/13/Z Institutional support: RVO:61388971 Keywords : HEAT-SHOCK-PROTEIN * MOLECULAR CHAPERONE DNAK * SUBSTRATE-BINDING DOMAIN * INVASIVE BREAST - CANCER Subject RIV: CE - Biochemistry Impact factor: 6.540, year: 2016

  3. Conformational dynamics and ligand binding in the multi-domain protein PDC109.

    Hyun Jin Kim

    2010-02-01

    Full Text Available PDC109 is a modular multi-domain protein with two fibronectin type II (Fn2 repeats joined by a linker. It plays a major role in bull sperm binding to the oviductal epithelium through its interactions with phosphorylcholines (PhCs, a head group of sperm cell membrane lipids. The crystal structure of the PDC109-PhC complex shows that each PhC binds to the corresponding Fn2 domain, while the two domains are on the same face of the protein. Long timescale explicit solvent molecular dynamics (MD simulations of PDC109, in the presence and absence of PhC, suggest that PhC binding strongly correlates with the relative orientation of choline-phospholipid binding sites of the two Fn2 domains; unless the two domains tightly bind PhCs, they tend to change their relative orientation by deforming the flexible linker. The effective PDC109-PhC association constant of 28 M(-1, estimated from their potential of mean force is consistent with the experimental result. Principal component analysis of the long timescale MD simulations was compared to the significantly less expensive normal mode analysis of minimized structures. The comparison indicates that difference between relative domain motions of PDC109 with bound and unbound PhC is captured by the first principal component in the principal component analysis as well as the three lowest normal modes in the normal mode analysis. The present study illustrates the use of detailed MD simulations to clarify the energetics of specific ligand-domain interactions revealed by a static crystallographic model, as well as their influence on relative domain motions in a multi-domain protein.

  4. Fast and Selective Modification of Thiol Proteins/Peptides by N-(Phenylseleno)phthalimide

    Wang, Zhengfang; Zhang, Yun; Zhang, Hao; Harrington, Peter B.; Chen, Hao

    2012-03-01

    We previously reported that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used to selectively derivatize thiols for mass spectrometric analysis, and the introduced selenium tags are useful as they could survive or removed with collision-induced dissociation (CID). Described herein is the further study of the reactivity of various protein/peptide thiols toward NPSP and its application to derivatize thiol peptides in protein digests. With a modified protocol (i.e., dissolving NPSP in acetonitrile instead of aqueous solvent), we found that quantitative conversion of thiols can be obtained in seconds, using NPSP in a slight excess amount (NPSP:thiol of 1.1-2:1). Further investigation shows that the thiol reactivity toward NPSP reflects its chemical environment and accessibility in proteins/peptides. For instance, adjacent basic amino acid residues increase the thiol reactivity, probably because they could stabilize the thiolate form to facilitate the nucleophilic attack of thiol on NPSP. In the case of creatine phosphokinase, the native protein predominately has one thiol reacted with NPSP while all of four thiol groups of the denatured protein can be derivatized, in accordance with the corresponding protein conformation. In addition, thiol peptides in protein/peptide enzymatic digests can be quickly and effectively tagged by NPSP following tri- n-butylphosphine (TBP) reduction. Notably, all three thiols of the peptide QCCASVCSL in the insulin peptic digest can be modified simultaneously by NPSP. These results suggest a novel and selective method for protecting thiols in the bottom-up approach for protein structure analysis.

  5. Identification of a conformational neutralizing epitope on the VP1 protein of type A foot-and-mouth disease virus.

    Liu, Wenming; Yang, Baolin; Wang, Mingxia; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Zhou, Guohui; Yu, Li

    2017-12-01

    Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. In recent years, outbreaks of serotype A FMD have occurred in many countries. High-affinity neutralizing antibodies against a conserved epitope could provide protective immunity against diverse subtypes of FMDV serotype A and protect against future pandemics. In this study, we generated a serotype A FMDV-specific potent neutralizing monoclonal antibody (MAb), 6C9, which recognizes a conformation-dependent epitope. MAb 6C9 potently neutralized FMDV A/XJBC/CHA/2010 with a 50% neutralization titer (NT 50 ) of 4096. Screening of a phage-displayed random 12-mer peptide library revealed that MAb 6C9 bound to phages displaying the consensus motif YxxPxGDLG, which is highly homologous to the 135 YxxPxxxxxGDLG 147 motif found in the serotype A FMDV virus-encoded structural protein VP1. To further verify the authentic epitope recognized by MAb 6C9, two FMDV A/XJBC/CHA/2010 mutant viruses, P138A and G144A, were generated using a reverse genetic system. Subsequent micro-neutralization assays and double-antibody sandwich (DAS) ELISA analyses revealed that the Pro 138 and Gly 144 residues of the conformational epitope that are recognized by 6C9 are important for MAb 6C9 binding. Importantly, the epitope 135 YxxPxxxxxGDLG 147 was highly conserved among different topotypes of serotype A FMDV strains in a sequence alignment analysis. Thus, the results of this study could have potential applications in the development of novel epitope-based vaccines and suitable a MAb-based diagnostic method for the detection of serotype A FMDV and the quantitation of antibodies against this serotype. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Differential regulation of lipid and protein metabolism in obese vs. lean subjects before and after a 72-h fast

    Bak, Ann Mosegaard; Møller, Andreas Buch; Vendelbo, Mikkel Holm

    2016-01-01

    release in obese subjects under basal and fasting conditions. We therefore studied nine lean and nine obese subjects twice, after 12 and 72 h of fasting, using measurements of mRNA and protein expression and phosphorylation of lipolytic and protein metabolic signaling molecules in fat and muscle together...... with whole body and forearm tracer techniques. Obese subjects displayed increased whole body lipolysis, decreased urea production rates, and decreased forearm muscle protein breakdown per 100 ml of forearm tissue, differences that persisted after 72 h of fasting. Lipolysis per fat mass unit was reduced...... in obese subjects and, correspondingly, adipose tissue hormone-sensitive lipase (HSL) phosphorylation and mRNA and protein levels of the adipose triglyceride lipase (ATGL) coactivator CGI58 were decreased. Fasting resulted in higher HSL phosphorylations and lower protein levels of the ATGL inhibitor G0S2...

  7. Interconverting conformations of variants of the human amyloidogenic protein beta2-microglobulin quantitatively characterized by dynamic capillary electrophoresis and computer simulation

    Heegaard, Niels H H; Jørgensen, Thomas J D; Cheng, Lei

    2006-01-01

    Capillary electrophoretic separation profiles of cleaved variants of beta2-microglobulin (beta2m) reflect the conformational equilibria existing in solutions of these proteins. The characterization of these equilibria is of interest since beta2m is responsible for amyloid formation in dialysis-re...

  8. The role of proteins and metal ions in the protection of chromatin DNA at fast neutrons action

    Radu, L.; Preoteasa, V.; Radulescu, I.; Constantinescu, B.

    1997-01-01

    The role of chromatin proteins and of some ions on the fast neutrons actions on chromatin DNA from rat Walker tumors was analysed. The DNA in chromatin is effectively protected against fast neutrons actions by DNA bound proteins and specially by histones, because of the limited accessibility of the condensed chromatin DNA to hydroxyl radicals and of the scavenging of radicals by the chromatin proteins. The ions utilised protect chromatin DNA against the damage produced ed by fast neutrons, through the induction of structural DNA changes with a less accessibility to OH radicals. (authors)

  9. Tetrahymena telomerase protein p65 induces conformational changes throughout telomerase RNA (TER) and rescues telomerase reverse transcriptase and TER assembly mutants.

    Berman, Andrea J; Gooding, Anne R; Cech, Thomas R

    2010-10-01

    The biogenesis of the Tetrahymena telomerase ribonucleoprotein particle (RNP) is enhanced by p65, a La family protein. Single-molecule and biochemical studies have uncovered a hierarchical assembly of the RNP, wherein the binding of p65 to stems I and IV of telomerase RNA (TER) causes a conformational change that facilitates the subsequent binding of telomerase reverse transcriptase (TERT) to TER. We used purified p65 and variants of TERT and TER to investigate the conformational rearrangements that occur during RNP assembly. Nuclease protection assays and mutational analysis revealed that p65 interacts with and stimulates conformational changes in regions of TER beyond stem IV. Several TER mutants exhibited telomerase activity only in the presence of p65, revealing the importance of p65 in promoting the correct RNP assembly pathway. In addition, p65 rescued TERT assembly mutants but not TERT activity mutants. Taken together, these results suggest that p65 stimulates telomerase assembly and activity in two ways. First, by sequestering stems I and IV, p65 limits the ensemble of structural conformations of TER, thereby presenting TERT with the active conformation of TER. Second, p65 acts as a molecular buttress within the assembled RNP, mutually stabilizing TER and TERT in catalytically active conformations.

  10. Calcium Sensing by Recoverin: Effect of Protein Conformation on Ion Affinity.

    Timr, Štěpán; Kadlec, Jan; Srb, Pavel; Ollila, O H Samuli; Jungwirth, Pavel

    2018-04-05

    The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.

  11. Fast and accurate protein substructure searching with simulated annealing and GPUs

    Stivala Alex D

    2010-09-01

    Full Text Available Abstract Background Searching a database of protein structures for matches to a query structure, or occurrences of a structural motif, is an important task in structural biology and bioinformatics. While there are many existing methods for structural similarity searching, faster and more accurate approaches are still required, and few current methods are capable of substructure (motif searching. Results We developed an improved heuristic for tableau-based protein structure and substructure searching using simulated annealing, that is as fast or faster and comparable in accuracy, with some widely used existing methods. Furthermore, we created a parallel implementation on a modern graphics processing unit (GPU. Conclusions The GPU implementation achieves up to 34 times speedup over the CPU implementation of tableau-based structure search with simulated annealing, making it one of the fastest available methods. To the best of our knowledge, this is the first application of a GPU to the protein structural search problem.

  12. Development of Conformation Independent Computational Models for the Early Recognition of Breast Cancer Resistance Protein Substrates

    Melisa Edith Gantner

    2013-01-01

    Full Text Available ABC efflux transporters are polyspecific members of the ABC superfamily that, acting as drug and metabolite carriers, provide a biochemical barrier against drug penetration and contribute to detoxification. Their overexpression is linked to multidrug resistance issues in a diversity of diseases. Breast cancer resistance protein (BCRP is the most expressed ABC efflux transporter throughout the intestine and the blood-brain barrier, limiting oral absorption and brain bioavailability of its substrates. Early recognition of BCRP substrates is thus essential to optimize oral drug absorption, design of novel therapeutics for central nervous system conditions, and overcome BCRP-mediated cross-resistance issues. We present the development of an ensemble of ligand-based machine learning algorithms for the early recognition of BCRP substrates, from a database of 262 substrates and nonsubstrates compiled from the literature. Such dataset was rationally partitioned into training and test sets by application of a 2-step clustering procedure. The models were developed through application of linear discriminant analysis to random subsamples of Dragon molecular descriptors. Simple data fusion and statistical comparison of partial areas under the curve of ROC curves were applied to obtain the best 2-model combination, which presented 82% and 74.5% of overall accuracy in the training and test set, respectively.

  13. Effects of toluene on protein synthesis and the interaction with ethanol in hepatocytes isolated from fed and fasted rats

    Smith-Kielland, A.; Ripel, Aa.; Gadeholt, G.

    1989-01-01

    The effects of three different concentrations (about 20, 100 and 1000 μM) of toluene on protein synthesis were studied in hepatocytes isolated from fed and fasted rats after 60 and 120 min. of incubation. The interaction between ethanol (60 mM) and the low and high toluene concentrations were also tested. To measure protein synthesis, 14 C-valine was used as the precursor amino acid. Total valine concentration was 2 mM to ensure near-constant specific radioactivity of precursor. Toluene concentrations were measured by head-space gas chromatography. Protein synthesis was unchanged in the presence of low toluene concentrations. Intermediate toluene concentration decreased protein synthesis by about 20% and high toluene concentration decreased protein synthesis by about 60%. Protein synthesis was similar in cells from fed and fasted rats. Ethanol alone inhibited protein synthesis by 20-30%, more in fasted than in fed rats. Toluene and ethanol in combination inhibited protein synthesis additively. The high toluene concentration with or without ethanol appeared to inhibit synthesis/secretion of export proteins in hepatocytes from fasted rats. In conclusion, our study indicates that toluene in relatively high concentrations inhibits general protein synthesis in isolated rat hepatocytes. Toluene and ethanol seems to inhibit protein synthesis additively. (author)

  14. Effects of toluene on protein synthesis and the interaction with ethanol in hepatocytes isolated from fed and fasted rats

    Smith-Kielland, A.; Ripel, Aa.; Gadeholt, G.

    1989-01-01

    The effects of three different concentrations (about 20, 100 and 1000 ..mu..M) of toluene on protein synthesis were studied in hepatocytes isolated from fed and fasted rats after 60 and 120 min. of incubation. The interaction between ethanol (60 mM) and the low and high toluene concentrations were also tested. To measure protein synthesis, /sup 14/C-valine was used as the precursor amino acid. Total valine concentration was 2 mM to ensure near-constant specific radioactivity of precursor. Toluene concentrations were measured by head-space gas chromatography. Protein synthesis was unchanged in the presence of low toluene concentrations. Intermediate toluene concentration decreased protein synthesis by about 20% and high toluene concentration decreased protein synthesis by about 60%. Protein synthesis was similar in cells from fed and fasted rats. Ethanol alone inhibited protein synthesis by 20-30%, more in fasted than in fed rats. Toluene and ethanol in combination inhibited protein synthesis additively. The high toluene concentration with or without ethanol appeared to inhibit synthesis/secretion of export proteins in hepatocytes from fasted rats. In conclusion, our study indicates that toluene in relatively high concentrations inhibits general protein synthesis in isolated rat hepatocytes. Toluene and ethanol seems to inhibit protein synthesis additively.

  15. 13CHD2–CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins

    Rennella, Enrico; Huang, Rui; Velyvis, Algirdas; Kay, Lewis E.

    2015-01-01

    An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of 13 CHD 2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is validated with an application to a G48A Fyn SH3 domain that exchanges between a folded conformation and a sparsely populated and transiently formed unfolded ensemble. Experiments on a number of different protein systems, including a 360 kDa half-proteasome, establish that the sensitivity of this 13 CHD 2 13 C–CEST technique can be upwards of a factor of 5 times higher than for a previously published 13 CH 3 13 C–CEST approach (Bouvignies and Kay in J Biomol NMR 53:303–310, 2012), suggesting that the methodology will be powerful for studies of conformational exchange in high molecular weight proteins

  16. "Invisible" conformers of an antifungal disulfide protein revealed by constrained cold and heat unfolding, CEST-NMR experiments, and molecular dynamics calculations.

    Fizil, Ádám; Gáspári, Zoltán; Barna, Terézia; Marx, Florentine; Batta, Gyula

    2015-03-23

    Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20-40 % at 298 K in a disulfide-rich protein. In addition, sensitive (15) N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR "dark matter". Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

  17. “Invisible” Conformers of an Antifungal Disulfide Protein Revealed by Constrained Cold and Heat Unfolding, CEST-NMR Experiments, and Molecular Dynamics Calculations

    Fizil, Ádám; Gáspári, Zoltán; Barna, Terézia; Marx, Florentine; Batta, Gyula

    2015-01-01

    Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20–40 % at 298 K in a disulfide-rich protein. In addition, sensitive 15N-CEST NMR experiments identified a low populated (0.15 %) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR “dark matter”. Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction. PMID:25676351

  18. Comparison of Palivizumab-Like Antibody Binding to Different Conformations of the RSV F Protein in RSV-Infected Adult Hematopoietic Cell Transplant Recipients.

    Ye, Xunyan; Iwuchukwu, Obinna P; Avadhanula, Vasanthi; Aideyan, Letisha O; McBride, Trevor J; Ferlic-Stark, Laura L; Patel, Kirtida D; Piedra, Felipe-Andres; Shah, Dimpy P; Chemaly, Roy F; Piedra, Pedro A

    2018-03-28

    Most respiratory syncytial virus (RSV) vaccine candidates include fusion (F) protein in different conformations. Antigenic site II found in the different F conformations is the target of palivizumab, the only US Food and Drug Administration approved monoclonal antibody (mAb). Serum palivizumab-like antibody (PLA) is a potential serologic correlate of immunity. Our objective was to determine if different conformations of F protein in a palivizumab competitive antibody (PCA) assay affect the PLA concentrations. Four PCA assays were standardized using mAbs. Each contained prefusion, postfusion, or intermediate F forms. PLA concentrations were measured in acute and convalescent sera from 22 RSV/A and 18 RSV/B-infected adult hematopoietic cell transplant (HCT) recipients. PLA concentrations were calculated using a 4-parameter logistic regression model and analyzed for statistical significance. PCA assays revealed significantly greater PLA concentrations in convalescent sera; comparable increases in PLA concentration in RSV/A and RSV/B-infected HCT recipients; and significantly reduced PLA concentrations in HCT recipients who shed RSV ≥14 days. A significant positive correlation was observed between PCA assays and RSV neutralizing antibody titers. F protein conformation does not appear to have a measurable impact on PCA assays for measuring PLA induced by RSV/A or RSV/B infection.

  19. Artificial receptor-functionalized nanoshell: facile preparation, fast separation and specific protein recognition

    Ouyang, Ruizhuo; Lei, Jianping; Ju, Huangxian

    2010-05-01

    This work combined molecular imprinting technology with superparamagnetic nanospheres as the core to prepare artificial receptor-functionalized magnetic nanoparticles for separation of homologous proteins. Using dopamine as a functional monomer, novel surface protein-imprinted superparamagnetic polydopamine (PDA) core-shell nanoparticles were successfully prepared in physiological conditions, which could maintain the natural structure of a protein template and achieved the development of molecularly imprinted polymers (MIPs) from one dimension to zero dimension for efficient recognition towards large biomolecules. The resultant nanoparticles could be used for convenient magnetic separation of homologous proteins with high specificity. The nanoparticles possessed good monodispersibility, uniform surface morphology and high saturation magnetization value. The bound amounts of template proteins measured by both indirect and direct methods were in good agreement. The maximum number of imprinted cavities on the surface of the bovine hemoglobin (Hb)-imprinted nanoshell was 2.21 × 1018 g - 1, which well matched their maximum binding capacity toward bovine Hb. Both the simple method for preparation of MIPs and the magnetic nanospheres showed good application potential in fast separation, effective concentration and selective biosensing of large protein molecules.

  20. Artificial receptor-functionalized nanoshell: facile preparation, fast separation and specific protein recognition

    Ouyang, Ruizhuo; Lei Jianping; Ju Huangxian, E-mail: jpl@nju.edu.cn, E-mail: hxju@nju.edu.cn [Key Laboratory of Analytical Chemistry for Life Science (Education Ministry of China), Department of Chemistry, Nanjing University, Nanjing 210093 (China)

    2010-05-07

    This work combined molecular imprinting technology with superparamagnetic nanospheres as the core to prepare artificial receptor-functionalized magnetic nanoparticles for separation of homologous proteins. Using dopamine as a functional monomer, novel surface protein-imprinted superparamagnetic polydopamine (PDA) core-shell nanoparticles were successfully prepared in physiological conditions, which could maintain the natural structure of a protein template and achieved the development of molecularly imprinted polymers (MIPs) from one dimension to zero dimension for efficient recognition towards large biomolecules. The resultant nanoparticles could be used for convenient magnetic separation of homologous proteins with high specificity. The nanoparticles possessed good monodispersibility, uniform surface morphology and high saturation magnetization value. The bound amounts of template proteins measured by both indirect and direct methods were in good agreement. The maximum number of imprinted cavities on the surface of the bovine hemoglobin (Hb)-imprinted nanoshell was 2.21 x 10{sup 18} g{sup -1}, which well matched their maximum binding capacity toward bovine Hb. Both the simple method for preparation of MIPs and the magnetic nanospheres showed good application potential in fast separation, effective concentration and selective biosensing of large protein molecules.

  1. Fast and anisotropic flexibility-rigidity index for protein flexibility and fluctuation analysis

    Opron, Kristopher [Department of Biochemistry and Molecular Biology, Michigan State University, Michigan 48824 (United States); Xia, Kelin [Department of Mathematics, Michigan State University, Michigan 48824 (United States); Wei, Guo-Wei, E-mail: wei@math.msu.edu [Department of Biochemistry and Molecular Biology, Michigan State University, Michigan 48824 (United States); Department of Mathematics, Michigan State University, Michigan 48824 (United States); Department of Electrical and Computer Engineering, Michigan State University, Michigan 48824 (United States)

    2014-06-21

    Protein structural fluctuation, typically measured by Debye-Waller factors, or B-factors, is a manifestation of protein flexibility, which strongly correlates to protein function. The flexibility-rigidity index (FRI) is a newly proposed method for the construction of atomic rigidity functions required in the theory of continuum elasticity with atomic rigidity, which is a new multiscale formalism for describing excessively large biomolecular systems. The FRI method analyzes protein rigidity and flexibility and is capable of predicting protein B-factors without resorting to matrix diagonalization. A fundamental assumption used in the FRI is that protein structures are uniquely determined by various internal and external interactions, while the protein functions, such as stability and flexibility, are solely determined by the structure. As such, one can predict protein flexibility without resorting to the protein interaction Hamiltonian. Consequently, bypassing the matrix diagonalization, the original FRI has a computational complexity of O(N{sup 2}). This work introduces a fast FRI (fFRI) algorithm for the flexibility analysis of large macromolecules. The proposed fFRI further reduces the computational complexity to O(N). Additionally, we propose anisotropic FRI (aFRI) algorithms for the analysis of protein collective dynamics. The aFRI algorithms permit adaptive Hessian matrices, from a completely global 3N × 3N matrix to completely local 3 × 3 matrices. These 3 × 3 matrices, despite being calculated locally, also contain non-local correlation information. Eigenvectors obtained from the proposed aFRI algorithms are able to demonstrate collective motions. Moreover, we investigate the performance of FRI by employing four families of radial basis correlation functions. Both parameter optimized and parameter-free FRI methods are explored. Furthermore, we compare the accuracy and efficiency of FRI with some established approaches to flexibility analysis, namely

  2. Application of time series analysis on molecular dynamics simulations of proteins: a study of different conformational spaces by principal component analysis.

    Alakent, Burak; Doruker, Pemra; Camurdan, Mehmet C

    2004-09-08

    Time series analysis is applied on the collective coordinates obtained from principal component analysis of independent molecular dynamics simulations of alpha-amylase inhibitor tendamistat and immunity protein of colicin E7 based on the Calpha coordinates history. Even though the principal component directions obtained for each run are considerably different, the dynamics information obtained from these runs are surprisingly similar in terms of time series models and parameters. There are two main differences in the dynamics of the two proteins: the higher density of low frequencies and the larger step sizes for the interminima motions of colicin E7 than those of alpha-amylase inhibitor, which may be attributed to the higher number of residues of colicin E7 and/or the structural differences of the two proteins. The cumulative density function of the low frequencies in each run conforms to the expectations from the normal mode analysis. When different runs of alpha-amylase inhibitor are projected on the same set of eigenvectors, it is found that principal components obtained from a certain conformational region of a protein has a moderate explanation power in other conformational regions and the local minima are similar to a certain extent, while the height of the energy barriers in between the minima significantly change. As a final remark, time series analysis tools are further exploited in this study with the motive of explaining the equilibrium fluctuations of proteins. Copyright 2004 American Institute of Physics

  3. Modular design of synthetic protein mimics. Characterization of the helical conformation of a 13-residue peptide in crystals

    Karle, I.L.; Flippen-Anderson, J.L.; Uma, K.; Balaram, P.

    1989-01-01

    The incorporation of α-aminoisobutyryl (Aib) residues into peptide sequences facilitates helical folding. Aib-containing sequences have been chosen for the design of rigid helical segments in a modular approach to the construction of a synthetic protein mimic. The helical conformation of the synthetic peptide Boc-Aib-(Val-Ala-Leu-Aib) 3 -OMe in crystals is established by X-ray diffraction. The 13-residue apolar peptide adopts a helical form in the crystal with seven α-type hydrogen bonds in the middle and 3 10 -type hydrogen bonds at either end. The helices stack in columns, zigzag rather than linear, by means of direct NH hor-ellipsis OC head to tail hydrogen bonds. Leucyl side chains are extended on one side of the helix and valyl side chains on the other side. Water molecules form hydrogen bonds with several backbone carbonyl oxygens that also participate in α-helix hydrogen bonds. There is no apparent distortion of the helix caused by hydration

  4. Fluorescent Protein-Based Ca2+ Sensor Reveals Global, Divalent Cation-Dependent Conformational Changes in Cardiac Troponin C.

    Myriam A Badr

    Full Text Available Cardiac troponin C (cTnC is a key effector in cardiac muscle excitation-contraction coupling as the Ca2+ sensing subunit responsible for controlling contraction. In this study, we generated several FRET sensors for divalent cations based on cTnC flanked by a donor fluorescent protein (CFP and an acceptor fluorescent protein (YFP. The sensors report Ca2+ and Mg2+ binding, and relay global structural information about the structural relationship between cTnC's N- and C-domains. The sensors were first characterized using end point titrations to decipher the response to Ca2+ binding in the presence or absence of Mg2+. The sensor that exhibited the largest responses in end point titrations, CTV-TnC, (Cerulean, TnC, and Venus was characterized more extensively. Most of the divalent cation-dependent FRET signal originates from the high affinity C-terminal EF hands. CTV-TnC reconstitutes into skinned fiber preparations indicating proper assembly of troponin complex, with only ~0.2 pCa unit rightward shift of Ca2+-sensitive force development compared to WT-cTnC. Affinity of CTV-TnC for divalent cations is in agreement with known values for WT-cTnC. Analytical ultracentrifugation indicates that CTV-TnC undergoes compaction as divalent cations bind. C-terminal sites induce ion-specific (Ca2+ versus Mg2+ conformational changes in cTnC. Our data also provide support for the presence of additional, non-EF-hand sites on cTnC for Mg2+ binding. In conclusion, we successfully generated a novel FRET-Ca2+ sensor based on full length cTnC with a variety of cellular applications. Our sensor reveals global structural information about cTnC upon divalent cation binding.

  5. Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissues regeneration.

    Kaufman, Gili; Whitescarver, Ryan; Nunes, Laiz; Palmer, Xavier-Lewis; Skrtic, Drago; Tutak, Wojtek

    2017-10-09

    Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by imageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within 3 days of incubation, fibroblast spheroids interacted with the fibers and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for

  6. Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissue regeneration.

    Kaufman, Gili; Whitescarver, Ryan A; Nunes, Laiz; Palmer, Xavier-Lewis; Skrtic, Drago; Tutak, Wojtek

    2018-01-24

    Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by ImageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within three days of incubation, fibroblast spheroids interacted with the fibers, and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for

  7. Conformal Nets II: Conformal Blocks

    Bartels, Arthur; Douglas, Christopher L.; Henriques, André

    2017-08-01

    Conformal nets provide a mathematical formalism for conformal field theory. Associated to a conformal net with finite index, we give a construction of the `bundle of conformal blocks', a representation of the mapping class groupoid of closed topological surfaces into the category of finite-dimensional projective Hilbert spaces. We also construct infinite-dimensional spaces of conformal blocks for topological surfaces with smooth boundary. We prove that the conformal blocks satisfy a factorization formula for gluing surfaces along circles, and an analogous formula for gluing surfaces along intervals. We use this interval factorization property to give a new proof of the modularity of the category of representations of a conformal net.

  8. Differential regulation of lipid and protein metabolism in obese vs. lean subjects before and after a 72-h fast.

    Bak, Ann Mosegaard; Møller, Andreas Buch; Vendelbo, Mikkel Holm; Nielsen, Thomas Svava; Viggers, Rikke; Rungby, Jørgen; Pedersen, Steen Bønløkke; Jørgensen, Jens Otto Lunde; Jessen, Niels; Møller, Niels

    2016-07-01

    Increased availability of lipids may conserve muscle protein during catabolic stress. Our study was designed to define 1) intracellular mechanisms leading to increased lipolysis and 2) whether this scenario is associated with decreased amino acid and urea fluxes, and decreased muscle amino acid release in obese subjects under basal and fasting conditions. We therefore studied nine lean and nine obese subjects twice, after 12 and 72 h of fasting, using measurements of mRNA and protein expression and phosphorylation of lipolytic and protein metabolic signaling molecules in fat and muscle together with whole body and forearm tracer techniques. Obese subjects displayed increased whole body lipolysis, decreased urea production rates, and decreased forearm muscle protein breakdown per 100 ml of forearm tissue, differences that persisted after 72 h of fasting. Lipolysis per fat mass unit was reduced in obese subjects and, correspondingly, adipose tissue hormone-sensitive lipase (HSL) phosphorylation and mRNA and protein levels of the adipose triglyceride lipase (ATGL) coactivator CGI58 were decreased. Fasting resulted in higher HSL phosphorylations and lower protein levels of the ATGL inhibitor G0S2. Muscle protein expressions of mammalian target of rapamycin (mTOR) and 4EBP1 were lower in obese subjects, and MuRf1 mRNA was higher with fasting in lean but not obese subjects. Phosphorylation and signaling of mTOR decreased with fasting in both groups, whereas ULK1 protein and mRNA levels increased. In summary, obese subjects exhibit increased lipolysis due to a large fat mass with blunted prolipolytic signaling, together with decreased urea and amino acid fluxes both in the basal and 72-h fasted state; this is compatible with preservation of muscle and whole body protein. Copyright © 2016 the American Physiological Society.

  9. Quantifying polypeptide conformational space: sensitivity to conformation and ensemble definition.

    Sullivan, David C; Lim, Carmay

    2006-08-24

    Quantifying the density of conformations over phase space (the conformational distribution) is needed to model important macromolecular processes such as protein folding. In this work, we quantify the conformational distribution for a simple polypeptide (N-mer polyalanine) using the cumulative distribution function (CDF), which gives the probability that two randomly selected conformations are separated by less than a "conformational" distance and whose inverse gives conformation counts as a function of conformational radius. An important finding is that the conformation counts obtained by the CDF inverse depend critically on the assignment of a conformation's distance span and the ensemble (e.g., unfolded state model): varying ensemble and conformation definition (1 --> 2 A) varies the CDF-based conformation counts for Ala(50) from 10(11) to 10(69). In particular, relatively short molecular dynamics (MD) relaxation of Ala(50)'s random-walk ensemble reduces the number of conformers from 10(55) to 10(14) (using a 1 A root-mean-square-deviation radius conformation definition) pointing to potential disconnections in comparing the results from simplified models of unfolded proteins with those from all-atom MD simulations. Explicit waters are found to roughen the landscape considerably. Under some common conformation definitions, the results herein provide (i) an upper limit to the number of accessible conformations that compose unfolded states of proteins, (ii) the optimal clustering radius/conformation radius for counting conformations for a given energy and solvent model, (iii) a means of comparing various studies, and (iv) an assessment of the applicability of random search in protein folding.

  10. Fast high-pressure freezing of protein crystals in their mother liquor

    Burkhardt, Anja; Warmer, Martin; Panneerselvam, Saravanan; Wagner, Armin; Zouni, Athina; Glöckner, Carina; Reimer, Rudolph; Hohenberg, Heinrich; Meents, Alke

    2012-01-01

    Protein crystals were vitrified using high-pressure freezing in their mother liquor at 210 MPa and 77 K without cryoprotectants or oil coating. The method was successfully applied to photosystem II, which is representative of a membrane protein with a large unit cell and weak crystal contacts. High-pressure freezing (HPF) is a method which allows sample vitrification without cryoprotectants. In the present work, protein crystals were cooled to cryogenic temperatures at a pressure of 210 MPa. In contrast to other HPF methods published to date in the field of cryocrystallography, this protocol involves rapid sample cooling using a standard HPF device. The fast cooling rates allow HPF of protein crystals directly in their mother liquor without the need for cryoprotectants or external reagents. HPF was first attempted with hen egg-white lysozyme and cubic insulin crystals, yielding good to excellent diffraction quality. Non-cryoprotected crystals of the membrane protein photosystem II have been successfully cryocooled for the first time. This indicates that the presented HPF method is well suited to the vitrification of challenging systems with large unit cells and weak crystal contacts

  11. Postprandial lipemia detects the effect of soy protein on cardiovascular disease risk compared with the fasting lipid profile.

    Santo, Antonio S; Santo, Ariana M; Browne, Richard W; Burton, Harold; Leddy, John J; Horvath, Steven M; Horvath, Peter J

    2010-12-01

    Studies examining the effect of soy protein on cardiovascular disease (CVD) risk factors have not taken advantage of the postprandial state as an adjunct to the fasting lipid profile. The American Heart Association has acknowledged the efficacy of soy protein in reducing CVD risk factors to be limited. We hypothesized that the postprandial state would be more sensitive to any favorable changes associated with consuming soy protein compared with the fasting lipid profile. Furthermore, the presence of isoflavones in soy would enhance this effect. Thirty sedentary males aged 18-30 years were randomly assigned to milk protein (Milk), isoflavone-poor soy (Soy-), or isoflavone-rich soy (Soy+). Usual diets were supplemented with 25 g/day of protein for 28 days. Serum samples were collected before and after supplementation in a fasted state and postprandially at 30, 60, 120, 240, and 360 min after a high-fat, 1,000 kcal shake. Triacylglycerol (TAG), total cholesterol, non-esterified fatty acids, apolipoproteins B-100 and A-I and glucose concentrations were quantified. Fasting concentrations were not different after any protein supplementation. Postprandial TAG and TAG AUC increased after Soy-consumption supporting the postprandial state as a more sensitive indicator of soy ingestion effects on CVD risk factors compared with the fasting lipid profile. Furthermore, the absence of isoflavones in soy protein may have deleterious consequences on purported cardio-protective effects.

  12. Probing the Mechanism of pH-Induced Large-Scale Conformational Changes in Dengue Virus Envelope Protein Using Atomistic Simulations

    Prakash, Meher K.; Barducci, Alessandro; Parrinello, Michele

    2010-01-01

    Abstract One of the key steps in the infection of the cell by dengue virus is a pH-induced conformational change of the viral envelope proteins. These envelope proteins undergo a rearrangement from a dimer to a trimer, with large conformational changes in the monomeric unit. In this article, metadynamics simulations were used to enable us to understand the mechanism of these large-scale changes in the monomer. By using all-atom, explicit solvent simulations of the monomers, the stability of the protein structure is studied under low and high pH conditions. Free energy profiles obtained along appropriate collective coordinates demonstrate that pH affects the domain interface in both the conformations of E monomer, stabilizing one and destabilizing the other. These simulations suggest a mechanism with an intermediate detached state between the two monomeric structures. Using further analysis, we comment on the key residue interactions responsible for the instability and the pH-sensing role of a histidine that could not otherwise be studied experimentally. The insights gained from this study and methodology can be extended for studying similar mechanisms in the E proteins of the other members of class II flavivirus family. PMID:20643078

  13. Variants of beta-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature

    Mimmi, Maria C; Jørgensen, Thomas J D; Pettirossi, Fabio

    2006-01-01

    -58 is removed. We find that the solution stability of both variants, especially of beta2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type beta2-microglobulin and is strongly dependent on temperature and protein concentration. 1H-NMR spectroscopy and amide hydrogen......Cleavage of the small amyloidogenic protein beta2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related beta2-microglobulin amyloidosis, since elevated levels of cleaved beta2-microglobulin may be found in the circulation...

  14. Preparation and properties of fast temperature-responsive soy protein/PNIPAAm IPN hydrogels

    Liu Yong

    2014-01-01

    Full Text Available The interpenetrating polymer network of fast temperature-responsive hydrogels based on soy protein and poly(N-isopropylacrylamide were successfully prepared using the sodium bicarbonate (NaHCO3 solutions as the reaction medium. The structure and properties of the hydrogels were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, differential scanning calorimetry and thermal gravimetric analysis. The swelling and deswelling kinetics were also investigated in detail. The results have shown that the proposed hydrogels had high porous structure, good miscibility and thermal stability, and fast temperature responsivity. The presence of NaHCO3 had little effect on the volume phase transition temperature (VPTT of the hydrogels, and the VPTTs were at about 32°C. Compared with the traditional hydrogels, the proposed hydrogels had much faster swelling and deswelling rate. The swelling mechanism of the hydrogels was the non-Fickian diffusion. This fast temperature-responsive hydrogels may have potential applications in the field of biomedical materials.

  15. Fast hydrogen exchange affects 15N relaxation measurements in intrinsically disordered proteins

    Kim, Seho; Wu, Kuen-Phon; Baum, Jean

    2013-01-01

    Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, 1 H– 15 N HSQC, is used to measure the 15 N transverse relaxation rate (R 2 ), the measured R 2 rate is convoluted with the HX rate (k HX ) and has higher apparent R 2 values. Since the 15 N R 2 measurement is important for analyzing protein backbone dynamics, the HX effect on the R 2 measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing 15 N R 2 CPMG experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R 2 CPMG can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D 2 O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R 2 CPMG values be obtained by methods described herein.

  16. Convergence of sampling in protein simulations

    Hess, B

    With molecular dynamics protein dynamics can be simulated in atomic detail. Current computers are not fast enough to probe all available conformations, but fluctuations around one conformation can be sampled to a reasonable extent. The motions with the largest fluctuations can be filtered out of a

  17. Conformal house

    Ryttov, Thomas Aaby; Sannino, Francesco

    2010-01-01

    fixed point. As a consistency check we recover the previously investigated bounds of the conformal windows when restricting to a single matter representation. The earlier conformal windows can be imagined to be part now of the new conformal house. We predict the nonperturbative anomalous dimensions...... at the infrared fixed points. We further investigate the effects of adding mass terms to the condensates on the conformal house chiral dynamics and construct the simplest instanton induced effective Lagrangian terms...

  18. Rapid measurement of residual dipolar couplings for fast fold elucidation of proteins

    Rasia, Rodolfo M. [Jean-Pierre Ebel CNRS/CEA/UJF, Institut de Biologie Structurale (France); Lescop, Ewen [CNRS, Institut de Chimie des Substances Naturelles (France); Palatnik, Javier F. [Universidad Nacional de Rosario, Instituto de Biologia Molecular y Celular de Rosario, Facultad de Ciencias Bioquimicas y Farmaceuticas (Argentina); Boisbouvier, Jerome, E-mail: jerome.boisbouvier@ibs.fr; Brutscher, Bernhard, E-mail: Bernhard.brutscher@ibs.fr [Jean-Pierre Ebel CNRS/CEA/UJF, Institut de Biologie Structurale (France)

    2011-11-15

    It has been demonstrated that protein folds can be determined using appropriate computational protocols with NMR chemical shifts as the sole source of experimental restraints. While such approaches are very promising they still suffer from low convergence resulting in long computation times to achieve accurate results. Here we present a suite of time- and sensitivity optimized NMR experiments for rapid measurement of up to six RDCs per residue. Including such an RDC data set, measured in less than 24 h on a single aligned protein sample, greatly improves convergence of the Rosetta-NMR protocol, allowing for overnight fold calculation of small proteins. We demonstrate the performance of our fast fold calculation approach for ubiquitin as a test case, and for two RNA-binding domains of the plant protein HYL1. Structure calculations based on simulated RDC data highlight the importance of an accurate and precise set of several complementary RDCs as additional input restraints for high-quality de novo structure determination.

  19. Direct infusion-SIM as fast and robust method for absolute protein quantification in complex samples

    Christina Looße

    2015-06-01

    Full Text Available Relative and absolute quantification of proteins in biological and clinical samples are common approaches in proteomics. Until now, targeted protein quantification is mainly performed using a combination of HPLC-based peptide separation and selected reaction monitoring on triple quadrupole mass spectrometers. Here, we show for the first time the potential of absolute quantification using a direct infusion strategy combined with single ion monitoring (SIM on a Q Exactive mass spectrometer. By using complex membrane fractions of Escherichia coli, we absolutely quantified the recombinant expressed heterologous human cytochrome P450 monooxygenase 3A4 (CYP3A4 comparing direct infusion-SIM with conventional HPLC-SIM. Direct-infusion SIM revealed only 14.7% (±4.1 (s.e.m. deviation on average, compared to HPLC-SIM and a decreased processing and analysis time of 4.5 min (that could be further decreased to 30 s for a single sample in contrast to 65 min by the LC–MS method. Summarized, our simplified workflow using direct infusion-SIM provides a fast and robust method for quantification of proteins in complex protein mixtures.

  20. Adjustable chain trees for proteins

    Winter, Pawel; Fonseca, Rasmus

    2012-01-01

    A chain tree is a data structure for changing protein conformations. It enables very fast detection of clashes and free energy potential calculations. A modified version of chain trees that adjust themselves to the changing conformations of folding proteins is introduced. This results in much...... tighter bounding volume hierarchies and therefore fewer intersection checks. Computational results indicate that the efficiency of the adjustable chain trees is significantly improved compared to the traditional chain trees....

  1. Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

    Deng, Jingren; Lazar, Iulia M.

    2016-04-01

    The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

  2. Calcium binding promotes prion protein fragment 90-231 conformational change toward a membrane destabilizing and cytotoxic structure.

    Sacha Sorrentino

    Full Text Available The pathological form of prion protein (PrP(Sc, as other amyloidogenic proteins, causes a marked increase of membrane permeability. PrP(Sc extracted from infected Syrian hamster brains induces a considerable change in membrane ionic conductance, although the contribution of this interaction to the molecular mechanism of neurodegeneration process is still controversial. We previously showed that the human PrP fragment 90-231 (hPrP₉₀₋₂₃₁ increases ionic conductance across artificial lipid bilayer, in a calcium-dependent manner, producing an alteration similar to that observed for PrP(Sc. In the present study we demonstrate that hPrP₉₀₋₂₃₁, pre-incubated with 10 mM Ca⁺⁺ and then re-suspended in physiological external solution increases not only membrane conductance but neurotoxicity as well. Furthermore we show the existence of a direct link between these two effects as demonstrated by a highly statistically significant correlation in several experimental conditions. A similar correlation between increased membrane conductance and cell degeneration has been observed assaying hPrP₉₀₋₂₃₁ bearing pathogenic mutations (D202N and E200K. We also report that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ induces a conformational change based on an alteration of secondary structure characterized by loss of alpha-helix content causing hydrophobic amino acid exposure and proteinase K resistance. These features, either acquired after controlled thermal denaturation or induced by D202N and E200K mutations were previously identified as responsible for hPrP₉₀₋₂₃₁ cytotoxicity. Finally, by in silico structural analysis, we propose that Ca⁺⁺ binding to hPrP₉₀₋₂₃₁ modifies amino acid orientation, in the same way induced by E200K mutation, thus suggesting a pathway for the structural alterations responsible of PrP neurotoxicity.

  3. Following DNA chain extension and protein conformational changes in crystals of a Y-family DNA polymerase via Raman crystallography.

    Espinoza-Herrera, Shirly J; Gaur, Vineet; Suo, Zucai; Carey, Paul R

    2013-07-23

    Y-Family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. (13)C- and (15)N-labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg(2+) and Mn(2+), nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of *C modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following a single d*CTP addition. Adding these two bases caused visible damage to the crystal that was possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn(2+) or Mg(2+), the primer extension reaction did not occur; instead, a ternary protein·template·d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features caused by ring modes from the template/primer bases being perturbed and from the DNA backbone appear, as well as features from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary complex than in the binary complex. Using standardized Raman intensities followed as a function of time, the C(*C)TP population in the crystal was maximal at ∼20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred.

  4. Time-resolved Fourier transform infrared spectrometry using a microfabricated continuous flow mixer: application to protein conformation study using the example of ubiquitin.

    Kakuta, Masaya; Hinsmann, Peter; Manz, Andreas; Lendl, Bernhard

    2003-05-01

    We report on the use of time-resolved Fourier transform infrared spectroscopy (FT-IR) to study chemically induced conformational changes of proteins using the example of ubiquitin. For this purpose a micromachined mixer is coupled to a conventional IR transmission cell with a pathlength of 25 microm and operated in both the continuous and the stopped-flow mode. This experimental set-up allows the elucidation of reaction pathways in the time frame of about 500 milliseconds to seconds with little reagent consumption and low pressure. For continuous flow measurements employed in the time frame from 0.5 to 1.4 s the reaction time is determined by the flow rate used as the connection between the point of confluence in the micromixer and the flow cell was kept constant in all experiments. For stopped-flow experiments (>1.4 s) the time is determined by data acquisition of the rapid scanning infrared spectrometer. Ubiquitin, a small well-known protein with 76 amino acid residues, changes its conformation from native to A-state with the addition of methanol under low pH conditions. We investigated the conformational change in the time frame from 0.5 to 10 s by mixing ubiquitin (20% methanol-d(4)) with an 80% methanol-d(4) solution at pD 2 by evaluating the time dependent changes in the amide I band of the protein.

  5. Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

    Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

    2017-05-01

    We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

  6. Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants.

    Thomas L Prince

    Full Text Available The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90β, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90β with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90β, while HSP90β bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states.

  7. FRAGSION: ultra-fast protein fragment library generation by IOHMM sampling.

    Bhattacharya, Debswapna; Adhikari, Badri; Li, Jilong; Cheng, Jianlin

    2016-07-01

    Speed, accuracy and robustness of building protein fragment library have important implications in de novo protein structure prediction since fragment-based methods are one of the most successful approaches in template-free modeling (FM). Majority of the existing fragment detection methods rely on database-driven search strategies to identify candidate fragments, which are inherently time-consuming and often hinder the possibility to locate longer fragments due to the limited sizes of databases. Also, it is difficult to alleviate the effect of noisy sequence-based predicted features such as secondary structures on the quality of fragment. Here, we present FRAGSION, a database-free method to efficiently generate protein fragment library by sampling from an Input-Output Hidden Markov Model. FRAGSION offers some unique features compared to existing approaches in that it (i) is lightning-fast, consuming only few seconds of CPU time to generate fragment library for a protein of typical length (300 residues); (ii) can generate dynamic-size fragments of any length (even for the whole protein sequence) and (iii) offers ways to handle noise in predicted secondary structure during fragment sampling. On a FM dataset from the most recent Critical Assessment of Structure Prediction, we demonstrate that FGRAGSION provides advantages over the state-of-the-art fragment picking protocol of ROSETTA suite by speeding up computation by several orders of magnitude while achieving comparable performance in fragment quality. Source code and executable versions of FRAGSION for Linux and MacOS is freely available to non-commercial users at http://sysbio.rnet.missouri.edu/FRAGSION/ It is bundled with a manual and example data. chengji@missouri.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. CATHEDRAL: a fast and effective algorithm to predict folds and domain boundaries from multidomain protein structures.

    Oliver C Redfern

    2007-11-01

    Full Text Available We present CATHEDRAL, an iterative protocol for determining the location of previously observed protein folds in novel multidomain protein structures. CATHEDRAL builds on the features of a fast secondary-structure-based method (using graph theory to locate known folds within a multidomain context and a residue-based, double-dynamic programming algorithm, which is used to align members of the target fold groups against the query protein structure to identify the closest relative and assign domain boundaries. To increase the fidelity of the assignments, a support vector machine is used to provide an optimal scoring scheme. Once a domain is verified, it is excised, and the search protocol is repeated in an iterative fashion until all recognisable domains have been identified. We have performed an initial benchmark of CATHEDRAL against other publicly available structure comparison methods using a consensus dataset of domains derived from the CATH and SCOP domain classifications. CATHEDRAL shows superior performance in fold recognition and alignment accuracy when compared with many equivalent methods. If a novel multidomain structure contains a known fold, CATHEDRAL will locate it in 90% of cases, with <1% false positives. For nearly 80% of assigned domains in a manually validated test set, the boundaries were correctly delineated within a tolerance of ten residues. For the remaining cases, previously classified domains were very remotely related to the query chain so that embellishments to the core of the fold caused significant differences in domain sizes and manual refinement of the boundaries was necessary. To put this performance in context, a well-established sequence method based on hidden Markov models was only able to detect 65% of domains, with 33% of the subsequent boundaries assigned within ten residues. Since, on average, 50% of newly determined protein structures contain more than one domain unit, and typically 90% or more of these

  9. Electrostatics effects on Ca(2+) binding and conformational changes in EF-hand domains: Functional implications for EF-hand proteins.

    Ababou, Abdessamad; Zaleska, Mariola

    2015-12-01

    Mutations of Gln41 and Lys75 with nonpolar residues in the N-terminal domain of calmodulin (N-Cam) revealed the importance of solvation energetics in conformational change of Ca(2+) sensor EF-hand domains. While in general these domains have polar residues at these corresponding positions yet the extent of their conformational response to Ca(2+) binding and their Ca(2+) binding affinity can be different from N-Cam. Consequently, here we address the charge state of the polar residues at these positions. The results show that the charge state of these polar residues can affect substantially the conformational change and the Ca(2+) binding affinity of our N-Cam variants. Since all the variants kept their conformational activity in the presence of Ca(2+) suggests that the differences observed among them mainly originate from the difference in their molecular dynamics. Hence we propose that the molecular dynamics of Ca(2+) sensor EF-hand domains is a key factor in the multifunctional aspect of EF-hand proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

    2014-01-01

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV F , we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy

  11. The novel 2Fe–2S outer mitochondrial protein mitoNEET displays conformational flexibility in its N-terminal cytoplasmic tethering domain

    Conlan, Andrea R.; Paddock, Mark L.; Axelrod, Herbert L.; Cohen, Aina E.; Abresch, Edward C.; Wiley, Sandra; Roy, Melinda; Nechushtai, Rachel; Jennings, Patricia A.

    2009-01-01

    The crystal structure of the anti-diabetic drug target mitoNEET obtained from a GFP fusion construct (1.4 Å resolution, R factor = 20.2%) shows that the CDGSH 2Fe–2S binding domains are superimposable with previously determined non-fused constructs. However, there is considerable flexibility in the position of the outer mitochondrial tethering arms resulting in two different conformations in the crystal structure. A primary role for mitochondrial dysfunction is indicated in the pathogenesis of insulin resistance. A widely used drug for the treatment of type 2 diabetes is pioglitazone, a member of the thiazolidinedione class of molecules. MitoNEET, a 2Fe–2S outer mitochondrial membrane protein, binds pioglitazone [Colca et al. (2004 ▶), Am. J. Physiol. Endocrinol. Metab.286, E252–E260]. The soluble domain of the human mitoNEET protein has been expressed C-terminal to the superfolder green fluorescent protein and the mitoNEET protein has been isolated. Comparison of the crystal structure of mitoNEET isolated from cleavage of the fusion protein (1.4 Å resolution, R factor = 20.2%) with other solved structures shows that the CDGSH domains are superimposable, indicating proper assembly of mitoNEET. Furthermore, there is considerable flexibility in the position of the cytoplasmic tethering arms, resulting in two different conformations in the crystal structure. This flexibility affords multiple orientations on the outer mitochondrial membrane

  12. Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

    Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

    2014-07-15

    Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

  13. (3,2)D GFT-NMR experiments for fast data collection from proteins

    Xia Youlin; Zhu Guang; Veeraraghavan, Sudha; Gao Xiaolian

    2004-01-01

    High throughput structure determination of proteins will contribute to the success of proteomics investigations. The G-Matrix Fourier Transformation NMR (GFT-NMR) method significantly shortens experimental time by reducing the number of the dimensions of data acquisition for isotopically labeled proteins (Kim, S. and Szyperski, T. (2003) J. Am. Chem. Soc.125, 1385). We demonstrate herein a suite of ten 3D → 2D or (3,2)D GFT-NMR experiments using 13 C/ 15 N-labeled ubiquitin. These experiments were completed within 18 hours, representing a 4- to 18-fold reduction in data acquisition time compared to the corresponding conventional 3D experiments. A subset of the GFT-NMR experiments, (3,2)D HNCO, HNCACB, HN(CO)CACB, and 2D 1 H- 15 N HSQC, which are necessary for backbone assignments, were carried out within 6 hours. To facilitate the analysis of the GFT-NMR spectra, we developed automated procedures for viewing and analyzing the GFT-NMR spectra. Our overall strategy allows (3,2)D GFT-NMR experiments to be readily performed and analyzed. Nevertheless, the increase in spectral overlap and the reduction in signal sensitivity in these fast NMR experiments presently limit their application to relatively small proteins

  14. FreeContact: fast and free software for protein contact prediction from residue co-evolution.

    Kaján, László; Hopf, Thomas A; Kalaš, Matúš; Marks, Debora S; Rost, Burkhard

    2014-03-26

    20 years of improved technology and growing sequences now renders residue-residue contact constraints in large protein families through correlated mutations accurate enough to drive de novo predictions of protein three-dimensional structure. The method EVfold broke new ground using mean-field Direct Coupling Analysis (EVfold-mfDCA); the method PSICOV applied a related concept by estimating a sparse inverse covariance matrix. Both methods (EVfold-mfDCA and PSICOV) are publicly available, but both require too much CPU time for interactive applications. On top, EVfold-mfDCA depends on proprietary software. Here, we present FreeContact, a fast, open source implementation of EVfold-mfDCA and PSICOV. On a test set of 140 proteins, FreeContact was almost eight times faster than PSICOV without decreasing prediction performance. The EVfold-mfDCA implementation of FreeContact was over 220 times faster than PSICOV with negligible performance decrease. EVfold-mfDCA was unavailable for testing due to its dependency on proprietary software. FreeContact is implemented as the free C++ library "libfreecontact", complete with command line tool "freecontact", as well as Perl and Python modules. All components are available as Debian packages. FreeContact supports the BioXSD format for interoperability. FreeContact provides the opportunity to compute reliable contact predictions in any environment (desktop or cloud).

  15. Fast-acting and nearly gratuitous induction of gene expression and protein depletion in Saccharomyces cerevisiae

    McIsaac, R. Scott; Silverman, Sanford J.; McClean, Megan N.; Gibney, Patrick A.; Macinskas, Joanna; Hickman, Mark J.; Petti, Allegra A.; Botstein, David

    2011-01-01

    We describe the development and characterization of a system that allows the rapid and specific induction of individual genes in the yeast Saccharomyces cerevisiae without changes in nutrients or temperature. The system is based on the chimeric transcriptional activator Gal4dbd.ER.VP16 (GEV). Upon addition of the hormone β-estradiol, cytoplasmic GEV localizes to the nucleus and binds to promoters containing Gal4p consensus binding sequences to activate transcription. With galactokinase Gal1p and transcriptional activator Gal4p absent, the system is fast-acting, resulting in readily detectable transcription within 5 min after addition of the inducer. β-Estradiol is nearly a gratuitous inducer, as indicated by genome-wide profiling that shows unintended induction (by GEV) of only a few dozen genes. Response to inducer is graded: intermediate concentrations of inducer result in production of intermediate levels of product protein in all cells. We present data illustrating several applications of this system, including a modification of the regulated degron method, which allows rapid and specific degradation of a specific protein upon addition of β-estradiol. These gene induction and protein degradation systems provide important tools for studying the dynamics and functional relationships of genes and their respective regulatory networks. PMID:21965290

  16. A constitutively activating mutation alters the dynamics and energetics of a key conformational change in a ligand-free G protein-coupled receptor.

    Tsukamoto, Hisao; Farrens, David L

    2013-09-27

    G protein-coupled receptors (GPCRs) undergo dynamic transitions between active and inactive conformations. Usually, these conversions are triggered when the receptor detects an external signal, but some so-called constitutively activating mutations, or CAMs, induce a GPCR to bind and activate G proteins in the absence of external stimulation, in ways still not fully understood. Here, we investigated how a CAM alters the structure of a GPCR and the dynamics involved as the receptor transitions between different conformations. Our approach used site-directed fluorescence labeling (SDFL) spectroscopy to compare opsin, the ligand-free form of the GPCR rhodopsin, with opsin containing the CAM M257Y, focusing specifically on key movements that occur in the sixth transmembrane helix (TM6) during GPCR activation. The site-directed fluorescence labeling data indicate opsin is constrained to an inactive conformation both in detergent micelles and lipid membranes, but when it contains the M257Y CAM, opsin is more dynamic and can interact with a G protein mimetic. Further study of these receptors using tryptophan-induced quenching (TrIQ) methods indicates that in detergent, the CAM significantly increases the population of receptors in the active state, but not in lipids. Subsequent Arrhenius analysis of the TrIQ data suggests that, both in detergent and lipids, the CAM lowers the energy barrier for TM6 movement, a key transition required for conversion between the inactive and active conformations. Together, these data suggest that the lowered energy barrier is a primary effect of the CAM on the receptor dynamics and energetics.

  17. Effect of Ramadan fasting on serum heat shock protein 70 and serum lipid profile.

    Zare, A; Hajhashemi, M; Hassan, Z M; Zarrin, S; Pourpak, Z; Moin, M; Salarilak, S; Masudi, S; Shahabi, S

    2011-07-01

    Ramadan, the holy month for the Islamic world, is a period every year when food and fluid intake is restricted to the pre-sunrise and post-sunset hours. The aim of this study was to evaluate the effect of Ramadan fasting on the serum concentration of heat shock protein 70 (HSP70) and serum lipid profile in healthy men. A total of 32 male volunteers with a mean age of 28.5 (range 23-37) years were selected for the study. Blood samples were obtained one day prior to Ramadan and on the 3rd and 25th days of fasting. Serum HSP70, triglyceride (TG), cholesterol (Chol), low-density lipoprotein (LDL) and high-density lipoprotein (HDL), LDL/HDL and Chol/HDL ratios were investigated. It was observed that the mean concentrations of serum HSP70 and HDL on the 25th day of Ramadan were significantly higher than those recorded one day before Ramadan and on the 3rd day of Ramadan, and the levels on the 3rd day of Ramadan was significantly higher than those recorded one day before Ramadan. Mean concentrations of serum TG, Chol, LDL, and LDL/HDL and Chol/HDL ratios on the 25th day of Ramadan were significantly lower than those recorded one day before Ramadan and on the 3rd day of Ramadan, and the levels found on the 3rd day of Ramadan were also significantly lower than those recorded one day before Ramadan. Ramadan fasting increases serum HSP70 and improves serum lipid profile.

  18. Network single-walled carbon nanotube biosensors for fast and highly sensitive detection of proteins

    Hu Pingan; Zhang Jia; Wen Zhenzhong; Zhang Can

    2011-01-01

    Detection of proteins is powerfully assayed in the diagnosis of diseases. A strategy for the development of an ultrahigh sensitivity biosensor based on a network single-walled carbon nanotube (SWNT) field-effect transistor (FET) has been demonstrated. Metallic SWNTs (m-SWNTs) in the network nanotube FET were selectively removed or cut via a carefully controlled procedure of electrical break-down (BD), and left non-conducting m-SWNTs which magnified the Schottky barrier (SB) area. This nanotube FET exhibited ultrahigh sensitivity and fast response to biomolecules. The lowest detection limit of 0.5 pM was achieved by exploiting streptavidin (SA) or a biotin/SA pair as the research model, and BD-treated nanotube biosensors had a 2 x 10 4 -fold lower minimum detectable concentration than the device without BD treatment. The response time is in the range of 0.3-3 min.

  19. Fast hydrogen exchange affects {sup 15}N relaxation measurements in intrinsically disordered proteins

    Kim, Seho; Wu, Kuen-Phon; Baum, Jean, E-mail: jean.baum@rutgers.edu [Rutgers University, Department of Chemistry and Chemical Biology (United States)

    2013-03-15

    Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, {sup 1}H-{sup 15}N HSQC, is used to measure the {sup 15}N transverse relaxation rate (R{sub 2}), the measured R{sub 2} rate is convoluted with the HX rate (k{sub HX}) and has higher apparent R{sub 2} values. Since the {sup 15}N R{sub 2} measurement is important for analyzing protein backbone dynamics, the HX effect on the R{sub 2} measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing {sup 15}N R{sub 2}{sup CPMG} experiments on {alpha}-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R{sub 2}{sup CPMG} can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D{sub 2}O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R{sub 2}{sup CPMG} values be obtained by methods described herein.

  20. Protein-protein interactions as a proxy to monitor conformational changes and activation states of the tomato resistance protein I-2

    Lukasik-Shreepaathy, E.; Vossen, J.H.; Tameling, W.I.L.; de Vroomen, M.J.; Cornelissen, B.J.C.; Takken, F.L.W.

    2012-01-01

    Plant resistance proteins (R) are involved in pathogen recognition and subsequent initiation of defence responses. Their activity is regulated by inter- and intramolecular interactions. In a yeast two-hybrid screen two clones (I2I-1 and I2I-2) specifically interacting with I-2, a Fusarium oxysporum

  1. Fast automated placement of polar hydrogen atoms in protein-ligand complexes

    Lippert Tobias

    2009-08-01

    Full Text Available Abstract Background Hydrogen bonds play a major role in the stabilization of protein-ligand complexes. The ability of a functional group to form them depends on the position of its hydrogen atoms. An accurate knowledge of the positions of hydrogen atoms in proteins is therefore important to correctly identify hydrogen bonds and their properties. The high mobility of hydrogen atoms introduces several degrees of freedom: Tautomeric states, where a hydrogen atom alters its binding partner, torsional changes where the position of the hydrogen atom is rotated around the last heavy-atom bond in a residue, and protonation states, where the number of hydrogen atoms at a functional group may change. Also, side-chain flips in glutamine and asparagine and histidine residues, which are common crystallographic ambiguities must be identified before structure-based calculations can be conducted. Results We have implemented a method to determine the most probable hydrogen atom positions in a given protein-ligand complex. Optimality of hydrogen bond geometries is determined by an empirical scoring function which is used in molecular docking. This allows to evaluate protein-ligand interactions with an established model. Also, our method allows to resolve common crystallographic ambiguities such as as flipped amide groups and histidine residues. To ensure high speed, we make use of a dynamic programming approach. Conclusion Our results were checked against selected high-resolution structures from an external dataset, for which the positions of the hydrogen atoms have been validated manually. The quality of our results is comparable to that of other programs, with the advantage of being fast enough to be applied on-the-fly for interactive usage or during score evaluation.

  2. Solid state NMR sequential resonance assignments and conformational analysis of the 2x10.4 kDa dimeric form of the Bacillus subtilis protein Crh

    Boeckmann, Anja [Institut de Biologie et Chimie des Proteines, C.N.R.S UMR 5086 (France)], E-mail: a.bockmann@ibcp.fr; Lange, Adam [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany); Galinier, Anne [Institut de Biologie Structurale et Microbiologie, C.N.R.S UPR 9043 (France); Luca, Sorin [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany); Giraud, Nicolas; Juy, Michel [Institut de Biologie et Chimie des Proteines, C.N.R.S UMR 5086 (France); Heise, Henrike [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany); Montserret, Roland; Penin, Francois [Institut de Biologie et Chimie des Proteines, C.N.R.S UMR 5086 (France); Baldus, Marc [Max-Planck-Institute for Biophysical Chemistry, Solid-state NMR (Germany)], E-mail: maba@mpibpc.mpg.de

    2003-12-15

    Solid state NMR sample preparation and resonance assignments of the U-[{sup 13}C,{sup 15}N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all {sup 15}N, {sup 13}C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.

  3. Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change.

    Hetrick, Byron; Han, Min Suk; Helgeson, Luke A; Nolen, Brad J

    2013-05-23

    Actin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. A chemometric analysis of ligand-induced changes in intrinsic fluorescence of folate binding protein indicates a link between altered conformational structure and physico-chemical characteristics

    Bruun, Susanne W; Holm, Jan; Hansen, Steen Ingemann

    2009-01-01

    Ligand binding alters the conformational structure and physico-chemical characteristics of bovine folate binding protein (FBP). For the purpose of achieving further information we analyzed ligand (folate and methotrexate)-induced changes in the fluorescence landscape of FBP. Fluorescence excitation...... of folate accords fairly well with the disappearance of strongly hydrophobic tryptophan residues from the solvent-exposed surface of FBP. The PARAFAC has thus proven useful to establish a hitherto unexplained link between parallel changes in conformational structure and physico-chemical characteristics...... of FBP induced by folate binding. Parameters for ligand binding derived from PARAFAC analysis of the fluorescence data were qualitatively and quantitatively similar to those obtained from binding of radiofolate to FBP. Herein, methotrexate exhibited a higher affinity for FBP than in competition...

  5. Transportation Conformity

    This section provides information on: current laws, regulations and guidance, policy and technical guidance, project-level conformity, general information, contacts and training, adequacy review of SIP submissions

  6. S-Nitrosylation and uncompetitive/fast off-rate (UFO) drug therapy in neurodegenerative disorders of protein misfolding.

    Nakamura, T; Lipton, S A

    2007-07-01

    Although activation of glutamate receptors is essential for normal brain function, excessive activity leads to a form of neurotoxicity known as excitotoxicity. Key mediators of excitotoxic damage include overactivation of N-methyl-D-aspartate (NMDA) receptors, resulting in excessive Ca(2+) influx with production of free radicals and other injurious pathways. Overproduction of free radical nitric oxide (NO) contributes to acute and chronic neurodegenerative disorders. NO can react with cysteine thiol groups to form S-nitrosothiols and thus change protein function. S-nitrosylation can result in neuroprotective or neurodestructive consequences depending on the protein involved. Many neurodegenerative diseases manifest conformational changes in proteins that result in misfolding and aggregation. Our recent studies have linked nitrosative stress to protein misfolding and neuronal cell death. Molecular chaperones - such as protein-disulfide isomerase, glucose-regulated protein 78, and heat-shock proteins - can provide neuroprotection by facilitating proper protein folding. Here, we review the effect of S-nitrosylation on protein function under excitotoxic conditions, and present evidence that NO contributes to degenerative conditions by S-nitrosylating-specific chaperones that would otherwise prevent accumulation of misfolded proteins and neuronal cell death. In contrast, we also review therapeutics that can abrogate excitotoxic damage by preventing excessive NMDA receptor activity, in part via S-nitrosylation of this receptor to curtail excessive activity.

  7. UFO: a web server for ultra-fast functional profiling of whole genome protein sequences.

    Meinicke, Peter

    2009-09-02

    Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address.

  8. Analysis of Low Frequency Protein Truncating Stop-Codon Variants and Fasting Concentration of Growth Hormone.

    Erik Hallengren

    Full Text Available The genetic background of Growth Hormone (GH secretion is not well understood. Mutations giving rise to a stop codon have a high likelihood of affecting protein function.To analyze likely functional stop codon mutations that are associated with fasting plasma concentration of Growth Hormone.We analyzed stop codon mutations in 5451 individuals in the Malmö Diet and Cancer study by genotyping the Illumina Exome Chip. To enrich for stop codon mutations with likely functional effects on protein function, we focused on those disrupting >80% of the predicted amino acid sequence, which were carried by ≥ 10 individuals. Such mutations were related to GH concentration, measured with a high sensitivity assay (hs-GH and, if nominally significant, to GH related phenotypes, using linear regression analysis.Two stop codon mutations were associated with the fasting concentration of hs-GH. rs121909305 (NP_005370.1:p.R93* [Minor Allele Frequency (MAF = 0.8%] in the Myosin 1A gene (MYO1A was associated with a 0.36 (95%CI, 0.04 to 0.54; p=0.02 increment of the standardized value of the natural logarithm of hs-GH per 1 minor allele and rs35699176 (NP_067040.1:p.Q100* in the Zink Finger protein 77 gene (ZNF77 (MAF = 4.8% was associated with a 0.12 (95%CI, 0.02 to 0.22; p = 0.02 increase of hs-GH. The mutated high hs-GH associated allele of MYO1A was related to lower BMI (β-coefficient, -0.22; p = 0.05, waist (β-coefficient, -0.22; p = 0.04, body fat percentage (β-coefficient, -0.23; p = 0.03 and with higher HDL (β-coefficient, 0.23; p = 0.04. The ZNF77 stop codon was associated with height (β-coefficient, 0.11; p = 0.02 but not with cardiometabolic risk factors.We here suggest that a stop codon of MYO1A, disrupting 91% of the predicted amino acid sequence, is associated with higher hs-GH and GH-related traits suggesting that MYO1A is involved in GH metabolism and possibly body fat distribution. However, our results are preliminary and need replication in

  9. Functional fluorescent protein insertions in herpes simplex virus gB report on gB conformation before and after execution of membrane fusion.

    John R Gallagher

    2014-09-01

    Full Text Available Entry of herpes simplex virus (HSV into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy.

  10. A Conformational Change in C-Reactive Protein Enhances Leukocyte Recruitment and Reactive Oxygen Species Generation in Ischemia/Reperfusion Injury

    Jan R. Thiele

    2018-04-01

    Full Text Available IntroductionC-reactive protein circulates as a pentameric protein (pCRP. pCRP is a well-established diagnostic marker as plasma levels rise in response to tissue injury and inflammation. We recently described pro-inflammatory properties of CRP, which are mediated by conformational changes from pCRP to bioactive isoforms expressing pro-inflammatory neo-epitopes [pCRP* and monomeric C-reactive protein (mCRP]. Here, we investigate the role of CRP isoforms in renal ischemia/reperfusion injury (IRI.MethodsRat kidneys in animals with and without intraperitoneally injected pCRP were subjected to IRI by the time of pCRP exposure and were subsequently analyzed for monocyte infiltration, caspase-3 expression, and tubular damage. Blood urea nitrogen (BUN was analyzed pre-ischemia and post-reperfusion. CRP effects on leukocyte recruitment were investigated via intravital imaging of rat-striated muscle IRI. Localized conformational CRP changes were analyzed by immunohistochemistry using conformation specific antibodies. 1,6-bis(phosphocholine-hexane (1,6-bisPC, which stabilizes CRP in its native pentameric form was used to validate CRP effects. Leukocyte activation was assessed by quantification of reactive oxygen species (ROS induction by CRP isoforms ex vivo and in vitro through electron spin resonance spectroscopy. Signaling pathways were analyzed by disrupting lipid rafts with nystatin and subsequent ROS detection. In order to confirm the translational relevance of our findings, biopsies of microsurgical human free tissue transfers before and after IRI were examined by immunofluorescence for CRP deposition and co-localization of CD68+ leukocytes.ResultsThe application of pCRP aggravates tissue damage in renal IRI. 1,6-bisPC reverses these effects via inhibition of the conformational change that leads to exposure of pro-inflammatory epitopes in CRP (pCRP* and mCRP. Structurally altered CRP induces leukocyte–endothelial interaction and induces ROS

  11. GOSSIP: a method for fast and accurate global alignment of protein structures.

    Kifer, I; Nussinov, R; Wolfson, H J

    2011-04-01

    The database of known protein structures (PDB) is increasing rapidly. This results in a growing need for methods that can cope with the vast amount of structural data. To analyze the accumulating data, it is important to have a fast tool for identifying similar structures and clustering them by structural resemblance. Several excellent tools have been developed for the comparison of protein structures. These usually address the task of local structure alignment, an important yet computationally intensive problem due to its complexity. It is difficult to use such tools for comparing a large number of structures to each other at a reasonable time. Here we present GOSSIP, a novel method for a global all-against-all alignment of any set of protein structures. The method detects similarities between structures down to a certain cutoff (a parameter of the program), hence allowing it to detect similar structures at a much higher speed than local structure alignment methods. GOSSIP compares many structures in times which are several orders of magnitude faster than well-known available structure alignment servers, and it is also faster than a database scanning method. We evaluate GOSSIP both on a dataset of short structural fragments and on two large sequence-diverse structural benchmarks. Our conclusions are that for a threshold of 0.6 and above, the speed of GOSSIP is obtained with no compromise of the accuracy of the alignments or of the number of detected global similarities. A server, as well as an executable for download, are available at http://bioinfo3d.cs.tau.ac.il/gossip/.

  12. Protein and lipid metabolism adjustments in silver catfish (Rhamdia quelen during different periods of fasting and refeeding

    A. Marqueze

    2017-10-01

    Full Text Available Abstract The fish may experience periods of food deprivation or starvation which produce metabolic changes. In this study, adult Rhamdia quelen males were subjected to fasting periods of 1, 7, 14, and 21 days and of refeeding 2, 4, 6, and 12 days. The results demonstrated that liver protein was depleted after 1 day of fasting, but recovered after 6 days of refeeding. After 14 days of fasting, mobilization in the lipids of the muscular tissue took place, and these reserves began to re-establish themselves after 4 days of refeeding. Plasmatic triglycerides increased after 1 day of fasting, and decreased following 2 days of refeeding. The glycerol in the plasma oscillated constantly during the different periods of fasting and refeeding. Changes in the metabolism of both protein and lipids during these periods can be considered as survival strategies used by R. quelen. The difference in the metabolic profile of the tissues, the influence of the period of fasting, and the type of reserves mobilized were all in evidence.

  13. Workers’ Conformism

    Nikolay Ivantchev

    2013-10-01

    Full Text Available Conformism was studied among 46 workers with different kinds of occupations by means of two modified scales measuring conformity by Santor, Messervey, and Kusumakar (2000 – scale for perceived peer pressure and scale for conformism in antisocial situations. The hypothesis of the study that workers’ conformism is expressed in a medium degree was confirmed partly. More than a half of the workers conform in a medium degree for taking risk, and for the use of alcohol and drugs, and for sexual relationships. More than a half of the respondents conform in a small degree for anti-social activities (like a theft. The workers were more inclined to conform for risk taking (10.9%, then – for the use of alcohol, drugs and for sexual relationships (8.7%, and in the lowest degree – for anti-social activities (6.5%. The workers who were inclined for the use of alcohol and drugs tended also to conform for anti-social activities.

  14. Protein covalent immobilization via its scarce thiol versus abundant amine groups: Effect on orientation, cell binding domain exposure and conformational lability.

    Ba, O M; Hindie, M; Marmey, P; Gallet, O; Anselme, K; Ponche, A; Duncan, A C

    2015-10-01

    Quantity, orientation, conformation and covalent linkage of naturally cell adhesive proteins adsorbed or covalently linked to a surface, are known to influence the preservation of their subsequent long term cell adhesion properties and bioactivity. In the present work, we explore two different strategies for the covalent linking of plasma fibronectin (pFN) - used as a cell adhesive model protein, onto a polystyrene (PS) surface. One is aimed at tethering the protein to the surface in a semi-oriented fashion (via one of the 4 free thiol reactive groups on the protein) with a heterofunctional coupling agent (SSMPB method). The other aims to immobilize the protein in a more random fashion by reaction between the abundant pendant primary amine bearing amino acids of the pFN and activated carboxylic surface functions obtained after glutaric anhydride surface treatment (GA method). The overall goal will be to verify the hypothesis of a correlation between covalent immobilization of a model cell adhesive protein to a PS surface in a semi-oriented configuration (versus randomly oriented) with promotion of enhanced exposure of the protein's cell binding domain. This in turn would lead to enhanced cell adhesion. Ideally the goal is to elaborate substrates exhibiting a long term stable protein monolayer with preserved cell adhesive properties and bioactivity for biomaterial and/or cell adhesion commercial plate applications. However, the initial restrictive objective of this paper is to first quantitatively and qualitatively investigate the reversibly (merely adsorbed) versus covalently irreversibly bound protein to the surface after the immobilization procedure. Although immobilized surface amounts were similar (close to the monolayer range) for all immobilization approaches, covalent grafting showed improved retention and stronger "tethering" of the pFN protein to the surface (roughly 40%) after SDS rinsing compared to that for mere adsorption (0%) suggesting an added value

  15. Handheld Chem/Biosensor Using Extreme Conformational Changes in Designed Binding Proteins to Enhance Surface Plasmon Resonance (SPR)

    2016-04-01

    characteristics similar to a random coil conformation. By increasing the solution ionic strength it is possible to screen the side chain charges and... characteristics of metallic thin films perforated with subwavelength-sized apertures, spawned the research field of metamaterials and anomalous transmission...Veldhoven, Netherlands). Following a post-exposure bake on a hotplate at 95 °C for 90 s, each wafer was developed in AZ®726 MIF (MicroChemicals GmbH

  16. CABS-flex 2.0: a web server for fast simulations of flexibility of protein structures.

    Kuriata, Aleksander; Gierut, Aleksandra Maria; Oleniecki, Tymoteusz; Ciemny, Maciej Pawel; Kolinski, Andrzej; Kurcinski, Mateusz; Kmiecik, Sebastian

    2018-05-14

    Classical simulations of protein flexibility remain computationally expensive, especially for large proteins. A few years ago, we developed a fast method for predicting protein structure fluctuations that uses a single protein model as the input. The method has been made available as the CABS-flex web server and applied in numerous studies of protein structure-function relationships. Here, we present a major update of the CABS-flex web server to version 2.0. The new features include: extension of the method to significantly larger and multimeric proteins, customizable distance restraints and simulation parameters, contact maps and a new, enhanced web server interface. CABS-flex 2.0 is freely available at http://biocomp.chem.uw.edu.pl/CABSflex2.

  17. Refeeding with a high-protein diet after a 48 h fast causes acute hepatocellular injury in mice.

    Oarada, Motoko; Tsuzuki, Tsuyoshi; Nikawa, Takeshi; Kohno, Shohei; Hirasaka, Katsuya; Gonoi, Tohru

    2012-05-01

    Elucidating the effects of refeeding a high-protein diet after fasting on disease development is of interest in relation to excessive protein ingestion and irregular eating habits in developed countries. The objective of the present study was to address the hepatic effects of refeeding a high-protein diet after fasting. Mice were fasted for 48 h and then refed with a test diet containing 3, 15, 35, 40, 45 or 50 % casein. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and liver immediate-early gene expression levels were sequentially measured for the first 24 h after initiation of refeeding. Refeeding with a 50 % casein diet after 48 h of fasting led to a rapid (within 2-3 h) and abnormal elevation in serum ALT (P = 0·006) and AST (P = 0·001) activities and a marked increase in liver Finkel-Biskis-Jinkins (FBJ) osteosarcoma oncogene (P = 0·007) and nuclear receptor subfamily 4, group A, member 1 (P = 0·002) mRNA levels. In contrast, refeeding of the 3, 15 or 35 % casein diets produced no substantial increases in serum ALT and AST activities in mice. Refeeding of 40, 45 or 50 % casein increased serum ALT and AST activities in proportion to this dietary casein content. In mice refed the 3, 15 or 35, but not 50 %, casein diets, liver heat shock protein 72 transcript levels greatly increased. We conclude from these data that the consumption of a high-protein diet after fasting causes acute hepatocellular injury in healthy animals, and propose that careful attention should be paid to the use of such diets.

  18. Small-angle reflectometry of milk protein (β -casein) at the air/serum interface and its conformational changes due to fat content and temperature

    Heidari, R.; White, J.W.

    2003-01-01

    Full text: The surface structure of dispersed emulsions play a key role in stability of the system. Proteins being one of the most important surface-active components in foods stabilise interfaces by self-interaction, resulting in a stiff visco-elastic adsorbed layer. These interactions are sensitive to disruptive effects of lipids. Previous kinetics studies by the group 1 using the X-ray reflectivity method to investigate the surface adsorption of milk proteins indicate that β -casein had a stronger affinity for the air-liquid interface compared to whey proteins. It has been shown that initially a dense protein layer, with the thickness of 20 Angstroms is formed then a second more diffuse layer with lower volume density of protein follows. Here we report the conformational changes (with particular emphasise on the β -casein tail) occurred at the air-milk serum interface due to the effects of milk fat content, temperature and the milk preparation technique (ie homogenisation vs microfluidisation). In the effect of fat content on the adsorption of protein into the interface the key conclusion is that at lower temperatures the surface composition remains unchanged. The compositional changes, however, become significant at room temperature indicating adsorption of less reflective-water-soluble components into the surface layer. Repulsive interactions between casein aggregates are also involved. Microfluidised samples having the advantage of smaller particle size prove to be more stable to fat or temperature effects compared to the corresponding homogenised milks

  19. UFO: a web server for ultra-fast functional profiling of whole genome protein sequences

    Meinicke Peter

    2009-09-01

    Full Text Available Abstract Background Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Description Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. Conclusion For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address.

  20. Membrane-bound conformation of M13 major coat protein : a structure validation through FRET-derived constraints

    Vos, W.L.; Koehorst, R.B.M.; Spruijt, R.B.; Hemminga, M.A.

    2005-01-01

    M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein

  1. Human serum albumin as protecting agent of silver nanoparticles: role of the protein conformation and amine groups in the nanoparticle stabilization

    Alarcon, Emilio I.; Bueno-Alejo, Carlos J.; Noel, Christopher W.; Stamplecoskie, Kevin G. [Centre for Catalysis Research and Innovation, University of Ottawa, Department of Chemistry (Canada); Pacioni, Natalia L. [Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, INFIQC, Departamento de Quimica Organica (Argentina); Poblete, Horacio [Center for Bioinformatics and Molecular Simulations, Universidad de Talca (Chile); Scaiano, J. C., E-mail: tito@photo.chem.uottawa.ca [Centre for Catalysis Research and Innovation, University of Ottawa, Department of Chemistry (Canada)

    2013-01-15

    Thermally denatured human serum albumin interacts with {approx}3.0 nm spherical AgNP enhancing the fluorescence of Trp-214 at large protein/nanoparticle ratios. However, using native HSA, no changes in the emission were observed. The observation is likely due to differences between native and denatured protein packing resulting from protein corona formation. We have also found that NH{sub 2} blocking of the protein strongly affects the ability of the protein to protect AgNP from different salts/ions such as NaCl, PBS, Hank's buffer, Tris-HCl, MES, and DMEM. Additionally, AgNP can be readily prepared in aqueous solutions by a photochemical approach employing HSA as an in situ protecting agent. The role of the protein in this case is beyond that of protecting agent; thus, Ag{sup +} ions and I-2959 complexation within the protein structure also affects the efficiency of AgNP formation. Blocking NH{sub 2} in HSA modified the AgNP growth profile, surface plasmon band shape, and long-term stability suggesting that amine groups are directly involved in the formation and post-stabilization of AgNP. In particular, AgNP size and shape are extensively influenced by NH{sub 2} blocking, leading primarily to cubes and plates with sizes around 5-15 nm; in contrast, spherical monodisperse 4.0 nm AgNP are observed for native HSA. The nanoparticles prepared by this protocol are non-toxic in primary cells and have remarkable antibacterial properties. Finally, surface plasmon excitation of native HSA-AgNP promoted loss of protein conformation in just 5 min, suggesting that plasmon heating causes protein denaturation using continuous light sources such as commercial LED.

  2. Improving binding mode and binding affinity predictions of docking by ligand-based search of protein conformations: evaluation in D3R grand challenge 2015

    Xu, Xianjin; Yan, Chengfei; Zou, Xiaoqin

    2017-08-01

    The growing number of protein-ligand complex structures, particularly the structures of proteins co-bound with different ligands, in the Protein Data Bank helps us tackle two major challenges in molecular docking studies: the protein flexibility and the scoring function. Here, we introduced a systematic strategy by using the information embedded in the known protein-ligand complex structures to improve both binding mode and binding affinity predictions. Specifically, a ligand similarity calculation method was employed to search a receptor structure with a bound ligand sharing high similarity with the query ligand for the docking use. The strategy was applied to the two datasets (HSP90 and MAP4K4) in recent D3R Grand Challenge 2015. In addition, for the HSP90 dataset, a system-specific scoring function (ITScore2_hsp90) was generated by recalibrating our statistical potential-based scoring function (ITScore2) using the known protein-ligand complex structures and the statistical mechanics-based iterative method. For the HSP90 dataset, better performances were achieved for both binding mode and binding affinity predictions comparing with the original ITScore2 and with ensemble docking. For the MAP4K4 dataset, although there were only eight known protein-ligand complex structures, our docking strategy achieved a comparable performance with ensemble docking. Our method for receptor conformational selection and iterative method for the development of system-specific statistical potential-based scoring functions can be easily applied to other protein targets that have a number of protein-ligand complex structures available to improve predictions on binding.

  3. Human serum albumin as protecting agent of silver nanoparticles: role of the protein conformation and amine groups in the nanoparticle stabilization

    Alarcon, Emilio I.; Bueno-Alejo, Carlos J.; Noel, Christopher W.; Stamplecoskie, Kevin G.; Pacioni, Natalia L.; Poblete, Horacio; Scaiano, J. C.

    2013-01-01

    Thermally denatured human serum albumin interacts with ∼3.0 nm spherical AgNP enhancing the fluorescence of Trp-214 at large protein/nanoparticle ratios. However, using native HSA, no changes in the emission were observed. The observation is likely due to differences between native and denatured protein packing resulting from protein corona formation. We have also found that NH 2 blocking of the protein strongly affects the ability of the protein to protect AgNP from different salts/ions such as NaCl, PBS, Hank’s buffer, Tris–HCl, MES, and DMEM. Additionally, AgNP can be readily prepared in aqueous solutions by a photochemical approach employing HSA as an in situ protecting agent. The role of the protein in this case is beyond that of protecting agent; thus, Ag + ions and I-2959 complexation within the protein structure also affects the efficiency of AgNP formation. Blocking NH 2 in HSA modified the AgNP growth profile, surface plasmon band shape, and long-term stability suggesting that amine groups are directly involved in the formation and post-stabilization of AgNP. In particular, AgNP size and shape are extensively influenced by NH 2 blocking, leading primarily to cubes and plates with sizes around 5–15 nm; in contrast, spherical monodisperse 4.0 nm AgNP are observed for native HSA. The nanoparticles prepared by this protocol are non-toxic in primary cells and have remarkable antibacterial properties. Finally, surface plasmon excitation of native HSA-AgNP promoted loss of protein conformation in just 5 min, suggesting that plasmon heating causes protein denaturation using continuous light sources such as commercial LED.

  4. Conformational control of the binding of the transactivation domain of the MLL protein and c-Myb to the KIX domain of CREB.

    Elif Nihal Korkmaz

    Full Text Available The KIX domain of CBP is a transcriptional coactivator. Concomitant binding to the activation domain of proto-oncogene protein c-Myb and the transactivation domain of the trithorax group protein mixed lineage leukemia (MLL transcription factor lead to the biologically active ternary MLL∶KIX∶c-Myb complex which plays a role in Pol II-mediated transcription. The binding of the activation domain of MLL to KIX enhances c-Myb binding. Here we carried out molecular dynamics (MD simulations for the MLL∶KIX∶c-Myb ternary complex, its binary components and KIX with the goal of providing a mechanistic explanation for the experimental observations. The dynamic behavior revealed that the MLL binding site is allosterically coupled to the c-Myb binding site. MLL binding redistributes the conformational ensemble of KIX, leading to higher populations of states which favor c-Myb binding. The key element in the allosteric communication pathways is the KIX loop, which acts as a control mechanism to enhance subsequent binding events. We tested this conclusion by in silico mutations of loop residues in the KIX∶MLL complex and by comparing wild type and mutant dynamics through MD simulations. The loop assumed MLL binding conformation similar to that observed in the KIX∶c-Myb state which disfavors the allosteric network. The coupling with c-Myb binding site faded, abolishing the positive cooperativity observed in the presence of MLL. Our major conclusion is that by eliciting a loop-mediated allosteric switch between the different states following the binding events, transcriptional activation can be regulated. The KIX system presents an example how nature makes use of conformational control in higher level regulation of transcriptional activity and thus cellular events.

  5. Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis.

    Gao, Yansong; Zhou, Yulian; Goldstein, Joseph L; Brown, Michael S; Radhakrishnan, Arun

    2017-05-26

    Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases cholesterol synthesis. Previously, we provided evidence that cholesterol inhibition is initiated when cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of cholesterol synthesis. These insights suggest a new structural model for cholesterol-mediated regulation of Scap activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Conformal Infinity

    Frauendiener Jörg

    2000-08-01

    Full Text Available The notion of conformal infinity has a long history within the research in Einstein's theory of gravity. Today, ``conformal infinity'' is related with almost all other branches of research in general relativity, from quantisation procedures to abstract mathematical issues to numerical applications. This review article attempts to show how this concept gradually and inevitably evolved out of physical issues, namely the need to understand gravitational radiation and isolated systems within the theory of gravitation and how it lends itself very naturally to solve radiation problems in numerical relativity. The fundamental concept of null-infinity is introduced. Friedrich's regular conformal field equations are presented and various initial value problems for them are discussed. Finally, it is shown that the conformal field equations provide a very powerful method within numerical relativity to study global problems such as gravitational wave propagation and detection.

  7. Conformal Infinity.

    Frauendiener, Jörg

    2004-01-01

    The notion of conformal infinity has a long history within the research in Einstein's theory of gravity. Today, "conformal infinity" is related to almost all other branches of research in general relativity, from quantisation procedures to abstract mathematical issues to numerical applications. This review article attempts to show how this concept gradually and inevitably evolved from physical issues, namely the need to understand gravitational radiation and isolated systems within the theory of gravitation, and how it lends itself very naturally to the solution of radiation problems in numerical relativity. The fundamental concept of null-infinity is introduced. Friedrich's regular conformal field equations are presented and various initial value problems for them are discussed. Finally, it is shown that the conformal field equations provide a very powerful method within numerical relativity to study global problems such as gravitational wave propagation and detection.

  8. Conformal Infinity

    Frauendiener Jörg

    2004-01-01

    Full Text Available The notion of conformal infinity has a long history within the research in Einstein's theory of gravity. Today, 'conformal infinity' is related to almost all other branches of research in general relativity, from quantisation procedures to abstract mathematical issues to numerical applications. This review article attempts to show how this concept gradually and inevitably evolved from physical issues, namely the need to understand gravitational radiation and isolated systems within the theory of gravitation, and how it lends itself very naturally to the solution of radiation problems in numerical relativity. The fundamental concept of null-infinity is introduced. Friedrich's regular conformal field equations are presented and various initial value problems for them are discussed. Finally, it is shown that the conformal field equations provide a very powerful method within numerical relativity to study global problems such as gravitational wave propagation and detection.

  9. General Conformity

    The General Conformity requirements ensure that the actions taken by federal agencies in nonattainment and maintenance areas do not interfere with a state’s plans to meet national standards for air quality.

  10. Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders*

    Li, Min; Quan, Chao; Toth, Rachel; Campbell, David G.; MacKintosh, Carol; Wang, Hong Yu; Chen, Shuai

    2015-01-01

    Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser858 of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease. PMID:26499801

  11. Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders.

    Li, Min; Quan, Chao; Toth, Rachel; Campbell, David G; MacKintosh, Carol; Wang, Hong Yu; Chen, Shuai

    2015-12-11

    Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser(858) of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Conformal Infinity

    Frauendiener, J?rg

    2000-01-01

    The notion of conformal infinity has a long history within the research in Einstein's theory of gravity. Today, 'conformal infinity' is related to almost all other branches of research in general relativity, from quantisation procedures to abstract mathematical issues to numerical applications. This review article attempts to show how this concept gradually and inevitably evolved from physical issues, namely the need to understand gravitational radiation and isolated systems within the theory...

  13. Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design

    Niefind, Karsten; Issinger, Olaf-Georg

    2010-01-01

    well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2alpha structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2alpha structures. In the latter a surprising...... plasticity of important ATP-binding elements - the interdomain hinge region and the glycine-rich loop - was discovered. In fully active CK2alpha the hinge region is open and does not anchor the ATP ribose, but alternatively it can adopt a closed conformation, form hydrogen bonds to the ribose moiety and thus...

  14. Variability of the Cyclin-Dependent Kinase 2 Flexibility Without Significant Change in the Initial Conformation of the Protein or Its Environment; a Computational Study.

    Taghizadeh, Mohammad; Goliaei, Bahram; Madadkar-Sobhani, Armin

    2016-06-01

    Protein flexibility, which has been referred as a dynamic behavior has various roles in proteins' functions. Furthermore, for some developed tools in bioinformatics, such as protein-protein docking software, considering the protein flexibility, causes a higher degree of accuracy. Through undertaking the present work, we have accomplished the quantification plus analysis of the variations in the human Cyclin Dependent Kinase 2 (hCDK2) protein flexibility without affecting a significant change in its initial environment or the protein per se. The main goal of the present research was to calculate variations in the flexibility for each residue of the hCDK2, analysis of their flexibility variations through clustering, and to investigate the functional aspects of the residues with high flexibility variations. Using Gromacs package (version 4.5.4), three independent molecular dynamics (MD) simulations of the hCDK2 protein (PDB ID: 1HCL) was accomplished with no significant changes in their initial environments, structures, or conformations, followed by Root Mean Square Fluctuations (RMSF) calculation of these MD trajectories. The amount of variations in these three curves of RMSF was calculated using two formulas. More than 50% of the variation in the flexibility (the distance between the maximum and the minimum amount of the RMSF) was found at the region of Val-154. As well, there are other major flexibility fluctuations in other residues. These residues were mostly positioned in the vicinity of the functional residues. The subsequent works were done, as followed by clustering all hCDK2 residues into four groups considering the amount of their variability with respect to flexibility and their position in the RMSF curves. This work has introduced a new class of flexibility aspect of the proteins' residues. It could also help designing and engineering proteins, with introducing a new dynamic aspect of hCDK2, and accordingly, for the other similar globular proteins. In

  15. Concentration of total protein and degree of acidity (pH of saliva when fasting and after breakfasting

    Gemella Nur Illahi

    2016-04-01

    Full Text Available Background: While fasting, the mouth does not work to eat and drink so that the salivary glands become less active so saliva production decreased and there was a change in eating timewhich is relation to the mastication process that impact on changes in the degree of acidity (pH Objectives: To determine the concentration of total protein and the degree of acidity (pH of saliva when fasting and after breakfasting. Materials and Methods: The study was observational analytic design with longitudinal (follow up study conducted in the Hj. Halima Dg. Sikati Dental Hospital inKandea in July 2015, the sampling method was purposive sampling. Population was 35 clinical students at the Department of Dental Public Health, Faculty of Dentistry Hasanuddin University with a total sample of 16 students who fit the criteria of the study subjects. To calculate the total protein of saliva concentration using Kyltecautoanalyzerand pH meter to measure the acidity of saliva. Data was analyzed was using SPSS version 17.0 (paired t-test, p <0.05. Results: The mean of total protein (% while fasting by 0135% ± 0.026 and the mean total protein (% after breakfasting at 0.179% ± 0.035, while the average degree of acidity (pH during fasting at 7.26 ± 0:24 and the average degree of acidity (pH after breakfasting at 7.66 ± 0.23 with p-value (0.000. Conclusions: An increase in the total protein concentration and acidity (pH after breakfasting.

  16. Conformation radiotherapy and conformal radiotherapy

    Morita, Kozo

    1999-01-01

    In order to coincide the high dose region to the target volume, the 'Conformation Radiotherapy Technique' using the multileaf collimator and the device for 'hollow-out technique' was developed by Prof. S. Takahashi in 1960. This technique can be classified a type of 2D-dynamic conformal RT techniques. By the clinical application of this technique, the late complications of the lens, the intestine and the urinary bladder after radiotherapy for the maxillary cancer and the cervical cancer decreased. Since 1980's the exact position and shape of the tumor and the surrounding normal tissues can be easily obtained by the tremendous development of the CT/MRI imaging technique. As a result, various kinds of new conformal techniques such as the 3D-CRT, the dose intensity modulation, the tomotherapy have been developed since the beginning of 1990'. Several 'dose escalation study with 2D-/3D conformal RT' is now under way to improve the treatment results. (author)

  17. Conformal Gravity

    Hooft, G.

    2012-01-01

    The dynamical degree of freedom for the gravitational force is the metric tensor, having 10 locally independent degrees of freedom (of which 4 can be used to fix the coordinate choice). In conformal gravity, we split this field into an overall scalar factor and a nine-component remainder. All unrenormalizable infinities are in this remainder, while the scalar component can be handled like any other scalar field such as the Higgs field. In this formalism, conformal symmetry is spontaneously broken. An imperative demand on any healthy quantum gravity theory is that black holes should be described as quantum systems with micro-states as dictated by the Hawking-Bekenstein theory. This requires conformal symmetry that may be broken spontaneously but not explicitly, and this means that all conformal anomalies must cancel out. Cancellation of conformal anomalies yields constraints on the matter sector as described by some universal field theory. Thus black hole physics may eventually be of help in the construction of unified field theories. (author)

  18. Native Alanine Substitution in the Glycine Hinge Modulates Conformational Flexibility of Heme Nitric Oxide/Oxygen (H-NOX) Sensing Proteins.

    Hespen, Charles W; Bruegger, Joel J; Guo, Yirui; Marletta, Michael A

    2018-06-15

    Heme nitric oxide/oxygen sensing (H-NOX) domains are direct NO sensors that regulate a variety of biological functions in both bacteria and eukaryotes. Previous work on H-NOX proteins has shown that upon NO binding, a conformational change occurs along two glycine residues on adjacent helices (termed the glycine hinge). Despite the apparent importance of the glycine hinge, it is not fully conserved in all H-NOX domains. Several H-NOX sensors from the family Flavobacteriaceae contain a native alanine substitution in one of the hinge residues. In this work, the effect of the increased steric bulk within the Ala-Gly hinge on H-NOX function was investigated. The hinge in Kordia algicida OT-1 ( Ka H-NOX) is composed of A71 and G145. Ligand-binding properties and signaling function for this H-NOX were characterized. The variant A71G was designed to convert the hinge region of Ka H-NOX to the typical Gly-Gly motif. In activity assays with its cognate histidine kinase (HnoK), the wild type displayed increased signal specificity compared to A71G. Increasing titrations of unliganded A71G gradually inhibits HnoK autophosphorylation, while increasing titrations of unliganded wild type H-NOX does not inhibit HnoK. Crystal structures of both wild type and A71G Ka H-NOX were solved to 1.9 and 1.6 Å, respectively. Regions of H-NOX domains previously identified as involved in protein-protein interactions with HnoK display significantly higher b-factors in A71G compared to wild-type H-NOX. Both biochemical and structural data indicate that the hinge region controls overall conformational flexibility of the H-NOX, affecting NO complex formation and regulation of its HnoK.

  19. Two global conformation states of a novel NAD(P) reductase like protein of the thermogenic appendix of the Sauromatum guttatum inflorescence.

    Skubatz, Hanna; Howald, William N

    2013-06-01

    A novel NAD(P) reductase like protein (RL) belonging to a class of reductases involved in phenylpropanoid synthesis was previously purified to homogeneity from the Sauromatum guttatum appendix. The Sauromatum appendix raises its temperature above ambient temperature to ~30 °C on the day of inflorescence opening (D-day). Changes in the charge state distribution of the protein in electrospray ionization-mass spectrometry spectra were observed during the development of the appendix. RL adopted two conformations, state A (an extended state) that appeared before heat-production (D - 4 to D - 2), and state B (a compact state) that began appearing on D - 1 and reached a maximum on D-day. RL in healthy leaves of Arabidopsis is present in state A, whereas in thermogenic sporophylls of male cones of Encephalartos ferox is present in state B. These conformational changes strongly suggest an involvement of RL in heat-production. The biophysical properties of this protein are remarkable. It is self-assembled in aqueous solutions into micrometer sizes of organized morphologies. The assembly produces a broad range of cyclic and linear morphologies that resemble micelles, rods, lamellar micelles, as well as vesicles. The assemblies could also form network structures. RL molecules entangle with each other and formed branched, interconnected networks. These unusual assemblies suggest that RL is an oligomer, and its oligomerization can provide additional information needed for thermoregulation. We hypothesize that state A controls the plant basal temperature and state B allows a shift in the temperature set point to above ambient temperature.

  20. Conversion of functionally undefined homopentameric protein PbaA into a proteasome activator by mutational modification of its C-terminal segment conformation.

    Yagi-Utsumi, Maho; Sikdar, Arunima; Kozai, Toshiya; Inoue, Rintaro; Sugiyama, Masaaki; Uchihashi, Takayuki; Yagi, Hirokazu; Satoh, Tadashi; Kato, Koichi

    2018-01-01

    Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallographically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. SLITHER: a web server for generating contiguous conformations of substrate molecules entering into deep active sites of proteins or migrating through channels in membrane transporters.

    Lee, Po-Hsien; Kuo, Kuei-Ling; Chu, Pei-Ying; Liu, Eric M; Lin, Jung-Hsin

    2009-07-01

    Many proteins use a long channel to guide the substrate or ligand molecules into the well-defined active sites for catalytic reactions or for switching molecular states. In addition, substrates of membrane transporters can migrate to another side of cellular compartment by means of certain selective mechanisms. SLITHER (http://bioinfo.mc.ntu.edu.tw/slither/or http://slither.rcas.sinica.edu.tw/) is a web server that can generate contiguous conformations of a molecule along a curved tunnel inside a protein, and the binding free energy profile along the predicted channel pathway. SLITHER adopts an iterative docking scheme, which combines with a puddle-skimming procedure, i.e. repeatedly elevating the potential energies of the identified global minima, thereby determines the contiguous binding modes of substrates inside the protein. In contrast to some programs that are widely used to determine the geometric dimensions in the ion channels, SLITHER can be applied to predict whether a substrate molecule can crawl through an inner channel or a half-channel of proteins across surmountable energy barriers. Besides, SLITHER also provides the list of the pore-facing residues, which can be directly compared with many genetic diseases. Finally, the adjacent binding poses determined by SLITHER can also be used for fragment-based drug design.

  2. Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

    Knejzlik, Zdenek; Ulbrich, Pavel; Strohalm, Martin; Lastuvkova, Hana; Kodicek, Milan; Sakalian, Michael; Ruml, Tomas

    2009-01-01

    The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibers is linked to a conformational transition from unfolded or another non-periodical state to α-helix during multimerization.

  3. A randomized trial of protein supplementation compared with extra fast food on the effects of resistance training to increase metabolism.

    Hambre, David; Vergara, Marta; Lood, Yvonne; Bachrach-Lindström, Margareta; Lindström, Torbjörn; Nystrom, Fredrik H

    2012-10-01

    To prospectively evaluate the effects of resistance training combined with increased energy intake or protein-supplementation on lean body-mass, resting metabolic-rate (RMR) and cardiovascular risk factors. Twenty-four healthy males (aged 19-32 years) performed resistance exercise for 12 weeks aiming for at least 1 hour training-sessions 3 times a week. The participants were randomized to consume extra protein (33 g whey protein/day) or a meal of fast-food/day (1350 kcal, 41 g protein). Body-composition was measured with Dual-Energy X-ray Absorptiometry (DEXA) and RMR by indirect calorimetry. Fasting blood samples were drawn before and after the 3-month training period and after 12 months. The body weight increased from 75.1 ± 6.9 kg to 78.7 ± 7.2 kg (p < 0.0001), without differences between the groups. RMR increased from 1787 ± 143 kcal/24 h to 1954 ± 187 kcal/24 h (p < 0.0001, N = 24), which was more than expected from the increase in lean body-mass (increase from 59.7 ± 4.3 kg to 61.8 ± 4.1 kg p = 0.004). Fasting serum-insulin levels increased in the fast-food group compared with the extra-protein group (p = 0.03). ApoB increased from 0.691 ± 0.14 g/L to 0.768 ± 0.17 g/L, p = 0.004, in the fast-food group only. Long-term follow up after 12 months showed that RMR, body weight, total fat and lean body-masses did not differ from baseline (n = 19). Resistance training for 12 weeks increased RMR and lean body-mass similarly when based on either an increased energy-intake or protein supplement. However, the increase in RMR was higher than expected from the increase in lean body-mass. Thus resistance training could potentially decrease the risk of obesity by induction of increased RMR.

  4. Multiscale Modeling of the Active Site of [Fe] Hydrogenase: The H2 Binding Site in Open and Closed Protein Conformations

    Hedegård, Erik D.; Kongsted, Jacob; Ryde, Ulf

    2015-01-01

    A series of QM/MM optimizations of the full protein of [Fe] hydrogenase were performed. The FeGP cofactor has been optimized in the water-bound resting state (1), with a side-on bound dihydrogen (2), or as a hydride intermediate (3). For inclusion of H4MPT in the closed structure, advanced multis...... that hydride transfer from 3 has a significantly higher barrier than found in previous studies neglecting the full protein environment....

  5. Exploring the diameter and surface dependent conformational changes in carbon nanotube-protein corona and the related cytotoxicity

    Zhao, Xingchen; Lu, Dawei; Hao, Fang; Liu, Rutao

    2015-01-01

    Highlights: • CNT diameter and surface area govern the stability of adsorbed proteins. • More BSA was loaded and destabilized on smaller CNTs. • Protein corona reduces the cytotoxicity of CNTs - Abstract: In this work, we investigated and compared carbon nanotubes (CNTs) of different diameters regarding their interaction with bovine serum albumin (BSA) and their ability to alter protein structure. BSA was exposed to CNT solutions, and the effects were assessed by utilizing fluorescence spectroscopy, UV–vis absorption spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), bichinchoninic acid (BCA) and zeta-potential measurement assays. We demonstrate that CNT diameter and surface area play key roles in influencing the stability of adsorbed proteins. Results showed that the secondary and tertiary structural stability of BSA decreased upon adsorption onto CNTs, with greater decrease on smaller-diametered nanotubes. Besides, more protein was loaded onto CNTs with small diameter, reducing the cytotoxicity. This study, therefore, provides fundamental information for the influence of CNT diameter and surface on protein behavior, which may be helpful to understand toxic effects of CNTs and prove beneficial for developing novel biomedical devices and safe use of nanomaterials

  6. Interactions between protein molecules and the virus removal membrane surface: Effects of immunoglobulin G adsorption and conformational changes on filter performance.

    Hamamoto, Ryo; Ito, Hidemi; Hirohara, Makoto; Chang, Ryongsok; Hongo-Hirasaki, Tomoko; Hayashi, Tomohiro

    2018-03-01

    Membrane fouling commonly occurs in all filter types during virus filtration in protein-based biopharmaceutical manufacturing. Mechanisms of decline in virus filter performance due to membrane fouling were investigated using a cellulose-based virus filter as a model membrane. Filter performance was critically dependent on solution conditions; specifically, ionic strength. To understand the interaction between immunoglobulin G (IgG) and cellulose, sensors coated with cellulose were fabricated for surface plasmon resonance and quartz crystal microbalance with energy dissipation measurements. The primary cause of flux decline appeared to be irreversible IgG adsorption on the surface of the virus filter membrane. In particular, post-adsorption conformational changes in the IgG molecules promoted further irreversible IgG adsorption, a finding that could not be adequately explained by DLVO theory. Analyses of adsorption and desorption and conformational changes in IgG molecules on cellulose surfaces mimicking cellulose-based virus removal membranes provide an effective approach for identifying ways of optimizing solution conditions to maximize virus filter performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:379-386, 2018. © 2017 American Institute of Chemical Engineers.

  7. Fast computational methods for predicting protein structure from primary amino acid sequence

    Agarwal, Pratul Kumar [Knoxville, TN

    2011-07-19

    The present invention provides a method utilizing primary amino acid sequence of a protein, energy minimization, molecular dynamics and protein vibrational modes to predict three-dimensional structure of a protein. The present invention also determines possible intermediates in the protein folding pathway. The present invention has important applications to the design of novel drugs as well as protein engineering. The present invention predicts the three-dimensional structure of a protein independent of size of the protein, overcoming a significant limitation in the prior art.

  8. A single amino acid of human immunodeficiency virus type 2 capsid protein affects conformation of two external loops and viral sensitivity to TRIM5α.

    Tadashi Miyamoto

    Full Text Available We previously reported that human immunodeficiency virus type 2 (HIV-2 carrying alanine or glutamine but not proline at position 120 of the capsid protein (CA could grow in the presence of anti-viral factor TRIM5α of cynomolgus monkey (CM. To elucidate details of the interaction between the CA and TRIM5α, we generated mutant HIV-2 viruses, each carrying one of the remaining 17 possible amino acid residues, and examined their sensitivity to CM TRIM5α-mediated restriction. Results showed that hydrophobic residues or those with ring structures were associated with sensitivity, while those with small side chains or amide groups conferred resistance. Molecular dynamics simulation study revealed a structural basis for the differential TRIM5α sensitivities. The mutations at position 120 in the loop between helices 6 and 7 (L6/7 affected conformation of the neighboring loop between helices 4 and 5 (L4/5, and sensitive viruses had a common L4/5 conformation. In addition, the common L4/5 structures of the sensitive viruses were associated with a decreased probability of hydrogen bond formation between the 97th aspartic acid in L4/5 and the 119th arginine in L6/7. When we introduced aspartic acid-to-alanine substitution at position 97 (D97A of the resistant virus carrying glutamine at position 120 to disrupt hydrogen bond formation, the resultant virus became moderately sensitive. Interestingly, the virus carrying glutamic acid at position 120 showed resistance, while its predicted L4/5 conformation was similar to those of sensitive viruses. The D97A substitution failed to alter the resistance of this particular virus, indicating that the 120th amino acid residue itself is also involved in sensitivity regardless of the L4/5 conformation. These results suggested that a hydrogen bond between the L4/5 and L6/7 modulates the overall structure of the exposed surface of the CA, but the amino acid residue at position 120 is also directly involved in CM TRIM5

  9. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  10. Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion.

    Baral, Pravas Kumar; Swayampakula, Mridula; Aguzzi, Adriano; James, Michael N G

    2018-05-01

    Conversion of the cellular prion protein PrP C into its pathogenic isoform PrP S c is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti-prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis-folding events. The structural data of moPrP:POM6 Fab complex are available in the PDB under the accession number www.rcsb.org/pdb/search/structidSearch.do?structureId=6AQ7. © 2018 Federation of European Biochemical Societies.

  11. Biophysical characterization of the feline immunodeficiency virus p24 capsid protein conformation and in vitro capsid assembly.

    Jennifer Serrière

    Full Text Available The Feline Immunodeficiency Virus (FIV capsid protein p24 oligomerizes to form a closed capsid that protects the viral genome. Because of its crucial role in the virion, FIV p24 is an interesting target for the development of therapeutic strategies, although little is known about its structure and assembly. We defined and optimized a protocol to overexpress recombinant FIV capsid protein in a bacterial system. Circular dichroism and isothermal titration calorimetry experiments showed that the structure of the purified FIV p24 protein was comprised mainly of α-helices. Dynamic light scattering (DLS and cross-linking experiments demonstrated that p24 was monomeric at low concentration and dimeric at high concentration. We developed a protocol for the in vitro assembly of the FIV capsid. As with HIV, an increased ionic strength resulted in FIV p24 assembly in vitro. Assembly appeared to be dependent on temperature, salt concentration, and protein concentration. The FIV p24 assembly kinetics was monitored by DLS. A limit end-point diameter suggested assembly into objects of definite shapes. This was confirmed by electron microscopy, where FIV p24 assembled into spherical particles. Comparison of FIV p24 with other retroviral capsid proteins showed that FIV assembly is particular and requires further specific study.

  12. Conformational coupling between receptor and kinase binding sites through a conserved salt bridge in a signaling complex scaffold protein.

    Davi R Ortega

    Full Text Available Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD, NMR spectroscopy, and circular dichroism (CD, we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.

  13. Probing the interaction of ferrocene containing hyperbranched poly-ester with model plasma protein: Effect on the interaction mechanism and conformational change

    Xiao, Fengjuan, E-mail: xfj66@126.com; Gu, Muqing; Liang, Ye; Li, Lanlan; Yu, Xiaolei; Wu, Xiangfeng

    2014-05-01

    Interaction mechanism and conformational change of model plasma protein-bovine serum albumin (BSA) induced by ferrocenyl-functionalized hyperbranched polyester (HBPE-Fc) were investigated using cyclicvoltammetry (CV), differential pulsed voltammetry (DPV), fluorescence, UV–vis absorption spectrometry and circular dichroism (CD). Some complicated interactions occurred between BSA and HBPE-Fc and the new redox centers appeared in the BSA/HBPE-Fc complex that changed and hindered the electron transfer of Fe/Fe{sup 2+}. Fluorescence quenching data showed that the fluorescence of BSA was statically quenched by HBPE-Fc, which implied that ground state complex formed between BSA and HBPE-Fc. van der Waals force and hydrogen bond played major roles in the interaction of HBPE-Fc with BSA. The binding constant Ka for HBPE-Fc–protein interaction is in the order of 10{sup 6} at room temperature indicates that there is a strong interaction between HBPE-Fc and BSA. Synchronous, three-dimensional fluorescence and CD studies indicated that the interaction of BSA with HBPE-Fc induced conformational changes in BSA with overall decrease in the α-helical structure and increase in β-pleated sheet structure. The molecular model of the interaction between HBPE-Fc and BSA was also presented according to the results in this study. - Highlights: • A novel ferrocenyl-functionalized hyperbranched polymer (HBPE-Fc) with potential anticancer effects. • New redox centers appear in the BSA/HBPE-Fc complex that changed and hindered the electron transfer of Fe/Fe{sup 2+}. • BSA fluorescence was statically quenched by HBPE-Fc. • BSA/HBPE-Fc ground state complex was mainly formed by the hydrogen bonds and van der Waals force. • HBPE-Fc induced conformational changes in BSA with overall decrease in the α-helical structure and increase in β-pleated sheet structure. • The molecular model of the interaction was presented according to the results in this study.

  14. Investigating the microstructure of keratin extracted from wool: peptide sequence (MALDI-TOF/TOF) and protein conformation (FTIR)

    Keratin was extracted from wool by reduction with 2-mercaptoethanol. It was isolated as intact keratin and characterized by its similar molecular weight, protein composition, and secondary structure to native keratin. Gel electrophoresis patterns and MALDI-TOF/TOF peptide sequences provided the ide...

  15. Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry

    Houde, Damian; Esmail Nazari, Zeinab; Bou-Assaf, George M

    2016-01-01

    When highly concentrated, an antibody solution can exhibit unusual behaviors, which can lead to unwanted properties, such as increased levels of protein aggregation and unusually high viscosity. Molecular modeling, along with many indirect biophysical measurements, has suggested that the cause fo...... in industry. Graphical Abstract ᅟ....

  16. Fast electron transfer through a single molecule natively structured redox protein

    Della Pia, Eduardo Antonio; Chi, Qijin; Macdonald, J. Emyr

    2012-01-01

    The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conduc...

  17. Lanthanide paramagnetic probes for NMR spectroscopic studies of fast molecular conformational dynamics and temperature control. Effective six-site proton exchange in 18-crown-6 by exchange spectroscopy.

    Babailov, Sergey P

    2012-02-06

    (1)H and (13)C NMR measurements are reported for the CDCl(3) and CD(2)Cl(2) solutions of [La(18-crown-6)(NO(3))(3)] (I), [Pr(18-crown-6) (NO(3))(3)] (II), [Ce(18-crown-6)(NO(3))(3)] (III), and [Nd(18-crown-6)(NO(3))(3)] (IV) complexes. Temperature dependencies of the (1)H NMR spectra of paramagnetic II-IV have been analyzed using the dynamic NMR (DNMR) methods for six-site exchange. Two types of conformational dynamic processes were identified (the first one is conditioned by interconversion of complex enantiomeric forms and pseudorotation of a macrocycle molecule upon the C(2) symmetry axis; the second one is conditioned by macrocycle molecule inversion). Application of exchange spectroscopy (2D-EXSY) of DNMR for investigation of this dynamic system (II-IV) simplifies the assignment of the NMR signals and represents the first experimental study of multisite exchange. In the present work, the methodology of paramagnetic 4f (Ce, Pr, and Nd) probe applications for the study of free-energy, enthalpy, and entropy changes in chemical exchange processes, as well as the advantages of this method in a comparison with DNMR studies of diamagnetic substances, is discussed. In particular, as a result of paramagnetic chemical shifts in 4f complexes, the range of measurable rate constants expands considerably compared to the analogous range in diamagnetic compounds. Coordination compounds investigated in the paper represent new types of thermometric NMR sensors and lanthanide paramagnetic probes for in situ temperature control in solution.

  18. Protein biomarker discovery and fast monitoring for the identification and detection of Anisakids by parallel reaction monitoring (PRM) mass spectrometry.

    Carrera, Mónica; Gallardo, José M; Pascual, Santiago; González, Ángel F; Medina, Isabel

    2016-06-16

    Anisakids are fish-borne parasites that are responsible for a large number of human infections and allergic reactions around the world. World health organizations and food safety authorities aim to control and prevent this emerging health problem. In the present work, a new method for the fast monitoring of these parasites is described. The strategy is divided in three steps: (i) purification of thermostable proteins from fish-borne parasites (Anisakids), (ii) in-solution HIFU trypsin digestion and (iii) monitoring of several peptide markers by parallel reaction monitoring (PRM) mass spectrometry. This methodology allows the fast detection of Anisakids in Biomarker Discovery and the Fast Monitoring for the identification and detection of Anisakids in fishery products. The strategy is based on the purification of thermostable proteins, the use of accelerated in-solution trypsin digestions under an ultrasonic field provided by High-Intensity Focused Ultrasound (HIFU) and the monitoring of several peptide biomarkers by Parallel Reaction Monitoring (PRM) Mass Spectrometry in a linear ion trap mass spectrometer. The workflow allows the unequivocal detection of Anisakids, in <2h. The present strategy constitutes the fastest method for Anisakids detection, whose application in the food quality control area, could provide to the authorities an effective and rapid method to guarantee the safety to the consumers. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster

    Katja E. Menger

    2015-06-01

    Full Text Available Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT, to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster.

  20. Surface-water interface induces conformational changes critical for protein adsorption: Implications for monolayer formation of EAS hydrophobin

    Kamron eLey

    2015-11-01

    Full Text Available The class I hydrophobin EAS is part of a family of small, amphiphilic fungal proteins best known for their ability to self-assemble into stable monolayers that modify the hydrophobicity of a surface to facilitate further microbial growth. These proteins have attracted increasing attention for industrial and biomedical applications, with the aim of designing surfaces that have the potential to maintain their clean state by resisting non-specific protein binding. To gain a better understanding of this process, we have employed all-atom molecular dynamics to study initial stages of the spontaneous adsorption of monomeric EAS hydrophobin on fully hydroxylated silica, a commonly used industrial and biomedical substrate. Particular interest has been paid to the Cys3-Cys4 loop, which has been shown to exhibit disruptive behavior in solution, and the Cys7-Cys8 loop, which is believed to be involved in the aggregation of EAS hydrophobin at interfaces. Specific and water mediated interactions with the surface were also analyzed. We have identified two possible binding motifs, one which allows unfolding of the Cys7-Cys8 loop due to the surfactant-like behavior of the Cys3-Cys4 loop, and another which has limited unfolding due to the Cys3-Cys4 loop remaining disordered in solution. We have also identified intermittent interactions with water which mediate the protein adsorption to the surface, as well as longer lasting interactions which control the diffusion of water around the adsorption site. These results have shown that EAS behaves in a similar way at the air-water and surface-water interfaces, and have also highlighted the need for hydrophilic ligand functionalization of the silica surface in order to prevent the adsorption of EAS hydrophobin.

  1. A radioimmunoassay to screen for antibodies to native conformational antigens and analyse ligand-induced structural states of antigenic proteins

    Bernotat-Danielowski, S.; Koepsell, H.

    1988-01-01

    A radioimmunoassay is described in which antigenic protein was immobilized by incubating nitrocellulose filters of defined diameter with antigen-containing solutions. Antigenic sites which are sensitive to protein denaturation by drying could be detected with the assay. The assay was also used to screen hybridoma supernatants for antibodies directed against Na + cotransport proteins from renal brush-border membranes. Monoclonal antibodies were selected which showed different binding charactertics depending on whether or not substrates of Na + cotransporters were present. One of the antibodies, which showed different antibody binding after addition of D-glucose or L-lactate, bound to a polypeptide component of the renal N + -D-glucose cotransporter and was able to inhibit Na + gradient-dependent. To investigate the effects of D-glucose and L-lactate on the binding of this antibody concentration dependence was measured. High and low affinity binding sites for D-glucose and L-lactate were characterized thereby demonstrating that the radioimmunoassay permits investigations of the properties of high and low affinity substrate binding sites. (author). refs.; 6 figs.; 2 tabs

  2. Conformality lost

    Kaplan, David B.; Lee, Jong-Wan; Son, Dam T.; Stephanov, Mikhail A.

    2009-01-01

    We consider zero-temperature transitions from conformal to nonconformal phases in quantum theories. We argue that there are three generic mechanisms for the loss of conformality in any number of dimensions: (i) fixed point goes to zero coupling, (ii) fixed point runs off to infinite coupling, or (iii) an IR fixed point annihilates with a UV fixed point and they both disappear into the complex plane. We give both relativistic and nonrelativistic examples of the last case in various dimensions and show that the critical behavior of the mass gap behaves similarly to the correlation length in the finite temperature Berezinskii-Kosterlitz-Thouless (BKT) phase transition in two dimensions, ξ∼exp(c/|T-T c | 1/2 ). We speculate that the chiral phase transition in QCD at large number of fermion flavors belongs to this universality class, and attempt to identify the UV fixed point that annihilates with the Banks-Zaks fixed point at the lower end of the conformal window.

  3. Space-related pharma-motifs for fast search of protein binding motifs and polypharmacological targets.

    Chiu, Yi-Yuan; Lin, Chun-Yu; Lin, Chih-Ta; Hsu, Kai-Cheng; Chang, Li-Zen; Yang, Jinn-Moon

    2012-01-01

    To discover a compound inhibiting multiple proteins (i.e. polypharmacological targets) is a new paradigm for the complex diseases (e.g. cancers and diabetes). In general, the polypharmacological proteins often share similar local binding environments and motifs. As the exponential growth of the number of protein structures, to find the similar structural binding motifs (pharma-motifs) is an emergency task for drug discovery (e.g. side effects and new uses for old drugs) and protein functions. We have developed a Space-Related Pharmamotifs (called SRPmotif) method to recognize the binding motifs by searching against protein structure database. SRPmotif is able to recognize conserved binding environments containing spatially discontinuous pharma-motifs which are often short conserved peptides with specific physico-chemical properties for protein functions. Among 356 pharma-motifs, 56.5% interacting residues are highly conserved. Experimental results indicate that 81.1% and 92.7% polypharmacological targets of each protein-ligand complex are annotated with same biological process (BP) and molecular function (MF) terms, respectively, based on Gene Ontology (GO). Our experimental results show that the identified pharma-motifs often consist of key residues in functional (active) sites and play the key roles for protein functions. The SRPmotif is available at http://gemdock.life.nctu.edu.tw/SRP/. SRPmotif is able to identify similar pharma-interfaces and pharma-motifs sharing similar binding environments for polypharmacological targets by rapidly searching against the protein structure database. Pharma-motifs describe the conservations of binding environments for drug discovery and protein functions. Additionally, these pharma-motifs provide the clues for discovering new sequence-based motifs to predict protein functions from protein sequence databases. We believe that SRPmotif is useful for elucidating protein functions and drug discovery.

  4. Study of conformational changes and protein aggregation of bovine serum albumin in presence of Sb(III) and Sb(V).

    Verdugo, Marcelo; Ruiz Encinar, Jorge; Costa-Fernández, José Manuel; Menendez-Miranda, Mario; Bouzas-Ramos, Diego; Bravo, Manuel; Quiroz, Waldo

    2017-01-01

    Antimony is a metalloid that affects biological functions in humans due to a mechanism still not understood. There is no doubt that the toxicity and physicochemical properties of Sb are strongly related with its chemical state. In this paper, the interaction between Sb(III) and Sb(V) with bovine serum albumin (BSA) was investigated in vitro by fluorescence spectroscopy, and circular dichroism (CD) under simulated physiological conditions. Moreover, the coupling of the separation technique, asymmetric flow field-flow fractionation, with elemental mass spectrometry to understand the interaction of Sb(V) and Sb(III) with the BSA was also used. Our results showed a different behaviour of Sb(III) vs. Sb(V) regarding their effects on the interaction with the BSA. The effects in terms of protein aggregates and conformational changes were higher in the presence of Sb(III) compared to Sb(V) which may explain the differences in toxicity between both Sb species in vivo. Obtained results demonstrated the protective effect of GSH that modifies the degree of interaction between the Sb species with BSA. Interestingly, in our experiments it was possible to detect an interaction between BSA and Sb species, which may be related with the presence of labile complex between the Sb and a protein for the first time.

  5. Biologically relevant conformational features of linear and cyclic proteolipid protein (PLP) peptide analogues obtained by high-resolution nuclear magnetic resonance and molecular dynamics

    Kordopati, Golfo G.; Tzoupis, Haralambos; Troganis, Anastassios N.; Tsivgoulis, Gerasimos M.; Golic Grdadolnik, Simona; Simal, Carmen; Tselios, Theodore V.

    2017-09-01

    Proteolipid protein (PLP) is one of the main proteins of myelin sheath that are destroyed during the progress of multiple sclerosis (MS). The immunodominant PLP139-151 epitope is known to induce experimental autoimmune encephalomyelitis (EAE, animal model of MS), wherein residues 144 and 147 are recognized by T cell receptor (TCR) during the formation of trimolecular complex with peptide-antigen and major histocompability complex. The conformational behavior of linear and cyclic peptide analogues of PLP, namely PLP139-151 and cyclic (139-151) (L144, R147) PLP139-151, have been studied in solution by means of nuclear magnetic resonance (NMR) methods in combination with unrestrained molecular dynamics simulations. The results indicate that the side chains of mutated amino acids in the cyclic analogue have different spatial orientation compared with the corresponding side chains of the linear analogue, which can lead to reduced affinity to TCR. NMR experiments combined with theoretical calculations pave the way for the design and synthesis of potent restricted peptides of immunodominant PLP139-151 epitope as well as non peptide mimetics that rises as an ultimate goal.

  6. Combined Spectroscopic and Calorimetric Studies to Reveal Absorption Mechanisms and Conformational Changes of Protein on Nanoporous Biomaterials

    Saharnaz Ahmadi

    2015-07-01

    Full Text Available In this study the effect of surface modification of mesoporous silica nanoparticles (MSNs on its adsorption capacities and protein stability after immobilization of beta-lactoglobulin B (BLG-B was investigated. For this purpose, non-functionalized (KIT-6 and aminopropyl-functionalized cubic Ia3d mesoporous silica ([n-PrNH2-KIT-6] nanoparticles were used as nanoporous supports. Aminopropyl-functionalized mesoporous nanoparticles exhibited more potential candidates for BLG-B adsorption and minimum BLG leaching than non-functionalized nanoparticles. It was observed that the amount of adsorbed BLG is dependent on the initial BLG concentration for both KIT-6 and [n-PrNH2-KIT-6] mesoporous nanoparticles. Also larger amounts of BLG-B on KIT-6 was immobilized upon raising the temperature of the medium from 4 to 55 °C while such increase was undetectable in the case of immobilization of BLG-B on the [n-PrNH2-KIT-6]. At temperatures above 55 °C the amounts of adsorbed BLG on both studied nanomaterials decreased significantly. By Differential scanning calorimetry or DSC analysis the heterogeneity of the protein solution and increase in Tm may indicate that immobilization of BLG-B onto the modified KIT-6 results in higher thermal stability compared to unmodified one. The obtained results provide several crucial factors in determining the mechanism(s of protein adsorption and stability on the nanostructured solid supports and the development of engineered nano-biomaterials for controlled drug-delivery systems and biomimetic interfaces for the immobilization of living cells.

  7. Conformationally selective multidimensional chemical shift ranges in proteins from a PACSY database purged using intrinsic quality criteria

    Fritzsching, Keith J.; Hong, Mei; Schmidt-Rohr, Klaus

    2016-01-01

    We have determined refined multidimensional chemical shift ranges for intra-residue correlations ( 13 C– 13 C, 15 N– 13 C, etc.) in proteins, which can be used to gain type-assignment and/or secondary-structure information from experimental NMR spectra. The chemical-shift ranges are the result of a statistical analysis of the PACSY database of >3000 proteins with 3D structures (1,200,207 13 C chemical shifts and >3 million chemical shifts in total); these data were originally derived from the Biological Magnetic Resonance Data Bank. Using relatively simple non-parametric statistics to find peak maxima in the distributions of helix, sheet, coil and turn chemical shifts, and without the use of limited “hand-picked” data sets, we show that ∼94 % of the 13 C NMR data and almost all 15 N data are quite accurately referenced and assigned, with smaller standard deviations (0.2 and 0.8 ppm, respectively) than recognized previously. On the other hand, approximately 6 % of the 13 C chemical shift data in the PACSY database are shown to be clearly misreferenced, mostly by ca. −2.4 ppm. The removal of the misreferenced data and other outliers by this purging by intrinsic quality criteria (PIQC) allows for reliable identification of secondary maxima in the two-dimensional chemical-shift distributions already pre-separated by secondary structure. We demonstrate that some of these correspond to specific regions in the Ramachandran plot, including left-handed helix dihedral angles, reflect unusual hydrogen bonding, or are due to the influence of a following proline residue. With appropriate smoothing, significantly more tightly defined chemical shift ranges are obtained for each amino acid type in the different secondary structures. These chemical shift ranges, which may be defined at any statistical threshold, can be used for amino-acid type assignment and secondary-structure analysis of chemical shifts from intra-residue cross peaks by inspection or by using a

  8. Conformationally selective multidimensional chemical shift ranges in proteins from a PACSY database purged using intrinsic quality criteria

    Fritzsching, Keith J., E-mail: kfritzsc@brandeis.edu [Brandeis University, Department of Chemistry (United States); Hong, Mei [Massachusetts Institute of Technology, Department of Chemistry (United States); Schmidt-Rohr, Klaus, E-mail: srohr@brandeis.edu [Brandeis University, Department of Chemistry (United States)

    2016-02-15

    We have determined refined multidimensional chemical shift ranges for intra-residue correlations ({sup 13}C–{sup 13}C, {sup 15}N–{sup 13}C, etc.) in proteins, which can be used to gain type-assignment and/or secondary-structure information from experimental NMR spectra. The chemical-shift ranges are the result of a statistical analysis of the PACSY database of >3000 proteins with 3D structures (1,200,207 {sup 13}C chemical shifts and >3 million chemical shifts in total); these data were originally derived from the Biological Magnetic Resonance Data Bank. Using relatively simple non-parametric statistics to find peak maxima in the distributions of helix, sheet, coil and turn chemical shifts, and without the use of limited “hand-picked” data sets, we show that ∼94 % of the {sup 13}C NMR data and almost all {sup 15}N data are quite accurately referenced and assigned, with smaller standard deviations (0.2 and 0.8 ppm, respectively) than recognized previously. On the other hand, approximately 6 % of the {sup 13}C chemical shift data in the PACSY database are shown to be clearly misreferenced, mostly by ca. −2.4 ppm. The removal of the misreferenced data and other outliers by this purging by intrinsic quality criteria (PIQC) allows for reliable identification of secondary maxima in the two-dimensional chemical-shift distributions already pre-separated by secondary structure. We demonstrate that some of these correspond to specific regions in the Ramachandran plot, including left-handed helix dihedral angles, reflect unusual hydrogen bonding, or are due to the influence of a following proline residue. With appropriate smoothing, significantly more tightly defined chemical shift ranges are obtained for each amino acid type in the different secondary structures. These chemical shift ranges, which may be defined at any statistical threshold, can be used for amino-acid type assignment and secondary-structure analysis of chemical shifts from intra

  9. Effects of Supplementation of Branched-Chain Amino Acids to Reduced-Protein Diet on Skeletal Muscle Protein Synthesis and Degradation in the Fed and Fasted States in a Piglet Model

    Liufeng Zheng

    2016-12-01

    Full Text Available Supplementation of branched-chain amino acids (BCAA has been demonstrated to promote skeletal muscle mass gain, but the mechanisms underlying this observation are still unknown. Since the regulation of muscle mass depends on a dynamic equilibrium (fasted losses–fed gains in protein turnover, the aim of this study was to investigate the effects of BCAA supplementation on muscle protein synthesis and degradation in fed/fasted states and the related mechanisms. Fourteen 26- (Experiment 1 and 28-day-old (Experiment 2 piglets were fed reduced-protein diets without or with supplemental BCAA. After a four-week acclimation period, skeletal muscle mass and components of anabolic and catabolic signaling in muscle samples after overnight fasting were determined in Experiment 1. Pigs in Experiment 2 were implanted with carotid arterial, jugular venous, femoral arterial and venous catheters, and fed once hourly along with the intravenous infusion of NaH13CO3 for 2 h, followed by a 6-h infusion of [1-13C]leucine. Muscle leucine kinetics were measured using arteriovenous difference technique. The mass of most muscles was increased by BCAA supplementation. During feeding, BCAA supplementation increased leucine uptake, protein synthesis, protein degradation and net transamination. The greater increase in protein synthesis than in protein degradation resulted in elevated protein deposition. Protein synthesis was strongly and positively correlated with the intramuscular net production of α-ketoisocaproate (KIC and protein degradation. Moreover, BCAA supplementation enhanced the fasted-state phosphorylation of protein translation initiation factors and inhibited the protein-degradation signaling of ubiquitin-proteasome and autophagy-lysosome systems. In conclusion, supplementation of BCAA to reduced-protein diet increases fed-state protein synthesis and inhibits fasted-state protein degradation, both of which could contribute to the elevation of skeletal muscle

  10. Intermittent fasting does not affect whole-body glucose, lipid, or protein metabolism

    Soeters, Maarten R.; Lammers, Nicolette M.; Dubbelhuis, Peter F.; Ackermans, Mariëtte T.; Jonkers-Schuitema, Cora F.; Fliers, Eric; Sauerwein, Hans P.; Aerts, Johannes M.; Serlie, Mireille J.

    2009-01-01

    Background: Intermittent fasting (IF) was shown to increase whole-body insulin sensitivity, but it is uncertain whether IF selectively influences intermediary metabolism. Such selectivity might be advantageous when adapting to periods of food abundance and food shortage. Objective: The objective was

  11. Anabolic effects of leucine-rich whey protein, carbohydrate, and soy protein with and without β-hydroxy-β-methylbutyrate (HMB) during fasting-induced catabolism: A human randomized crossover trial.

    Rittig, Nikolaj; Bach, Ermina; Thomsen, Henrik H; Møller, Andreas B; Hansen, Jakob; Johannsen, Mogens; Jensen, Erik; Serena, Anja; Jørgensen, Jens O; Richelsen, Bjørn; Jessen, Niels; Møller, Niels

    2017-06-01

    Protein-rich beverages are widely used clinically to preserve muscle protein and improve physical performance. Beverages with high contents of leucine or its keto-metabolite β-hydroxy-β-methylbutyrate (HMB) are especially anabolic in muscle, but it is uncertain whether this also applies to catabolic conditions such as fasting and whether common or separate intracellular signaling cascades are involved. To compare a specific leucine-rich whey protein beverage (LWH) with isocaloric carbohydrate- (CHO), soy protein (SOY), and soy protein +3 g HMB (HMB) during fasting-induced catabolic conditions. Eight healthy lean male subjects underwent four interventions (LWH, CHO, SOY, and HMB) using a randomized crossover design. Each trial included a 36 h fast and consisted of a 3 h basal fasting period and a 4 h 'sipping' period. Forearm net balances of phenylalanine (NB phe , measure of net protein loss) improved for all groups (p HMB compared with SOY (p HMB have superior anabolic effects on muscle protein kinetics after 36 h of fasting, and LWH distinctly activates the mTOR pathway. These novel findings suggest that leucine-rich whey protein and/or HMB are specifically beneficial during fasting-induced catabolic conditions. Copyright © 2016 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  12. A compact, multifunctional fusion module directs cholesterol-dependent homomultimerization and syncytiogenic efficiency of reovirus p10 FAST proteins.

    Tim Key

    2014-03-01

    Full Text Available The homologous p10 fusion-associated small transmembrane (FAST proteins of the avian (ARV and Nelson Bay (NBV reoviruses are the smallest known viral membrane fusion proteins, and are virulence determinants of the fusogenic reoviruses. The small size of FAST proteins is incompatible with the paradigmatic membrane fusion pathway proposed for enveloped viral fusion proteins. Understanding how these diminutive viral fusogens mediate the complex process of membrane fusion is therefore of considerable interest, from both the pathogenesis and mechanism-of-action perspectives. Using chimeric ARV/NBV p10 constructs, the 36-40-residue ectodomain was identified as the major determinant of the differing fusion efficiencies of these homologous p10 proteins. Extensive mutagenic analysis determined the ectodomain comprises two distinct, essential functional motifs. Syncytiogenesis assays, thiol-specific surface biotinylation, and liposome lipid mixing assays identified an ∼25-residue, N-terminal motif that dictates formation of a cystine loop fusion peptide in both ARV and NBV p10. Surface immunofluorescence staining, FRET analysis and cholesterol depletion/repletion studies determined the cystine loop motif is connected through a two-residue linker to a 13-residue membrane-proximal ectodomain region (MPER. The MPER constitutes a second, independent motif governing reversible, cholesterol-dependent assembly of p10 multimers in the plasma membrane. Results further indicate that: (1 ARV and NBV homomultimers segregate to distinct, cholesterol-dependent microdomains in the plasma membrane; (2 p10 homomultimerization and cholesterol-dependent microdomain localization are co-dependent; and (3 the four juxtamembrane MPER residues present in the multimerization motif dictate species-specific microdomain association and homomultimerization. The p10 ectodomain therefore constitutes a remarkably compact, multifunctional fusion module that directs syncytiogenic

  13. Quantum efficiency and two-photon absorption cross-section of conjugated polyelectrolytes used for protein conformation measurements with applications on amyloid structures

    Stabo-Eeg, Frantz [Norwegian University of Science and Technology, N-7491 Trondheim (Norway)], E-mail: Frantz.Stabo-Eeg@phys.ntnu.no; Lindgren, Mikael [Norwegian University of Science and Technology, N-7491 Trondheim (Norway); Nilsson, K. Peter R.; Inganaes, Olle; Hammarstroem, Per [IFM Department of Physics, Chemistry and Biology Linkoeping University, S-581 83 Linkoeping (Sweden)

    2007-07-27

    Amyloid diseases such as Alzheimer's and spongiform encephalopathies evolve from aggregation of proteins due to misfolding of the protein structure. Early disease handling require sophisticated but yet simple techniques to follow the complex properties of the aggregation process. Conjugated polyelectrolytes (CPEs) have shown promising capabilities acting as optical biological sensors, since they can specifically bind to polypeptides both in solution and in solid phase. The structural changes in biomolecules can be monitored by changes of the optical spectra of the CPEs, both in absorption and emission modes. Notably, the studied CPEs possess multi-photon excitation capability, making them potential for in vivo imaging using laser scanning microscopy. Aggregation of proteins depends on concentration, temperature and pH. The optical effect on the molecular probe in various environments must also be investigated if applied in these environments. Here we present the results of quantum efficiency and two-photon absorption cross-section of three CPEs: POMT, POWT and PTAA in three different pH buffer systems. The extinction coefficient and quantum efficiency were measured. POMT was found to have the highest quantum efficiency being approximately 0.10 at pH 2.0. The two-photon absorption cross-section was measured for POMT and POWT and was found to be more than 18-25 times and 7-11 times that of Fluorescein, respectively. We also show how POMT fluorescence can be used to distinguish conformational differences between amyloid fibrils formed from reduced and non-reduced insulin in spectrally resolved images recorded with a laser scanning microscope using both one- and two-photon excitation.

  14. Ligand Binding Induces Conformational Changes in Human Cellular Retinol-binding Protein 1 (CRBP1) Revealed by Atomic Resolution Crystal Structures.

    Silvaroli, Josie A; Arne, Jason M; Chelstowska, Sylwia; Kiser, Philip D; Banerjee, Surajit; Golczak, Marcin

    2016-04-15

    Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP1 remain unclear. Here we report the first structure of CRBP1 in a ligand-free form as well as ultra-high resolution structures of this protein bound to either all-trans-retinol or retinylamine, the latter a therapeutic retinoid that prevents light-induced retinal degeneration. Superpositioning of human apo- and holo-CRBP1 revealed major differences within segments surrounding the entrance to the retinoid-binding site. These included α-helix II and hairpin turns between β-strands βC-βD and βE-βF as well as several side chains, such as Phe-57, Tyr-60, and Ile-77, that change their orientations to accommodate the ligand. Additionally, we mapped hydrogen bond networks inside the retinoid-binding cavity and demonstrated their significance for the ligand affinity. Analyses of the crystallographic B-factors indicated several regions with higher backbone mobility in the apoprotein that became more rigid upon retinoid binding. This conformational flexibility of human apo-CRBP1 facilitates interaction with the ligands, whereas the more rigid holoprotein structure protects the labile retinoid moiety during vitamin A transport. These findings suggest a mechanism of induced fit upon ligand binding by mammalian cellular retinol-binding proteins. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Fast and accurate non-sequential protein structure alignment using a new asymmetric linear sum assignment heuristic.

    Brown, Peter; Pullan, Wayne; Yang, Yuedong; Zhou, Yaoqi

    2016-02-01

    The three dimensional tertiary structure of a protein at near atomic level resolution provides insight alluding to its function and evolution. As protein structure decides its functionality, similarity in structure usually implies similarity in function. As such, structure alignment techniques are often useful in the classifications of protein function. Given the rapidly growing rate of new, experimentally determined structures being made available from repositories such as the Protein Data Bank, fast and accurate computational structure comparison tools are required. This paper presents SPalignNS, a non-sequential protein structure alignment tool using a novel asymmetrical greedy search technique. The performance of SPalignNS was evaluated against existing sequential and non-sequential structure alignment methods by performing trials with commonly used datasets. These benchmark datasets used to gauge alignment accuracy include (i) 9538 pairwise alignments implied by the HOMSTRAD database of homologous proteins; (ii) a subset of 64 difficult alignments from set (i) that have low structure similarity; (iii) 199 pairwise alignments of proteins with similar structure but different topology; and (iv) a subset of 20 pairwise alignments from the RIPC set. SPalignNS is shown to achieve greater alignment accuracy (lower or comparable root-mean squared distance with increased structure overlap coverage) for all datasets, and the highest agreement with reference alignments from the challenging dataset (iv) above, when compared with both sequentially constrained alignments and other non-sequential alignments. SPalignNS was implemented in C++. The source code, binary executable, and a web server version is freely available at: http://sparks-lab.org yaoqi.zhou@griffith.edu.au. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. Benchmarking Commercial Conformer Ensemble Generators.

    Friedrich, Nils-Ole; de Bruyn Kops, Christina; Flachsenberg, Florian; Sommer, Kai; Rarey, Matthias; Kirchmair, Johannes

    2017-11-27

    We assess and compare the performance of eight commercial conformer ensemble generators (ConfGen, ConfGenX, cxcalc, iCon, MOE LowModeMD, MOE Stochastic, MOE Conformation Import, and OMEGA) and one leading free algorithm, the distance geometry algorithm implemented in RDKit. The comparative study is based on a new version of the Platinum Diverse Dataset, a high-quality benchmarking dataset of 2859 protein-bound ligand conformations extracted from the PDB. Differences in the performance of commercial algorithms are much smaller than those observed for free algorithms in our previous study (J. Chem. Inf. 2017, 57, 529-539). For commercial algorithms, the median minimum root-mean-square deviations measured between protein-bound ligand conformations and ensembles of a maximum of 250 conformers are between 0.46 and 0.61 Å. Commercial conformer ensemble generators are characterized by their high robustness, with at least 99% of all input molecules successfully processed and few or even no substantial geometrical errors detectable in their output conformations. The RDKit distance geometry algorithm (with minimization enabled) appears to be a good free alternative since its performance is comparable to that of the midranked commercial algorithms. Based on a statistical analysis, we elaborate on which algorithms to use and how to parametrize them for best performance in different application scenarios.

  17. Fast axonal transport of 3H-leucin-labelled proteins in the unhurt and isolated optical nerve of rats

    Wagner, H.E.

    1981-01-01

    The distribution of radioactivity of amino acid molecules incorporated in protein after injection of 3 H-Leucin into the right bulb was investigated and determined along optical nerve after 1, 2, and 4 h. A slightly increased radioactivity at the point of entrance of the optical nerves into the optical duct was found. A slightly reduced axon diameter was discussed as a possible cause. The radioactivity brought into the optical nerve via the vascular system was determined by measuring the contralateral optical nerve. In relation to the axonally transported activity, it was low. The speed of the fast axonal transport is 168 mm/d. If the processes ruling the amino acids in the perikaryon are taken into consideration, the transport speed is 240 mm/d. The application of the protein synthesis prohibitor, Cycloheximide, 5 minutes after the injection of Leucinin completely prevented the appearance of axonally transported labelled proteins. When cycloheximide was administered 2 h after Leucin, a significantly loner radioactivity than in the nerve could be determined after another 2 h; i.e. the incorporation of Leucin was not completed yet after 2 h. The profile of active compounds was the same as in the control group. In other experiments, the axonal transport of labelled proteins in isolated optical nerve fibres was tested. If the separation was carried out 2 h after the injection of Leucin an extreme reduction in activity could be determined after 1 or 2 h. The continued distribution of activity after cycloheximide treatment and removal of perikarya in comparison with the control indicate the continuation of the transport, also after separation of the axon from the perikaryon. This means that, during the time of the experiment, the mechanism of the fast axonal transport functions independently of the perikaryon. (orig./MG) [de

  18. Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes.

    Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A; Schechtman, Deborah

    2016-02-25

    Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

  19. GLUCOSE AND TOTAL PROTEIN LEVEL IN LABORATORY RATS UNDER CONDITIONS OF SHORT-TERM FASTING

    Damir Suljević

    2013-09-01

    Full Text Available Glucose level (UV enzymatic method and total protein level (Biuret method were measured in the blood samples of the rats exposed to short-term starvation. We found a statistically significant increase in the glucose level in experimental animals during starvation, which is also evident in males and females in the experimental group (p <0.05, while decrease in the total protein level was not statistically significant. During starvation, more significant weight loss was observed in females compared to males.Key words: glucose, total protein, serum, Rattus

  20. A method for fast energy estimation and visualization of protein-ligand interaction

    Tomioka, Nobuo; Itai, Akiko; Iitaka, Yoichi

    1987-10-01

    A new computational and graphical method for facilitating ligand-protein docking studies is developed on a three-dimensional computer graphics display. Various physical and chemical properties inside the ligand binding pocket of a receptor protein, whose structure is elucidated by X-ray crystal analysis, are calculated on three-dimensional grid points and are stored in advance. By utilizing those tabulated data, it is possible to estimate the non-bonded and electrostatic interaction energy and the number of possible hydrogen bonds between protein and ligand molecules in real time during an interactive docking operation. The method also provides a comprehensive visualization of the local environment inside the binding pocket. With this method, it becomes easier to find a roughly stable geometry of ligand molecules, and one can therefore make a rapid survey of the binding capability of many drug candidates. The method will be useful for drug design as well as for the examination of protein-ligand interactions.

  1. Analysis of Protein Content in Rices Using Fast Neutron Reaction 14N(n,2 n) N13

    Sri-Sulamdari; Elin-Nuraini; Chotimah

    2000-01-01

    Protein content in rices such as IR 33 , Cisadane, and Rojolele has beendetermined using fast neutron activation analysis (FNAA). The existence ofprotein is characterized using E γ = 0.511 MeV from nuclear reaction 14 N (n,2 n) N 13 . Two methods of FNAA for quantification were used. Inabsolute method, the protein content was determined by measuring the neutronflux using Al foil, and in comparative additive method it was determined bycomparing to the known N standard which was additive to the samples. Theexperimental results show that the protein content in those rices ranges from(6.33 ± 0.05) % to (6.9 ± 0.11) % in weight. From the reference, thegrained rices contained 6.8 % in weight of protein. The value of the proteinstandard from the reference was in range of the experimental result. Howeverthere were still differences due to nuclear data stability of flux neutron,flux sample composition and the utilization of detector. (author)

  2. Characterization of mu s-ms dynamics of proteins using a combined analysis of N-15 NMR relaxation and chemical shift: Conformational exchange in plastocyanin induced by histidine protonations

    Hass, M. A. S.; Thuesen, Marianne Hallberg; Christensen, Hans Erik Mølager

    2004-01-01

    of the exchanging species can be determined independently of the relaxation rates. The applicability of the approach is demonstrated by a detailed analysis of the conformational exchange processes previously observed in the reduced form of the blue copper protein, plastocyanin from the cyanobacteria Anabaena......An approach is presented that allows a detailed, quantitative characterization of conformational exchange processes in proteins on the mus-ms time scale. The approach relies on a combined analysis of NMR relaxation rates and chemical shift changes and requires that the chemical shift...... quantitatively by the correlation between the R-ex terms and the corresponding chemical shift differences of the exchanging species. By this approach, the R-ex terms of N-15 nuclei belonging to contiguous regions in the protein could be assigned to the same exchange process. Furthermore, the analysis...

  3. Development of a biosensor protein bullet as a fluorescent method for fast detection of Escherichia coli in drinking water.

    Ignacio Gutiérrez-Del-Río

    Full Text Available Drinking water can be exposed to different biological contaminants from the source, through the pipelines, until reaching the final consumer or industry. Some of these are pathogenic bacteria and viruses which may cause important gastrointestinal or systemic diseases. The microbiological quality of drinking water relies mainly in monitoring three indicator bacteria of faecal origin, Escherichia coli, Enterococcus faecalis and Clostridium perfringens, which serve as early sentinels of potential health hazards for the population. Here we describe the analysis of three chimeric fluorescent protein bullets as biosensor candidates for fast detection of E. coli in drinking water. Two of the chimeric proteins (based on GFP-hadrurin and GFP-pb5 chimera proteins failed with respect to specificity and/or sensitivity, but the GFP-colS4 chimera protein was able to carry out specific detection of E. coli in drinking water samples in a procedure encompassing about 8 min for final result and this biosensor protein was able to detect in a linear way between 20 and 103 CFU of this bacterium. Below 20 CFU, the system cannot differentiate presence or absence of the target bacterium. The fluorescence in this biosensor system is provided by the GFP subunit of the chimeric protein, which, in the case of the better performing sensor bullet, GFP-colS4 chimera, is covalently bound to a flexible peptide bridge and to a bacteriocin binding specifically to E. coli cells. Once bound to the target bacteria, the excitation step with 395 nm LED light causes emission of fluorescence from the GFP domain, which is amplified in a photomultiplier tube, and finally this signal is converted into an output voltage which can be associated with a CFU value and these data distributed along mobile phone networks, for example. This method, and the portable fluorimeter which has been developed for it, may contribute to reduce the analysis time for detecting E. coli presence in drinking

  4. High-throughput fractionation of human plasma for fast enrichment of low- and high-abundance proteins.

    Breen, Lucas; Cao, Lulu; Eom, Kirsten; Srajer Gajdosik, Martina; Camara, Lila; Giacometti, Jasminka; Dupuy, Damian E; Josic, Djuro

    2012-05-01

    Fast, cost-effective and reproducible isolation of IgM from plasma is invaluable to the study of IgM and subsequent understanding of the human immune system. Additionally, vast amounts of information regarding human physiology and disease can be derived from analysis of the low abundance proteome of the plasma. In this study, methods were optimized for both the high-throughput isolation of IgM from human plasma, and the high-throughput isolation and fractionation of low abundance plasma proteins. To optimize the chromatographic isolation of IgM from human plasma, many variables were examined including chromatography resin, mobile phases, and order of chromatographic separations. Purification of IgM was achieved most successfully through isolation of immunoglobulin from human plasma using Protein A chromatography with a specific resin followed by subsequent fractionation using QA strong anion exchange chromatography. Through these optimization experiments, an additional method was established to prepare plasma for analysis of low abundance proteins. This method involved chromatographic depletion of high-abundance plasma proteins and reduction of plasma proteome complexity through further chromatographic fractionation. Purification of IgM was achieved with high purity as confirmed by SDS-PAGE and IgM-specific immunoblot. Isolation and fractionation of low abundance protein was also performed successfully, as confirmed by SDS-PAGE and mass spectrometry analysis followed by label-free quantitative spectral analysis. The level of purity of the isolated IgM allows for further IgM-specific analysis of plasma samples. The developed fractionation scheme can be used for high throughput screening of human plasma in order to identify low and high abundance proteins as potential prognostic and diagnostic disease biomarkers.

  5. Development of a biosensor protein bullet as a fluorescent method for fast detection of Escherichia coli in drinking water.

    Gutiérrez-Del-Río, Ignacio; Marín, Laura; Fernández, Javier; Álvarez San Millán, María; Ferrero, Francisco Javier; Valledor, Marta; Campo, Juan Carlos; Cobián, Natalia; Méndez, Ignacio; Lombó, Felipe

    2018-01-01

    Drinking water can be exposed to different biological contaminants from the source, through the pipelines, until reaching the final consumer or industry. Some of these are pathogenic bacteria and viruses which may cause important gastrointestinal or systemic diseases. The microbiological quality of drinking water relies mainly in monitoring three indicator bacteria of faecal origin, Escherichia coli, Enterococcus faecalis and Clostridium perfringens, which serve as early sentinels of potential health hazards for the population. Here we describe the analysis of three chimeric fluorescent protein bullets as biosensor candidates for fast detection of E. coli in drinking water. Two of the chimeric proteins (based on GFP-hadrurin and GFP-pb5 chimera proteins) failed with respect to specificity and/or sensitivity, but the GFP-colS4 chimera protein was able to carry out specific detection of E. coli in drinking water samples in a procedure encompassing about 8 min for final result and this biosensor protein was able to detect in a linear way between 20 and 103 CFU of this bacterium. Below 20 CFU, the system cannot differentiate presence or absence of the target bacterium. The fluorescence in this biosensor system is provided by the GFP subunit of the chimeric protein, which, in the case of the better performing sensor bullet, GFP-colS4 chimera, is covalently bound to a flexible peptide bridge and to a bacteriocin binding specifically to E. coli cells. Once bound to the target bacteria, the excitation step with 395 nm LED light causes emission of fluorescence from the GFP domain, which is amplified in a photomultiplier tube, and finally this signal is converted into an output voltage which can be associated with a CFU value and these data distributed along mobile phone networks, for example. This method, and the portable fluorimeter which has been developed for it, may contribute to reduce the analysis time for detecting E. coli presence in drinking water.

  6. Protein metabolism in slow- and fast-twitch skeletal muscle during turpentine-induced inflammation.

    Muthny, Tomas; Kovarik, Miroslav; Sispera, Ludek; Tilser, Ivan; Holecek, Milan

    2008-02-01

    The aim of our study was to evaluate the differences in protein and amino acid metabolism after subcutaneous turpentine administration in the soleus muscle (SOL), predominantly composed of red fibres, and the extensor digitorum longus muscle (EDL) composed of white fibres. Young rats (40-60 g) were injected subcutaneously with 0.2 ml of turpentine oil/100 g body weight (inflammation) or with the same volume of saline solution (control). Twenty-four hours later SOL and EDL were dissected and incubated in modified Krebs-Heinseleit buffer to estimate total and myofibrillar proteolysis, chymotrypsin-like activity of proteasome (CHTLA), leucine oxidation, protein synthesis and amino acid release into the medium. The data obtained demonstrate that in intact rats, all parameters measured except protein synthesis are significantly higher in SOL than in EDL. In turpentine treated animals, CHTLA increased and protein synthesis decreased significantly more in EDL. Release of leucine was inhibited significantly more in SOL. We conclude that turpentine-induced inflammation affects more CHTLA, protein synthesis and leucine release in EDL compared to SOL.

  7. BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR

    Aguirre, Clémentine; ten Brink, Tim; Walker, Olivier; Guillière, Florence; Davesne, Dany; Krimm, Isabelle

    2013-01-01

    Protein-protein interactions represent difficult but increasingly important targets for the design of therapeutic compounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approaches for the elaboration of protein-protein surface inhibitors from fragment-like molecules. One major challenge in targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are therefore required at the early steps of the drug design process. In this report we present a fast NMR method able to probe subtle conformational changes upon fragment binding. The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding. We illustrate the method by the retrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 4′-fluoro-[1,1′-biphenyl]-4-carboxylic acid that was previously shown to bind one of the Bcl-xL hot spots. The CSP-based approach shows that the protein undergoes a subtle conformational rearrangement upon interaction, for residues located in helices 2, 3 and the very beginning of 5. Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound forms of the Bcl-xL protein. These NMR-based results are in total agreement with previous molecular dynamic calculations that evidenced a high flexibility of Bcl-xL around the binding site. Here we show that CSP of protein amine protons are useful and reliable structural probes. Therefore, we propose to use CSP simulation to assess protein conformational changes upon ligand binding in the fragment-based drug design approach. PMID:23717610

  8. A new method for protein estimation in large seeds using fast-neutron-activation analysis

    Gupta, U.C.; Misra, S.C.; Rao, U.S.

    1974-01-01

    A new method was developed for the determination of protein content of large seeds, using powders of different N content. The powders were obtained by mixing glucose with amino acids in different proportions and were irradiated with and without the seeds in the MeV neutron flux. The irradiated samples were counted under identical conditions and their activities were used to calculate the protein content of the seeds. The results were compared with those obtained by conventional activation technique and were found to be in good agreement. This new method has the advantage of being non-destructive. (author)

  9. Intermittent fasting reduces body fat but exacerbates hepatic insulin resistance in young rats regardless of high protein and fat diets.

    Park, Sunmin; Yoo, Kyung Min; Hyun, Joo Suk; Kang, Suna

    2017-02-01

    Intermittent fasting (IMF) is a relatively new dietary approach to weight management, although the efficacy and adverse effects have not been full elucidated and the optimal diets for IMF are unknown. We tested the hypothesis that a one-meal-per-day intermittent fasting with high fat (HF) or protein (HP) diets can modify energy, lipid, and glucose metabolism in normal young male Sprague-Dawley rats with diet-induced obesity or overweight. Male rats aged 5 weeks received either HF (40% fat) or HP (26% protein) diets ad libitum (AL) or for 3 h at the beginning of the dark cycle (IMF) for 5 weeks. Epidydimal fat pads and fat deposits in the leg and abdomen were lower with HP and IMF. Energy expenditure at the beginning of the dark cycle, especially from fat oxidation, was higher with IMF than AL, possibly due to greater activity levels. Brown fat content was higher with IMF. Serum ghrelin levels were higher in HP-IMF than other groups, and accordingly, cumulative food intake was also higher in HP-IMF than HF-IMF. HF-IMF exhibited higher area under the curve (AUC) of serum glucose at the first part (0-40 min) during oral glucose tolerance test, whereas AUC of serum insulin levels in both parts were higher in IMF and HF. During intraperitoneal insulin tolerance test, serum glucose levels were higher with IMF than AL. Consistently, hepatic insulin signaling (GLUT2, pAkt) was attenuated and PEPCK expression was higher with IMF and HF than other groups, and HOMA-IR revealed significantly impaired attenuated insulin sensitivity in the IMF groups. However, surprisingly, hepatic and skeletal muscle glycogen storage was higher in IMF groups than AL. The higher glycogen storage in the IMF groups was associated with the lower expression of glycogen phosphorylase than the AL groups. In conclusion, IMF especially with HF increased insulin resistance, possibly by attenuating hepatic insulin signaling, and lowered glycogen phosphorylase expression despite decreased fat mass in young

  10. Fast and efficient detection of tuberculosis antigens using liposome encapsulated secretory proteins of Mycobacterium tuberculosis

    Dileep Tiwari

    2017-04-01

    Conclusion: Our study demonstrated that the newly developed liposome tuberculosis antigen card test detected antigens in our study population with approximately 97.48% sensitivity and 95.79% specificity. This is the first study to report the liposomal encapsulation of culture filtrate proteins from M. tuberculosis for diagnostic application.

  11. FuncPatch: a web server for the fast Bayesian inference of conserved functional patches in protein 3D structures.

    Huang, Yi-Fei; Golding, G Brian

    2015-02-15

    A number of statistical phylogenetic methods have been developed to infer conserved functional sites or regions in proteins. Many methods, e.g. Rate4Site, apply the standard phylogenetic models to infer site-specific substitution rates and totally ignore the spatial correlation of substitution rates in protein tertiary structures, which may reduce their power to identify conserved functional patches in protein tertiary structures when the sequences used in the analysis are highly similar. The 3D sliding window method has been proposed to infer conserved functional patches in protein tertiary structures, but the window size, which reflects the strength of the spatial correlation, must be predefined and is not inferred from data. We recently developed GP4Rate to solve these problems under the Bayesian framework. Unfortunately, GP4Rate is computationally slow. Here, we present an intuitive web server, FuncPatch, to perform a fast approximate Bayesian inference of conserved functional patches in protein tertiary structures. Both simulations and four case studies based on empirical data suggest that FuncPatch is a good approximation to GP4Rate. However, FuncPatch is orders of magnitudes faster than GP4Rate. In addition, simulations suggest that FuncPatch is potentially a useful tool complementary to Rate4Site, but the 3D sliding window method is less powerful than FuncPatch and Rate4Site. The functional patches predicted by FuncPatch in the four case studies are supported by experimental evidence, which corroborates the usefulness of FuncPatch. The software FuncPatch is freely available at the web site, http://info.mcmaster.ca/yifei/FuncPatch golding@mcmaster.ca Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Conformational variability of the stationary phase survival protein E from Xylella fastidiosa revealed by X-ray crystallography, small-angle X-ray scattering studies, and normal mode analysis.

    Machado, Agnes Thiane Pereira; Fonseca, Emanuella Maria Barreto; Reis, Marcelo Augusto Dos; Saraiva, Antonio Marcos; Santos, Clelton Aparecido Dos; de Toledo, Marcelo Augusto Szymanski; Polikarpov, Igor; de Souza, Anete Pereira; Aparicio, Ricardo; Iulek, Jorge

    2017-10-01

    Xylella fastidiosa is a xylem-limited bacterium that infects a wide variety of plants. Stationary phase survival protein E is classified as a nucleotidase, which is expressed when bacterial cells are in the stationary growth phase and subjected to environmental stresses. Here, we report four refined X-ray structures of this protein from X. fastidiosa in four different crystal forms in the presence and/or absence of the substrate 3'-AMP. In all chains, the conserved loop verified in family members assumes a closed conformation in either condition. Therefore, the enzymatic mechanism for the target protein might be different of its homologs. Two crystal forms exhibit two monomers whereas the other two show four monomers in the asymmetric unit. While the biological unit has been characterized as a tetramer, differences of their sizes and symmetry are remarkable. Four conformers identified by Small-Angle X-ray Scattering (SAXS) in a ligand-free solution are related to the low frequency normal modes of the crystallographic structures associated with rigid body-like protomer arrangements responsible for the longitudinal and symmetric adjustments between tetramers. When the substrate is present in solution, only two conformers are selected. The most prominent conformer for each case is associated to a normal mode able to elongate the protein by moving apart two dimers. To our knowledge, this work was the first investigation based on the normal modes that analyzed the quaternary structure variability for an enzyme of the SurE family followed by crystallography and SAXS validation. The combined results raise new directions to study allosteric features of XfSurE protein. © 2017 Wiley Periodicals, Inc.

  13. Conformal Tachyons

    Tomaschitz, R

    2000-01-01

    We study tachyons conformally coupled to the background geometry of a Milne universe. The causality of superluminal signal transfer is scrutinized in this context. The cosmic time of the comoving frame determines a distinguished time order for events connected by superluminal signals. An observer can relate his rest frame to the galaxy frame, and compare so the time order of events in his proper time to the cosmic time order. All observers can in this way arrive at identical conclusions on the causality of events connected by superluminal signals. An unambiguous energy concept for tachyonic rays is defined by means of the cosmic time of the comoving reference frame, without resorting to an antiparticle interpretation. On that basis we give an explicit proof that no signals can be sent into the past of observers. Causality violating signals are energetically forbidden, as they would have negative energy in the rest frame of the emitting observer. If an observer emits a superluminal signal, the tachyonic respon...

  14. Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature

    Soichiro Tsujino; Takashi Tomizaki

    2016-01-01

    Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinn...

  15. Flavodiiron Proteins Promote Fast and Transient O2 Photoreduction in Chlamydomonas.

    Chaux, Frédéric; Burlacot, Adrien; Mekhalfi, Malika; Auroy, Pascaline; Blangy, Stéphanie; Richaud, Pierre; Peltier, Gilles

    2017-07-01

    During oxygenic photosynthesis, the reducing power generated by light energy conversion is mainly used to reduce carbon dioxide. In bacteria and archae, flavodiiron (Flv) proteins catalyze O 2 or NO reduction, thus protecting cells against oxidative or nitrosative stress. These proteins are found in cyanobacteria, mosses, and microalgae, but have been lost in angiosperms. Here, we used chlorophyll fluorescence and oxygen exchange measurement using [ 18 O]-labeled O 2 and a membrane inlet mass spectrometer to characterize Chlamydomonas reinhardtii flvB insertion mutants devoid of both FlvB and FlvA proteins. We show that Flv proteins are involved in a photo-dependent electron flow to oxygen, which drives most of the photosynthetic electron flow during the induction of photosynthesis. As a consequence, the chlorophyll fluorescence patterns are strongly affected in flvB mutants during a light transient, showing a lower PSII operating yield and a slower nonphotochemical quenching induction. Photoautotrophic growth of flvB mutants was indistinguishable from the wild type under constant light, but severely impaired under fluctuating light due to PSI photo damage. Remarkably, net photosynthesis of flv mutants was higher than in the wild type during the initial hour of a fluctuating light regime, but this advantage vanished under long-term exposure, and turned into PSI photo damage, thus explaining the marked growth retardation observed in these conditions. We conclude that the C. reinhardtii Flv participates in a Mehler-like reduction of O 2 , which drives a large part of the photosynthetic electron flow during a light transient and is thus critical for growth under fluctuating light regimes. © 2017 American Society of Plant Biologists. All Rights Reserved.

  16. Divergent flow isoelectric focusing: fast and efficient method for protein sample preparation for mass spectrometry

    Mazanec, Karel; Bobálová, Janette; Šlais, Karel

    2009-01-01

    Roč. 393, 6-7 (2009), s. 1769-1778 ISSN 1618-2642 R&D Projects: GA MŠk 1M0570; GA AV ČR IAAX00310701 Institutional research plan: CEZ:AV0Z40310501 Keywords : Isoelectric focusing * proteins * MALDI-TOF/TOF MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.480, year: 2009

  17. fLPS: Fast discovery of compositional biases for the protein universe.

    Harrison, Paul M

    2017-11-13

    Proteins often contain regions that are compositionally biased (CB), i.e., they are made from a small subset of amino-acid residue types. These CB regions can be functionally important, e.g., the prion-forming and prion-like regions that are rich in asparagine and glutamine residues. Here I report a new program fLPS that can rapidly annotate CB regions. It discovers both single-residue and multiple-residue biases. It works through a process of probability minimization. First, contigs are constructed for each amino-acid type out of sequence windows with a low degree of bias; second, these contigs are searched exhaustively for low-probability subsequences (LPSs); third, such LPSs are iteratively assessed for merger into possible multiple-residue biases. At each of these stages, efficiency measures are taken to avoid or delay probability calculations unless/until they are necessary. On a current desktop workstation, the fLPS algorithm can annotate the biased regions of the yeast proteome (>5700 sequences) in 65 million sequences) in as little as ~1 h, which is >2 times faster than the commonly used program SEG, using default parameters. fLPS discovers both shorter CB regions (of the sort that are often termed 'low-complexity sequence'), and milder biases that may only be detectable over long tracts of sequence. fLPS can readily handle very large protein data sets, such as might come from metagenomics projects. It is useful in searching for proteins with similar CB regions, and for making functional inferences about CB regions for a protein of interest. The fLPS package is available from: http://biology.mcgill.ca/faculty/harrison/flps.html , or https://github.com/pmharrison/flps , or is a supplement to this article.

  18. Fast and simple protein-alignment-guided assembly of orthologous gene families from microbiome sequencing reads.

    Huson, Daniel H; Tappu, Rewati; Bazinet, Adam L; Xie, Chao; Cummings, Michael P; Nieselt, Kay; Williams, Rohan

    2017-01-25

    Microbiome sequencing projects typically collect tens of millions of short reads per sample. Depending on the goals of the project, the short reads can either be subjected to direct sequence analysis or be assembled into longer contigs. The assembly of whole genomes from metagenomic sequencing reads is a very difficult problem. However, for some questions, only specific genes of interest need to be assembled. This is then a gene-centric assembly where the goal is to assemble reads into contigs for a family of orthologous genes. We present a new method for performing gene-centric assembly, called protein-alignment-guided assembly, and provide an implementation in our metagenome analysis tool MEGAN. Genes are assembled on the fly, based on the alignment of all reads against a protein reference database such as NCBI-nr. Specifically, the user selects a gene family based on a classification such as KEGG and all reads binned to that gene family are assembled. Using published synthetic community metagenome sequencing reads and a set of 41 gene families, we show that the performance of this approach compares favorably with that of full-featured assemblers and that of a recently published HMM-based gene-centric assembler, both in terms of the number of reference genes detected and of the percentage of reference sequence covered. Protein-alignment-guided assembly of orthologous gene families complements whole-metagenome assembly in a new and very useful way.

  19. Maxwell equations in conformal invariant electrodynamics

    Fradkin, E.S.; AN SSSR, Novosibirsk. Inst. Avtomatiki i Ehlektrometrii); Kozhevnikov, A.A.; Palchik, M.Ya.; Pomeransky, A.A.

    1983-01-01

    We consider a conformal invariant formulation of quantum electrodynamics. Conformal invariance is achieved with a specific mathematical construction based on the indecomposable representations of the conformal group associated with the electromagnetic potential and current. As a corolary of this construction modified expressions for the 3-point Green functions are obtained which both contain transverse parts. They make it possible to formulate a conformal invariant skeleton perturbation theory. It is also shown that the Euclidean Maxwell equations in conformal electrodynamics are manifestations of its kinematical structure: in the case of the 3-point Green functions these equations follow (up to constants) from the conformal invariance while in the case of higher Green functions they are equivalent to the equality of the kernels of the partial wave expansions. This is the manifestation of the mathematical fast of a (partial) equivalence of the representations associated with the potential, current and the field tensor. (orig.)

  20. Focused conformational sampling in proteins

    Bacci, M.; Langini, C.; Vymětal, Jiří; Caflisch, A.; Vitalis, A.

    2017-01-01

    Roč. 147, č. 19 (2017), č. článku 195102. ISSN 0021-9606 Institutional support: RVO:61388963 Keywords : molecular dynamics simulations * histone recognition * fibril elongation Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 2.965, year: 2016

  1. Conformational Clusters of Phosphorylated Tyrosine.

    Abdelrasoul, Maha; Ponniah, Komala; Mao, Alice; Warden, Meghan S; Elhefnawy, Wessam; Li, Yaohang; Pascal, Steven M

    2017-12-06

    Tyrosine phosphorylation plays an important role in many cellular and intercellular processes including signal transduction, subcellular localization, and regulation of enzymatic activity. In 1999, Blom et al., using the limited number of protein data bank (PDB) structures available at that time, reported that the side chain structures of phosphorylated tyrosine (pY) are partitioned into two conserved conformational clusters ( Blom, N.; Gammeltoft, S.; Brunak, S. J. Mol. Biol. 1999 , 294 , 1351 - 1362 ). We have used the spectral clustering algorithm to cluster the increasingly growing number of protein structures with pY sites, and have found that the pY residues cluster into three distinct side chain conformations. Two of these pY conformational clusters associate strongly with a narrow range of tyrosine backbone conformation. The novel cluster also highly correlates with the identity of the n + 1 residue, and is strongly associated with a sequential pYpY conformation which places two adjacent pY side chains in a specific relative orientation. Further analysis shows that the three pY clusters are associated with distinct distributions of cognate protein kinases.

  2. Global conformational dynamics of a Y-family DNA polymerase during catalysis.

    Cuiling Xu

    2009-10-01

    Full Text Available Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4 from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.

  3. A simple three-dimensional-focusing, continuous-flow mixer for the study of fast protein dynamics.

    Burke, Kelly S; Parul, Dzmitry; Reddish, Michael J; Dyer, R Brian

    2013-08-07

    We present a simple, yet flexible microfluidic mixer with a demonstrated mixing time as short as 80 μs that is widely accessible because it is made of commercially available parts. To simplify the study of fast protein dynamics, we have developed an inexpensive continuous-flow microfluidic mixer, requiring no specialized equipment or techniques. The mixer uses three-dimensional, hydrodynamic focusing of a protein sample stream by a surrounding sheath solution to achieve rapid diffusional mixing between the sample and sheath. Mixing initiates the reaction of interest. Reactions can be spatially observed by fluorescence or absorbance spectroscopy. We characterized the pixel-to-time calibration and diffusional mixing experimentally. We achieved a mixing time as short as 80 μs. We studied the kinetics of horse apomyoglobin (apoMb) unfolding from the intermediate (I) state to its completely unfolded (U) state, induced by a pH jump from the initial pH of 4.5 in the sample stream to a final pH of 2.0 in the sheath solution. The reaction time was probed using the fluorescence of 1-anilinonaphthalene-8-sulfonate (1,8-ANS) bound to the folded protein. We observed unfolding of apoMb within 760 μs, without populating additional intermediate states under these conditions. We also studied the reaction kinetics of the conversion of pyruvate to lactate catalyzed by lactate dehydrogenase using the intrinsic tryptophan emission of the enzyme. We observe sub-millisecond kinetics that we attribute to Michaelis complex formation and loop domain closure. These results demonstrate the utility of the three-dimensional focusing mixer for biophysical studies of protein dynamics.

  4. Conformational Ensembles of an Intrinsically Disordered Protein pKID with and without a KIX Domain in Explicit Solvent Investigated by All-Atom Multicanonical Molecular Dynamics

    Haruki Nakamura

    2012-02-01

    Full Text Available The phosphorylated kinase-inducible activation domain (pKID adopts a helix–loop–helix structure upon binding to its partner KIX, although it is unstructured in the unbound state. The N-terminal and C-terminal regions of pKID, which adopt helices in the complex, are called, respectively, αA and αB. We performed all-atom multicanonical molecular dynamics simulations of pKID with and without KIX in explicit solvents to generate conformational ensembles. Although the unbound pKID was disordered overall, αA and αB exhibited a nascent helix propensity; the propensity of αA was stronger than that of αB, which agrees with experimental results. In the bound state, the free-energy landscape of αB involved two low free-energy fractions: native-like and non-native fractions. This result suggests that αB folds according to the induced-fit mechanism. The αB-helix direction was well aligned as in the NMR complex structure, although the αA helix exhibited high flexibility. These results also agree quantitatively with experimental observations. We have detected that the αB helix can bind to another site of KIX, to which another protein MLL also binds with the adopting helix. Consequently, MLL can facilitate pKID binding to the pKID-binding site by blocking the MLL-binding site. This also supports experimentally obtained results.

  5. Adaptive GDDA-BLAST: fast and efficient algorithm for protein sequence embedding.

    Yoojin Hong

    2010-10-01

    Full Text Available A major computational challenge in the genomic era is annotating structure/function to the vast quantities of sequence information that is now available. This problem is illustrated by the fact that most proteins lack comprehensive annotations, even when experimental evidence exists. We previously theorized that embedded-alignment profiles (simply "alignment profiles" hereafter provide a quantitative method that is capable of relating the structural and functional properties of proteins, as well as their evolutionary relationships. A key feature of alignment profiles lies in the interoperability of data format (e.g., alignment information, physio-chemical information, genomic information, etc.. Indeed, we have demonstrated that the Position Specific Scoring Matrices (PSSMs are an informative M-dimension that is scored by quantitatively measuring the embedded or unmodified sequence alignments. Moreover, the information obtained from these alignments is informative, and remains so even in the "twilight zone" of sequence similarity (<25% identity. Although our previous embedding strategy was powerful, it suffered from contaminating alignments (embedded AND unmodified and high computational costs. Herein, we describe the logic and algorithmic process for a heuristic embedding strategy named "Adaptive GDDA-BLAST." Adaptive GDDA-BLAST is, on average, up to 19 times faster than, but has similar sensitivity to our previous method. Further, data are provided to demonstrate the benefits of embedded-alignment measurements in terms of detecting structural homology in highly divergent protein sequences and isolating secondary structural elements of transmembrane and ankyrin-repeat domains. Together, these advances allow further exploration of the embedded alignment data space within sufficiently large data sets to eventually induce relevant statistical inferences. We show that sequence embedding could serve as one of the vehicles for measurement of low

  6. Fast large-scale clustering of protein structures using Gauss integrals

    Harder, Tim; Borg, Mikael; Boomsma, Wouter

    2011-01-01

    trajectories. Results: We present Pleiades, a novel approach to clustering protein structures with a rigorous mathematical underpinning. The method approximates clustering based on the root mean square deviation by rst mapping structures to Gauss integral vectors – which were introduced by Røgen and co......-workers – and subsequently performing K-means clustering. Conclusions: Compared to current methods, Pleiades dramatically improves on the time needed to perform clustering, and can cluster a signicantly larger number of structures, while providing state-ofthe- art results. The number of low energy structures generated...

  7. Conformal field theory in conformal space

    Preitschopf, C.R.; Vasiliev, M.A.

    1999-01-01

    We present a new framework for a Lagrangian description of conformal field theories in various dimensions based on a local version of d + 2-dimensional conformal space. The results include a true gauge theory of conformal gravity in d = (1, 3) and any standard matter coupled to it. An important feature is the automatic derivation of the conformal gravity constraints, which are necessary for the analysis of the matter systems

  8. Integrated Solid-Phase Extraction-Capillary Liquid Chromatography (speLC) Interfaced to ESI-MS/MS for Fast Characterization and Quantification of Protein and Proteomes

    Falkenby, Lasse Gaarde; Such-Sanmartín, Gerard; Larsen, Martin Røssel

    2014-01-01

    min speLC-MS/MS experiment. Analysis by selected reaction monitoring by speLC-SRM-MS/MS of distinct peptides derived from the blood proteins IGF1, IGF2, IBP2, and IBP3 demonstrated protein quantification with CV values below 10% across 96 replicates. The speLC-MS/MS system is ideally suited for fast......The high peptide sequencing speed provided by modern hybrid tandem mass spectrometers enables the utilization of fast liquid chromatographic (LC) separation techniques. We present a robust solid-phase extraction/capillary LC system (speLC) for 5-10 min separation of semicomplex peptide mixtures...

  9. Ultrasonic acoustic levitation for fast frame rate X-ray protein crystallography at room temperature

    Tsujino, Soichiro; Tomizaki, Takashi

    2016-05-01

    Increasing the data acquisition rate of X-ray diffraction images for macromolecular crystals at room temperature at synchrotrons has the potential to significantly accelerate both structural analysis of biomolecules and structure-based drug developments. Using lysozyme model crystals, we demonstrated the rapid acquisition of X-ray diffraction datasets by combining a high frame rate pixel array detector with ultrasonic acoustic levitation of protein crystals in liquid droplets. The rapid spinning of the crystal within a levitating droplet ensured an efficient sampling of the reciprocal space. The datasets were processed with a program suite developed for serial femtosecond crystallography (SFX). The structure, which was solved by molecular replacement, was found to be identical to the structure obtained by the conventional oscillation method for up to a 1.8-Å resolution limit. In particular, the absence of protein crystal damage resulting from the acoustic levitation was carefully established. These results represent a key step towards a fully automated sample handling and measurement pipeline, which has promising prospects for a high acquisition rate and high sample efficiency for room temperature X-ray crystallography.

  10. Fast evaluation of protein dynamics from deficient 15N relaxation data

    Jaremko, Łukasz

    2018-03-28

    Simple and convenient method of protein dynamics evaluation from the insufficient experimental N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N–H vectors on two different time scales, S and R, can be elucidated. The generalized order parameter, S, describes motions on the time scale faster than the overall tumbling correlation time (pico- to nanoseconds), while the chemical exchange term, R, identifies processes slower than the overall tumbling correlation time (micro- to milliseconds). Advantages and disadvantages of different methods of data handling are thoroughly discussed.

  11. Fast evaluation of protein dynamics from deficient 15N relaxation data

    Jaremko, Łukasz; Jaremko, Mariusz; Ejchart, Andrzej; Nowakowski, Michał

    2018-01-01

    Simple and convenient method of protein dynamics evaluation from the insufficient experimental N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N–H vectors on two different time scales, S and R, can be elucidated. The generalized order parameter, S, describes motions on the time scale faster than the overall tumbling correlation time (pico- to nanoseconds), while the chemical exchange term, R, identifies processes slower than the overall tumbling correlation time (micro- to milliseconds). Advantages and disadvantages of different methods of data handling are thoroughly discussed.

  12. PatternQuery: web application for fast detection of biomacromolecular structural patterns in the entire Protein Data Bank.

    Sehnal, David; Pravda, Lukáš; Svobodová Vařeková, Radka; Ionescu, Crina-Maria; Koča, Jaroslav

    2015-07-01

    Well defined biomacromolecular patterns such as binding sites, catalytic sites, specific protein or nucleic acid sequences, etc. precisely modulate many important biological phenomena. We introduce PatternQuery, a web-based application designed for detection and fast extraction of such patterns. The application uses a unique query language with Python-like syntax to define the patterns that will be extracted from datasets provided by the user, or from the entire Protein Data Bank (PDB). Moreover, the database-wide search can be restricted using a variety of criteria, such as PDB ID, resolution, and organism of origin, to provide only relevant data. The extraction generally takes a few seconds for several hundreds of entries, up to approximately one hour for the whole PDB. The detected patterns are made available for download to enable further processing, as well as presented in a clear tabular and graphical form directly in the browser. The unique design of the language and the provided service could pave the way towards novel PDB-wide analyses, which were either difficult or unfeasible in the past. The application is available free of charge at http://ncbr.muni.cz/PatternQuery. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis.

    Antonioli, Paolo; Mendieta, Martha E; Sebastiano, Roberto; Citterio, Attilio; Peltre, Gabriel; Busnel, Jean-Marc; Descroix, Stephanie; Candiano, Giovanni; Righetti, Pier Giorgio

    2006-03-20

    Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.

  14. Genetic analyses for conformation traits in South African Jersey and ...

    JACO

    Genetic trends for conformation traits of the South African Holstein show that ... conformation traits can be used to improve stayability, fertility and disease resistance (Rogers et al., 1999; .... Genetic correlations among protein yield, productive.

  15. A Critical Role of Fatty Acid Binding Protein 4 and 5 (FABP4/5) in the Systemic Response to Fasting

    Syamsunarno, Mas Rizky A. A.; Iso, Tatsuya; Hanaoka, Hirofumi; Yamaguchi, Aiko; Obokata, Masaru; Koitabashi, Norimichi; Goto, Kosaku; Hishiki, Takako; Nagahata, Yoshiko; Matsui, Hiroki; Sano, Motoaki; Kobayashi, Masaki; Kikuchi, Osamu; Sasaki, Tsutomu; Maeda, Kazuhisa; Murakami, Masami; Kitamura, Tadahiro; Suematsu, Makoto; YoshitoTsushima; Endo, Keigo; Hotamisligil, Gökhan S.; Kurabayashi, Masahiko

    2013-01-01

    During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the 125I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the

  16. Entropy in molecular recognition by proteins.

    Caro, José A; Harpole, Kyle W; Kasinath, Vignesh; Lim, Jackwee; Granja, Jeffrey; Valentine, Kathleen G; Sharp, Kim A; Wand, A Joshua

    2017-06-20

    Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein-ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein-ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein-ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or "entropy meter" also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water-protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins.

  17. Progress in proton-detected solid-state NMR (SSNMR): Super-fast 2D SSNMR collection for nano-mole-scale proteins

    Ishii, Yoshitaka; Wickramasinghe, Ayesha; Matsuda, Isamu; Endo, Yuki; Ishii, Yuji; Nishiyama, Yusuke; Nemoto, Takahiro; Kamihara, Takayuki

    2018-01-01

    Proton-detected solid-state NMR (SSNMR) spectroscopy has attracted much attention due to its excellent sensitivity and effectiveness in the analysis of trace amounts of amyloid proteins and other important biological systems. In this perspective article, we present the recent sensitivity limit of 1H-detected SSNMR using "ultra-fast" magic-angle spinning (MAS) at a spinning rate (νR) of 80-100 kHz. It was demonstrated that the high sensitivity of 1H-detected SSNMR at νR of 100 kHz and fast recycling using the paramagnetic-assisted condensed data collection (PACC) approach permitted "super-fast" collection of 1H-detected 2D protein SSNMR. A 1H-detected 2D 1H-15N correlation SSNMR spectrum for ∼27 nmol of a uniformly 13C- and 15N-labeled GB1 protein sample in microcrystalline form was acquired in only 9 s with 50% non-uniform sampling and short recycle delays of 100 ms. Additional data suggests that it is now feasible to detect as little as 1 nmol of the protein in 5.9 h by 1H-detected 2D 1H-15N SSNMR at a nominal signal-to-noise ratio of five. The demonstrated sensitivity is comparable to that of modern solution protein NMR. Moreover, this article summarizes the influence of ultra-fast MAS and 1H-detection on the spectral resolution and sensitivity of protein SSNMR. Recent progress in signal assignment and structural elucidation by 1H-detected protein SSNMR is outlined with both theoretical and experimental aspects.

  18. Comparison of High-Protein, Intermittent Fasting Low-Calorie Diet and Heart Healthy Diet for Vascular Health of the Obese.

    Zuo, Li; He, Feng; Tinsley, Grant M; Pannell, Benjamin K; Ward, Emery; Arciero, Paul J

    2016-01-01

    It has been debated whether different diets are more or less effective in long-term weight loss success and cardiovascular disease prevention among men and women. To further explore these questions, the present study evaluated the combined effects of a high-protein, intermittent fasting, low-calorie diet plan compared with a heart healthy diet plan during weight loss, and weight loss maintenance on blood lipids and vascular compliance of obese individuals. The experiment involved 40 obese adults (men, n = 21; women, n = 19) and was divided into two phases: (a) 12-week high-protein, intermittent fasting, low-calorie weight loss diet comparing men and women (Phase 1) and (b) a 1-year weight maintenance phase comparing high-protein, intermittent fasting with a heart healthy diet (Phase 2). Body weight, body mass index (BMI), blood lipids, and arterial compliance outcomes were assessed at weeks 1 (baseline control), 12 (weight loss), and 64 (12 + 52 week; weight loss maintenance). At the end of weight loss intervention, concomitant reductions in body weight, BMI and blood lipids were observed, as well as enhanced arterial compliance. No sex-specific differences in responses were observed. During phase 2, the high-protein, intermittent fasting group demonstrated a trend for less regain in BMI, low-density lipoprotein (LDL), and aortic pulse wave velocity than the heart healthy group. Our results suggest that a high-protein, intermittent fasting and low-calorie diet is associated with similar reductions in BMI and blood lipids in obese men and women. This diet also demonstrated an advantage in minimizing weight regain as well as enhancing arterial compliance as compared to a heart healthy diet after 1 year.

  19. Comparison of High-Protein, Intermittent-Fasting Low-Calorie Diet and Heart Healthy Diet for Vascular Health of the Obese

    Li Zuo

    2016-08-01

    Full Text Available Aim: It has been debated whether different diets are more or less effective in long-term weight loss success and cardiovascular diseases among men and women. Thus, the present study evaluated the combined effects of a high-protein, intermittent-fasting, low-calorie diet plan compared with heart healthy diet plan during weight loss maintenance on blood lipids and vascular compliance of individuals with obesity. Methods: The experiment involved 40 adults with obesity (men, n = 21; women, n = 19 and was divided into two phases: (a 12-week high-protein, intermittent-fasting, low-calorie weight loss diet comparing men and women (Phase 1 and (b a 1-year (52-week weight loss maintenance comparing high-protein, intermittent-fasting with a heart healthy diet (Phase 2. Body weight, body mass index, blood lipids, and arterial compliance outcomes were assessed at weeks 1 (baseline control, 12 (weight loss, and 64 (12+52 week; weight loss maintenance.Results: At the end of weight loss intervention, concomitant with reductions in body weight, body mass index, blood lipids, and arterial compliance was enhanced (p < 0.05. No sex-specific differences were observed. During phase 2, high-protein, intermittent-fasting, low-calorie group demonstrated less weight regain and percentage change in aortic pulse wave velocity than heart healthy group (p < 0.05. Conclusion: Our results suggest that high-protein, intermittent-fasting and low-calorie diet is associated with body weight loss and reduction in blood lipids. This diet also demonstrated a potential advantage in minimizing weight gain relapse as well as enhancing arterial compliance compared to the heart healthy diet in the long term.

  20. FAST: FAST Analysis of Sequences Toolbox

    Travis J. Lawrence

    2015-05-01

    Full Text Available FAST (FAST Analysis of Sequences Toolbox provides simple, powerful open source command-line tools to filter, transform, annotate and analyze biological sequence data. Modeled after the GNU (GNU’s Not Unix Textutils such as grep, cut, and tr, FAST tools such as fasgrep, fascut, and fastr make it easy to rapidly prototype expressive bioinformatic workflows in a compact and generic command vocabulary. Compact combinatorial encoding of data workflows with FAST commands can simplify the documentation and reproducibility of bioinformatic protocols, supporting better transparency in biological data science. Interface self-consistency and conformity with conventions of GNU, Matlab, Perl, BioPerl, R and GenBank help make FAST easy and rewarding to learn. FAST automates numerical, taxonomic, and text-based sorting, selection and transformation of sequence records and alignment sites based on content, index ranges, descriptive tags, annotated features, and in-line calculated analytics, including composition and codon usage. Automated content- and feature-based extraction of sites and support for molecular population genetic statistics makes FAST useful for molecular evolutionary analysis. FAST is portable, easy to install and secure thanks to the relative maturity of its Perl and BioPerl foundations, with stable releases posted to CPAN. Development as well as a publicly accessible Cookbook and Wiki are available on the FAST GitHub repository at https://github.com/tlawrence3/FAST. The default data exchange format in FAST is Multi-FastA (specifically, a restriction of BioPerl FastA format. Sanger and Illumina 1.8+ FastQ formatted files are also supported. FAST makes it easier for non-programmer biologists to interactively investigate and control biological data at the speed of thought.

  1. Fast Atomic Charge Calculation for Implementation into a Polarizable Force Field and Application to an Ion Channel Protein

    Raiker Witter

    2015-01-01

    Full Text Available Polarization of atoms plays a substantial role in molecular interactions. Class I and II force fields mostly calculate with fixed atomic charges which can cause inadequate descriptions for highly charged molecules, for example, ion channels or metalloproteins. Changes in charge distributions can be included into molecular mechanics calculations by various methods. Here, we present a very fast computational quantum mechanical method, the Bond Polarization Theory (BPT. Atomic charges are obtained via a charge calculation method that depend on the 3D structure of the system in a similar way as atomic charges of ab initio calculations. Different methods of population analysis and charge calculation methods and their dependence on the basis set were investigated. A refined parameterization yielded excellent correlation of R=0.9967. The method was implemented in the force field COSMOS-NMR and applied to the histidine-tryptophan-complex of the transmembrane domain of the M2 protein channel of influenza A virus. Our calculations show that moderate changes of side chain torsion angle χ1 and small variations of χ2 of Trp-41 are necessary to switch from the inactivated into the activated state; and a rough two-side jump model of His-37 is supported for proton gating in accordance with a flipping mechanism.

  2. Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements.

    Lundby, Alicia; Mutoh, Hiroki; Dimitrov, Dimitar; Akemann, Walther; Knöpfel, Thomas

    2008-06-25

    Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs.

  3. Extracting rate coefficients from single-molecule photon trajectories and FRET efficiency histograms for a fast-folding protein.

    Chung, Hoi Sung; Gopich, Irina V; McHale, Kevin; Cellmer, Troy; Louis, John M; Eaton, William A

    2011-04-28

    Recently developed statistical methods by Gopich and Szabo were used to extract folding and unfolding rate coefficients from single-molecule Förster resonance energy transfer (FRET) data for proteins with kinetics too fast to measure waiting time distributions. Two types of experiments and two different analyses were performed. In one experiment bursts of photons were collected from donor and acceptor fluorophores attached to a 73-residue protein, α(3)D, freely diffusing through the illuminated volume of a confocal microscope system. In the second, the protein was immobilized by linkage to a surface, and photons were collected until one of the fluorophores bleached. Folding and unfolding rate coefficients and mean FRET efficiencies for the folded and unfolded subpopulations were obtained from a photon by photon analysis of the trajectories using a maximum likelihood method. The ability of the method to describe the data in terms of a two-state model was checked by recoloring the photon trajectories with the extracted parameters and comparing the calculated FRET efficiency histograms with the measured histograms. The sum of the rate coefficients for the two-state model agreed to within 30% with the relaxation rate obtained from the decay of the donor-acceptor cross-correlation function, confirming the high accuracy of the method. Interestingly, apparently reliable rate coefficients could be extracted using the maximum likelihood method, even at low (rate coefficients and mean FRET efficiencies were also obtained in an approximate procedure by simply fitting the FRET efficiency histograms, calculated by binning the donor and acceptor photons, with a sum of three-Gaussian functions. The kinetics are exposed in these histograms by the growth of a FRET efficiency peak at values intermediate between the folded and unfolded peaks as the bin size increases, a phenomenon with similarities to NMR exchange broadening. When comparable populations of folded and unfolded

  4. Epigenetic dominance of prion conformers.

    Eri Saijo

    2013-10-01

    Full Text Available Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer's and Parkinson's diseases. To address the poorly understood mechanism by which host prion protein (PrP primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A at (OvPrP-A136 infected with SSBP/1 scrapie prions propagated a relatively stable (S prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V at 136 (OvPrP-V136 infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U, diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to

  5. Protein mechanics: a route from structure to function

    PRAKASH KUMAR

    and how fast individual amino acid side chains change their conformational ... within the overall protein structure, we could simply analyze the fluctuations of the mean ... value simply acts as an overall scale factor on the final results). In this case .... database (Porter et al 2004) or in an earlier elastic network study (Yang and ...

  6. Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced rhodobacter capsulatus cytochrome c(2) to the cytochrome bc(1) complex mediated by the conformation of the rieske iron-sulfur protein

    Devanathan, S.; Salamon, Z.; Tollin, G.; Fitch, J.C.; Meyer, T.E.; Berry, E.A.; Cusanovich, M.A.

    2007-01-01

    The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 M) but binds much more weakly to the oxidized form (Kd = 3.1 M). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 M. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 M) and reduced cytochrome c2 binds less strongly (Kd = 0.11 M) but ∼30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c

  7. Viscous conformal gauge theories

    Toniato, Arianna; Sannino, Francesco; Rischke, Dirk H.

    2017-01-01

    We present the conformal behavior of the shear viscosity-to-entropy density ratio and the fermion-number diffusion coefficient within the perturbative regime of the conformal window for gauge-fermion theories.......We present the conformal behavior of the shear viscosity-to-entropy density ratio and the fermion-number diffusion coefficient within the perturbative regime of the conformal window for gauge-fermion theories....

  8. Conformal Einstein spaces

    Kozameh, C.N.; Newman, E.T.; Tod, K.P.

    1985-01-01

    Conformal transformations in four-dimensional. In particular, a new set of two necessary and sufficient conditions for a space to be conformal to an Einstein space is presented. The first condition defines the class of spaces conformal to C spaces, whereas the last one (the vanishing of the Bach tensor) gives the particular subclass of C spaces which are conformally related to Einstein spaces. (author)

  9. Superspace conformal field theory

    Quella, Thomas [Koeln Univ. (Germany). Inst. fuer Theoretische Physik; Schomerus, Volker [Deutsches Elektronen-Synchrotron (DESY), Hamburg (Germany)

    2013-07-15

    Conformal sigma models and WZW models on coset superspaces provide important examples of logarithmic conformal field theories. They possess many applications to problems in string and condensed matter theory. We review recent results and developments, including the general construction of WZW models on type I supergroups, the classification of conformal sigma models and their embedding into string theory.

  10. Superspace conformal field theory

    Quella, Thomas

    2013-07-01

    Conformal sigma models and WZW models on coset superspaces provide important examples of logarithmic conformal field theories. They possess many applications to problems in string and condensed matter theory. We review recent results and developments, including the general construction of WZW models on type I supergroups, the classification of conformal sigma models and their embedding into string theory.

  11. Sorting protein lists with nwCompare: a simple and fast algorithm for n-way comparison of proteomic data files.

    Pont, Frédéric; Fournié, Jean Jacques

    2010-03-01

    MS, the reference technology for proteomics, routinely produces large numbers of protein lists whose fast comparison would prove very useful. Unfortunately, most softwares only allow comparisons of two to three lists at once. We introduce here nwCompare, a simple tool for n-way comparison of several protein lists without any query language, and exemplify its use with differential and shared cancer cell proteomes. As the software compares character strings, it can be applied to any type of data mining, such as genomic or metabolomic datalists.

  12. Generative Models of Conformational Dynamics

    Langmead, Christopher James

    2014-01-01

    Atomistic simulations of the conformational dynamics of proteins can be performed using either Molecular Dynamics or Monte Carlo procedures. The ensembles of three-dimensional structures produced during simulation can be analyzed in a number of ways to elucidate the thermodynamic and kinetic properties of the system. The goal of this chapter is to review both traditional and emerging methods for learning generative models from atomistic simulation data. Here, the term ‘generative’ refers to a...

  13. Limited proteolysis combined with isotope labeling and quantitative LC-MALDI MS for monitoring protein conformational changes: a study on calcium-binding sites of cardiac Troponin C

    McDonald, Chris; Li Liang

    2005-01-01

    Studies of protein-protein and protein-ligand interactions are important for understanding biological functions of proteins. A new technique based on the partial proteolysis of proteins combined with quantitative mass spectrometry is developed as a means of tracking structural changes after the formation of a protein-ligand complex. In this technique, a protein of interest with and without the binding of a ligand is digested with an enzyme to generate a set of peptides, followed by separation of the peptides by liquid chromatography. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is used to identify chromatographically separated peptides, and locate their sequence alignments in the parent protein. Using an isotopically labeled protein as a sample against an unlabeled protein standard, quantitative information can be gathered. This overcomes the inherent lack of quantitative capability of MALDI MS. The utility of the technique to investigate protein-ligand interactions is demonstrated in a model system involving calcium binding to cardiac Troponin C (cTnC). Using this technique, the general location of the three calcium-binding sites of cTnC can be determined by using several different enzymes to generate overlapping peptide maps of cTnC

  14. Non-conformable, partial and conformable transposition

    König, Thomas; Mäder, Lars Kai

    2013-01-01

    and the Commission regarding a directive’s outcome, play a much more strategic role than has to date acknowledged in the transposition literature. Whereas disagreement of a member state delays conformable transposition, it speeds up non-conformable transposition. Disagreement of the Commission only prolongs...... the transposition process. We therefore conclude that a stronger focus on an effective sanctioning mechanism is warranted for safeguarding compliance with directives....

  15. Fasting Induces Nuclear Factor E2-Related Factor 2 and ATP-Binding Cassette Transporters via Protein Kinase A and Sirtuin-1 in Mouse and Human

    Kulkarni, Supriya R.; Donepudi, Ajay C.; Xu, Jialin; Wei, Wei; Cheng, Qiuqiong C.; Driscoll, Maureen V.; Johnson, Delinda A.; Johnson, Jeffrey A.; Li, Xiaoling

    2014-01-01

    Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046

  16. Conformations of cationized linear oligosaccharides revealed by FTMS combined with in-ESI H/D exchange.

    Kostyukevich, Yury; Kononikhin, Alexey; Popov, Igor; Nikolaev, Eugene

    2015-10-01

    Previously (Kostyukevich et al. Anal Chem 2014, 86, 2595), we have reported that oligosaccharides anions are produced in the electrospray in two different conformations, which differ by the rate of gas phase hydrogen/deuterium (H/D) exchange reaction. In the present paper, we apply the in-electrospray ionization (ESI) source H/D exchange approach for the investigation of the oligosaccharides cations formed by attaching of metal ions (Na, K) to the molecule. It was observed that the formation of different conformers can be manipulated by varying the temperature of the desolvating capillary of the ESI interphase. Separation of the conformers was performed using gas phase H/D approach. Because the conformers have different rates of the H/D exchange reaction, the deuterium distribution spectrum becomes bimodal. It was found that the conformation corresponding to the slow H/D exchange rate dominates in the spectrum when the capillary temperature is low (~200 °C), and the conformation corresponding to the fast H/D exchange rate dominates at high (~400 °C) temperatures. In the intermediate temperature region, two conformers are present simultaneously. It was also observed that large oligosaccharide requires higher temperature for the formation of another conformer. It was found that the presence of the conformers considerably depends on the solvent used for ESI and the pH. We have compared these results with the previously performed in-ESI source H/D exchange experiments with peptides and proteins. Copyright © 2015 John Wiley & Sons, Ltd.

  17. A reassessment of synchronous fluorescence in the separation of Trp and Tyr contributions in protein emission and in the determination of conformational changes

    Bobone, Sara; van de Weert, Marco; Stella, Lorenzo

    2014-01-01

    solvents, as well as a real protein (bovine serum albumin). Unfortunately, synchronous spectra were found to be unreliable in the separation of Trp and Tyr emission components in proteins. A simple alternative approach based on the deconvolution of emission spectra is presented. In addition, an equation...

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

    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.

  19. One and two-dimensional electrophoresis of fast axonally-transported proteins in rat nerves following acrylamide and 2,5-hexanedione exposure

    Sickles, D.W.

    1990-01-01

    Transient and repeated deficiencies in protein delivery to the axon are observed following injections of acrylamide (ACR) and 2,5-hexanedione (2,5-HD) (Sickles DW, Neurotoxicology 10: 91;103, 1989; Neurosci Abstr 14:1219, 1988). We have furthered these studies by measuring the effects of single 50 mg/kg ACR and 4 nmole/kg 2,5-HD injections on the quantity of select fast-transported proteins. Proteins were radiolabelled with 3H-leucine injections of the DRG; 1 and 2 dimensional gels were used for separation of the sciatic nerve (9-45mm distal to the ganglion) homogenates. Scintillation counting demonstrated that transport of all proteins studied were affected by both toxicants. Some variation in effect was observed; a direct correlation between molecular weight (r=0.71) and original quantity of radiolabel (r=0.80) with the percent reduction in transport was observed. Some apparent increases in transport of certain proteins were observed on the 2D gels; but this may indicate a change in the isoelectric points of these transported proteins

  20. Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow

    Trabjerg, Esben; Jakobsen, Rasmus Uffe; Mysling, Simon

    2015-01-01

    Analysis of disulfide-bonded proteins by HDX-MS requires effective and rapid reduction of disulfide bonds before enzymatic digestion in order to increase sequence coverage. In a conventional HDX-MS workflow, disulfide bonds are reduced chemically by addition of a reducing agent to the quench......-antibody, respectively. The presented results demonstrate the successful electrochemical reduction during HDX-MS analysis of both a small exceptional tightly disulfide-bonded protein (NGF) as well as the largest protein attempted to date (IgG1-antibody). We envision that online electrochemical reduction...... the electrochemical reduction efficiency during HDX-MS analysis of two particularly challenging disulfide stabilized proteins: a therapeutic IgG1-antibody and Nerve Growth Factor-β (NGF). Several different parameters (flow rate, applied square wave potential as well as the type of labeling- and quench buffer) were...

  1. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.

    Stanger, Simone J; Law, Estelle A; Jamsai, Duangporn; O'Bryan, Moira K; Nixon, Brett; McLaughlin, Eileen A; Aitken, R John; Roman, Shaun D

    2016-08-01

    Spermatozoa require the process of capacitation to enable them to fertilize an egg. PKA is crucial to capacitation and the development of hyperactivated motility. Sperm PKA is activated by cAMP generated by the germ cell-enriched adenylyl cyclase encoded by Adcy10 Male mice lacking Adcy10 are sterile, because their spermatozoa are immotile. The current study was designed to identify binding partners of the sperm-specific (Cα2) catalytic subunit of PKA (PRKACA) by using it as the "bait" in a yeast 2-hybrid system. This approach was used to identify a novel germ cell-enriched protein, sperm PKA interacting factor (SPIF), in 25% of the positive clones. Homozygous Spif-null mice were embryonically lethal. SPIF was coexpressed and coregulated with PRKACA and with t-complex protein (TCP)-11, a protein associated with PKA signaling. We established that these 3 proteins form part of a novel complex in mouse spermatozoa. Upon capacitation, the SPIF protein becomes tyrosine phosphorylated in >95% of sperm. An apparent molecular rearrangement in the complex occurs, bringing PRKACA and TCP11 into proximity. Taken together, these results suggest a role for the novel complex of SPIF, PRKACA, and TCP11 during sperm capacitation, fertilization, and embryogenesis.-Stanger, S. J., Law, E. A., Jamsai, D., O'Bryan, M. K., Nixon, B., McLaughlin, E. A., Aitken, R. J., Roman, S. D. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation. © FASEB.

  2. Venom allergen-like protein 28 in Clonorchis sinensis: four epitopes on its surface and the potential role of Cys124 for its conformational stability.

    Lee, Myoung-Ro; Yoo, Won Gi; Kim, Yu Jung; Chung, Eun Ju; Cho, Shin-Hyeong; Ju, Jung-Won

    2018-06-06

    Venom allergen-like (VAL) proteins are important to host-parasite interactions. We previously demonstrated that a Clonorchis sinensis VAL (CsVAL) protein-derived synthetic peptide suppresses allergic and inflammatory responses. However, little is known regarding the physicochemical and antigenic properties of CsVAL proteins. Here, we identified a novel 194 amino acid VAL protein, named C. sinensis VAL 28 (CsVAL28), and characterized its functional motifs and structural details as a new member of the CAP superfamily. Unlike members of the Schistosoma mansoni VAL (SmVAL) family, CsVAL28 has a single CAP1 motif and six highly conserved disulfide bond-forming cysteines. Tertiary models of wild-type CsVAL28 and mutants were built using SmVAL4 as template via homology modeling. Normal mode analysis predicted that disulfide bond breaking by mutation of cysteine 124 to serine would greatly affect protein mobility. Four major immunoreactive linear epitopes were identified in the surface-exposed region or its vicinity via epitope mapping, using sera from clonorchiasis patients and healthy controls. Our findings provide in-depth knowledge on the structure-function properties of VAL proteins and may help determine highly antigenic regions for developing new diagnostic approaches.

  3. Effect of dietary protease supplementation and sex on dressing percentage and body conformation in broilers

    Dosković Vladimir; Bogosavljević-Bošković Snežana; Lukić Miloš; Škrbić Zdenka; Rakonjac Simeon; Petričević Veselin

    2016-01-01

    This paper presents research results on the effect of protease on the dressing percentage of conventionally dressed carcass and body conformation in broiler chickens. Broiler diet was supplemented with 0.2% protease (group E-I) and 0.3% protease (group E-II), and protein content in the feed was reduced by 4% (E-I) and 6% (E-II) through a decrease in soybean meal content. Fast-growing Cobb 500 broilers were used for a 63-day fattening trial. Body conformatio...

  4. Dietary changes in fasting levels of factor VII coagulant activity (FVII:C) are accompanied by changes in factor VII protein and other vitamin K-dependent proteins

    Bladbjerg, Else-Marie; Tholstrup, T; Marckmann, P

    1995-01-01

    The mechanisms behind dietary effects on fasting coagulant activity of factor VII (FVII:C) are not clarified. In the present study of 15 young volunteers, two experimental diets differing in composition of saturated fatty acids (C18:0 [diet S] or C12:0 + C14:0 [diet ML]) were served for 3 weeks...

  5. A new approach to protein enzymatic digestion for fast protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

    Dyčka, Filip; Laštovičková, Markéta; Bobálová, Janette

    2008-01-01

    Roč. 102, č. 15 (2008), s967-s968 ISSN 1803-2389. [Meeting on Chemistry and Life /4./. Brno, 09.09.2008-11.09.2008] R&D Projects: GA AV ČR IAA600040701 Institutional research plan: CEZ:AV0Z40310501 Keywords : enzymatic digest ion * fast approach Subject RIV: CB - Analytical Chemistry, Separation

  6. Nonsynonymous substitution rate (Ka is a relatively consistent parameter for defining fast-evolving and slow-evolving protein-coding genes

    Wang Lei

    2011-02-01

    Full Text Available Abstract Background Mammalian genome sequence data are being acquired in large quantities and at enormous speeds. We now have a tremendous opportunity to better understand which genes are the most variable or conserved, and what their particular functions and evolutionary dynamics are, through comparative genomics. Results We chose human and eleven other high-coverage mammalian genome data–as well as an avian genome as an outgroup–to analyze orthologous protein-coding genes using nonsynonymous (Ka and synonymous (Ks substitution rates. After evaluating eight commonly-used methods of Ka and Ks calculation, we observed that these methods yielded a nearly uniform result when estimating Ka, but not Ks (or Ka/Ks. When sorting genes based on Ka, we noticed that fast-evolving and slow-evolving genes often belonged to different functional classes, with respect to species-specificity and lineage-specificity. In particular, we identified two functional classes of genes in the acquired immune system. Fast-evolving genes coded for signal-transducing proteins, such as receptors, ligands, cytokines, and CDs (cluster of differentiation, mostly surface proteins, whereas the slow-evolving genes were for function-modulating proteins, such as kinases and adaptor proteins. In addition, among slow-evolving genes that had functions related to the central nervous system, neurodegenerative disease-related pathways were enriched significantly in most mammalian species. We also confirmed that gene expression was negatively correlated with evolution rate, i.e. slow-evolving genes were expressed at higher levels than fast-evolving genes. Our results indicated that the functional specializations of the three major mammalian clades were: sensory perception and oncogenesis in primates, reproduction and hormone regulation in large mammals, and immunity and angiotensin in rodents. Conclusion Our study suggests that Ka calculation, which is less biased compared to Ks and Ka

  7. Hepatic protein phosphatase 1 regulatory subunit 3B (Ppp1r3b) promotes hepatic glycogen synthesis and thereby regulates fasting energy homeostasis.

    Mehta, Minal B; Shewale, Swapnil V; Sequeira, Raymond N; Millar, John S; Hand, Nicholas J; Rader, Daniel J

    2017-06-23

    Maintenance of whole-body glucose homeostasis is critical to glycemic function. Genetic variants mapping to chromosome 8p23.1 in genome-wide association studies have been linked to glycemic traits in humans. The gene of known function closest to the mapped region, PPP1R3B (protein phosphatase 1 regulatory subunit 3B), encodes a protein (G L ) that regulates glycogen metabolism in the liver. We therefore sought to test the hypothesis that hepatic PPP1R3B is associated with glycemic traits. We generated mice with either liver-specific deletion ( Ppp1r3b Δ hep ) or liver-specific overexpression of Ppp1r3b The Ppp1r3b deletion significantly reduced glycogen synthase protein abundance, and the remaining protein was predominantly phosphorylated and inactive. As a consequence, glucose incorporation into hepatic glycogen was significantly impaired, total hepatic glycogen content was substantially decreased, and mice lacking hepatic Ppp1r3b had lower fasting plasma glucose than controls. The concomitant loss of liver glycogen impaired whole-body glucose homeostasis and increased hepatic expression of glycolytic enzymes in Ppp1r3b Δ hep mice relative to controls in the postprandial state. Eight hours of fasting significantly increased the expression of two critical gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, above the levels in control livers. Conversely, the liver-specific overexpression of Ppp1r3b enhanced hepatic glycogen storage above that of controls and, as a result, delayed the onset of fasting-induced hypoglycemia. Moreover, mice overexpressing hepatic Ppp1r3b upon long-term fasting (12-36 h) were protected from blood ketone-body accumulation, unlike control and Ppp1r3b Δ hep mice. These findings indicate a major role for Ppp1r3b in regulating hepatic glycogen stores and whole-body glucose/energy homeostasis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Induced quantum conformal gravity

    Novozhilov, Y.V.; Vassilevich, D.V.

    1988-11-01

    Quantum gravity is considered as induced by matter degrees of freedom and related to the symmetry breakdown in the low energy region of a non-Abelian gauge theory of fundamental fields. An effective action for quantum conformal gravity is derived where both the gravitational constant and conformal kinetic term are positive. Relation with induced classical gravity is established. (author). 15 refs

  9. Thickenings and conformal gravity

    Lebrun, Claude

    1991-07-01

    A twistor correspondence is given for complex conformal space-times with vanishing Bach and Eastwood-Dighton tensors; when the Weyl curvature is algebraically general, these equations are precisely the conformal version of Einstein's vacuum equations with cosmological constant. This gives a fully curved version of the linearized correspondence of Baston and Mason [B-M].

  10. Thickenings and conformal gravity

    LeBrun, C. (State Univ. of New York, Stony Brook, NY (USA). Dept. of Mathematics)

    1991-07-01

    A twistor correspondence is given for complex conformal space-times with vanishing Bach and Eastwood-Dighton tensors; when the Weyl curvature is algebraically general, these equations are precisely the conformal version of Einstein's vacuum equations with cosmological constant. This gives a fully curved version of the linearized correspondence of Baston and Mason (B-M). (orig.).

  11. Thickenings and conformal gravity

    LeBrun, C.

    1991-01-01

    A twistor correspondence is given for complex conformal space-times with vanishing Bach and Eastwood-Dighton tensors; when the Weyl curvature is algebraically general, these equations are precisely the conformal version of Einstein's vacuum equations with cosmological constant. This gives a fully curved version of the linearized correspondence of Baston and Mason [B-M]. (orig.)

  12. Conformal transformations in superspace

    Dao Vong Duc

    1977-01-01

    The spinor extension of the conformal algebra is investigated. The transformation law of superfields under the conformal coordinate inversion R defined in the superspace is derived. Using R-technique, the superconformally covariant two-point and three-point correlation functions are found

  13. Conformational stability of calreticulin

    Jørgensen, C.S.; Trandum, C.; Larsen, N.

    2005-01-01

    The conformational stability of calreticulin was investigated. Apparent unfolding temperatures (T-m) increased from 31 degrees C at pH 5 to 51 degrees C at pH 9, but electrophoretic analysis revealed that calreticulin oligomerized instead of unfolding. Structural analyses showed that the single C......-terminal a-helix was of major importance to the conformational stability of calreticulin....

  14. Low-molecular weight protein profiling of genetically modified maize using fast liquid chromatography electrospray ionization and time-of-flight mass spectrometry.

    Koc, Anna; Cañuelo, Ana; Garcia-Reyes, Juan F; Molina-Diaz, Antonio; Trojanowicz, Marek

    2012-06-01

    In this work, the use of liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC-TOFMS) has been evaluated for the profiling of relatively low-molecular weight protein species in both genetically modified (GM) and non-GM maize. The proposed approach consisted of a straightforward sample fractionation with different water and ethanol-based buffer solutions followed by separation and detection of the protein species using liquid chromatography with a small particle size (1.8 μm) C(18) column and electrospray-time-of-flight mass spectrometry detection in the positive ionization mode. The fractionation of maize reference material containing different content of transgenic material (from 0 to 5% GM) led to five different fractions (albumins, globulins, zeins, zein-like glutelins, and glutelins), all of them containing different protein species (from 2 to 52 different species in each fraction). Some relevant differences in the quantity and types of protein species were observed in the different fractions of the reference material (with different GM contents) tested, thus revealing the potential use of the proposed approach for fast protein profiling and to detect tentative GMO markers in maize. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Conformity index: A review

    Feuvret, Loic; Noel, Georges; Mazeron, Jean-Jacques; Bey, Pierre

    2006-01-01

    We present a critical analysis of the conformity indices described in the literature and an evaluation of their field of application. Three-dimensional conformal radiotherapy, with or without intensity modulation, is based on medical imaging techniques, three-dimensional dosimetry software, compression accessories, and verification procedures. It consists of delineating target volumes and critical healthy tissues to select the best combination of beams. This approach allows better adaptation of the isodose to the tumor volume, while limiting irradiation of healthy tissues. Tools must be developed to evaluate the quality of proposed treatment plans. Dosimetry software provides the dose distribution in each CT section and dose-volume histograms without really indicating the degree of conformity. The conformity index is a complementary tool that attributes a score to a treatment plan or that can compare several treatment plans for the same patient. The future of conformal index in everyday practice therefore remains unclear