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Sample records for determining protein backbone

  1. Inferential protein structure determination and refinement using fast, electronic structure based backbone amide chemical shift predictions

    CERN Document Server

    Christensen, Anders S

    2015-01-01

    This report covers the development of a new, fast method for calculating the backbone amide proton chemical shifts in proteins. Through quantum chemical calculations, structure-based forudsiglese the chemical shift for amidprotonen in protein has been parameterized. The parameters are then implemented in a computer program called Padawan. The program has since been implemented in protein folding program Phaistos, wherein the method andvendes to de novo folding of the protein structures and to refine the existing protein structures.

  2. The determinants of bond angle variability in protein/peptide backbones: A comprehensive statistical/quantum mechanics analysis.

    Science.gov (United States)

    Improta, Roberto; Vitagliano, Luigi; Esposito, Luciana

    2015-11-01

    The elucidation of the mutual influence between peptide bond geometry and local conformation has important implications for protein structure refinement, validation, and prediction. To gain insights into the structural determinants and the energetic contributions associated with protein/peptide backbone plasticity, we here report an extensive analysis of the variability of the peptide bond angles by combining statistical analyses of protein structures and quantum mechanics calculations on small model peptide systems. Our analyses demonstrate that all the backbone bond angles strongly depend on the peptide conformation and unveil the existence of regular trends as function of ψ and/or φ. The excellent agreement of the quantum mechanics calculations with the statistical surveys of protein structures validates the computational scheme here employed and demonstrates that the valence geometry of protein/peptide backbone is primarily dictated by local interactions. Notably, for the first time we show that the position of the H(α) hydrogen atom, which is an important parameter in NMR structural studies, is also dependent on the local conformation. Most of the trends observed may be satisfactorily explained by invoking steric repulsive interactions; in some specific cases the valence bond variability is also influenced by hydrogen-bond like interactions. Moreover, we can provide a reliable estimate of the energies involved in the interplay between geometry and conformations.

  3. Orientation-dependent backbone-only residue pair scoring functions for fixed backbone protein design

    Directory of Open Access Journals (Sweden)

    Bordner Andrew J

    2010-04-01

    Full Text Available Abstract Background Empirical scoring functions have proven useful in protein structure modeling. Most such scoring functions depend on protein side chain conformations. However, backbone-only scoring functions do not require computationally intensive structure optimization and so are well suited to protein design, which requires fast score evaluation. Furthermore, scoring functions that account for the distinctive relative position and orientation preferences of residue pairs are expected to be more accurate than those that depend only on the separation distance. Results Residue pair scoring functions for fixed backbone protein design were derived using only backbone geometry. Unlike previous studies that used spherical harmonics to fit 2D angular distributions, Gaussian Mixture Models were used to fit the full 3D (position only and 6D (position and orientation distributions of residue pairs. The performance of the 1D (residue separation only, 3D, and 6D scoring functions were compared by their ability to identify correct threading solutions for a non-redundant benchmark set of protein backbone structures. The threading accuracy was found to steadily increase with increasing dimension, with the 6D scoring function achieving the highest accuracy. Furthermore, the 3D and 6D scoring functions were shown to outperform side chain-dependent empirical potentials from three other studies. Next, two computational methods that take advantage of the speed and pairwise form of these new backbone-only scoring functions were investigated. The first is a procedure that exploits available sequence data by averaging scores over threading solutions for homologs. This was evaluated by applying it to the challenging problem of identifying interacting transmembrane alpha-helices and found to further improve prediction accuracy. The second is a protein design method for determining the optimal sequence for a backbone structure by applying Belief Propagation

  4. Three-Dimensional Protein Fold Determination from Backbone Amide Pseudocontact Shifts Generated by Lanthanide Tags at Multiple Sites

    KAUST Repository

    Yagi, Hiromasa

    2013-06-01

    Site-specific attachment of paramagnetic lanthanide ions to a protein generates pseudocontact shifts (PCS) in the nuclear magnetic resonance (NMR) spectra of the protein that are easily measured as changes in chemical shifts. By labeling the protein with lanthanide tags at four different sites, PCSs are observed for most amide protons and accurate information is obtained about their coordinates in three-dimensional space. The approach is demonstrated with the chaperone ERp29, for which large differences have been reported between X-ray and NMR structures of the C-terminal domain, ERp29-C. The results unambiguously show that the structure of rat ERp29-C in solution is similar to the crystal structure of human ERp29-C. PCSs of backbone amides were the only structural restraints required. Because these can be measured for more dilute protein solutions than other NMR restraints, the approach greatly widens the range of proteins amenable to structural studies in solution. © 2013 Elsevier Ltd. All rights reserved.

  5. Pyridoxamine Protects Protein Backbone from Oxidative Fragmentation

    Science.gov (United States)

    Chetyrkin, Sergei; Mathis, Missy; McDonald, W. Hayes; Shackelford, Xavier; Hudson, Billy; Voziyan, Paul

    2011-01-01

    Oxidative damage to proteins is one of the major pathogenic mechanisms in many chronic diseases. Therefore, inhibition of this oxidative damage can be an important part of therapeutic strategies. Pyridoxamine (PM), a prospective drug for treatment of diabetic nephropathy, has been previously shown to inhibit several oxidative and glycoxidative pathways, thus protecting amino acid side chains of the proteins from oxidative damage. Here, we demonstrated that PM can also protect protein backbone from fragmentation induced via different oxidative mechanisms including autoxidation of glucose. This protection was due to hydroxyl radical scavenging by PM and may contribute to PM therapeutic effects shown in clinical trials. PMID:21763683

  6. Changing the topology of protein backbone: the effect of backbone cyclization on the structure and dynamics of a SH3 domain

    Directory of Open Access Journals (Sweden)

    Frank H Schumann

    2015-04-01

    Full Text Available Understanding of the effects of the backbone cyclization on the structure and dynamics of a protein is essential for using protein topology engineering to alter protein stability and function. Here we have determined, for the first time, the structure and dynamics of the linear and various circular constructs of the N-SH3 domain from protein c-Crk. These constructs differ in the length and amino acid composition of the cyclization region. The backbone cyclization was carried out using intein-mediated intramolecular chemical ligation between the juxtaposed N- and the C-termini. The structure and backbone dynamics studies were performed using solution NMR. Our data suggest that the backbone cyclization has little effect on the overall three-dimensional structure of the SH3 domain: besides the termini, only minor structural changes were found in the proximity of the cyclization region. In contrast to the structure, backbone dynamics are significantly affected by the cyclization. On the subnanosecond time scale, the backbone of all circular constructs on average appears more rigid than that of the linear SH3 domain; this effect is observed over the entire backbone and is not limited to the cyclization site. The backbone mobility of the circular constructs becomes less restricted with increasing length of the circularization loop. In addition, significant conformational exchange motions (on the sub-millisecond time scale were found in the N-Src loop and in the adjacent β-strands in all circular constructs studied in this work. These effects of backbone cyclization on protein dynamics have potential implications for the stability of the protein fold and for ligand binding.

  7. The orientational preferences of backbones of proteins

    Institute of Scientific and Technical Information of China (English)

    CHEN Ying; WANG Jun; XUE Bin; WANG Wei

    2006-01-01

    The orientation between the backboneresidues of proteins is defined based on the local configurations and the corresponding preferences are analyzed by statistics. It is found that all the residue pairs have some specific preferences of orientations. The statistical analysis is mainly concentrated in the orientational distributions for two kinds of groupings of residues based on the hydrophobicity and secondary structural features. The statistics for such two types of groupings shows different orientational preferences. It is found that for the former grouping the orientational preference is rather weak,while for the later a kind of strong orientational preferences. This suggests that the formation of local structures and of secondary structures are highly related to the orientational preferences.

  8. Variation of protein backbone amide resonance by electrostatic field

    OpenAIRE

    Sharley, John N.

    2015-01-01

    Amide resonance is found to be sensitive to electrostatic field with component parallel or antiparallel the amide C-N bond. This effect is linear and without threshold in the biologically plausible electrostatic field range -0.005 to 0.005 au. Variation of amide resonance varies Resonance-Assisted Hydrogen Bonding such as occurs in the hydrogen bonded chains of backbone amides of protein secondary structures such as beta sheet and alpha helix, varying the stability of the secondary structure....

  9. Reconstruction of Protein Backbones from the BriX Collection of Canonical Protein Fragments

    OpenAIRE

    Lies Baeten; Joke Reumers; Vicente Tur; François Stricher; Tom Lenaerts; Luis Serrano; Frederic Rousseau; Joost Schymkowitz

    2008-01-01

    As modeling of changes in backbone conformation still lacks a computationally efficient solution, we developed a discretisation of the conformational states accessible to the protein backbone similar to the successful rotamer approach in side chains. The BriX fragment database, consisting of fragments from 4 to 14 residues long, was realized through identification of recurrent backbone fragments from a non-redundant set of high-resolution protein structures. BriX contains an alphabet of more ...

  10. Predicting the tolerated sequences for proteins and protein interfaces using RosettaBackrub flexible backbone design.

    Directory of Open Access Journals (Sweden)

    Colin A Smith

    Full Text Available Predicting the set of sequences that are tolerated by a protein or protein interface, while maintaining a desired function, is useful for characterizing protein interaction specificity and for computationally designing sequence libraries to engineer proteins with new functions. Here we provide a general method, a detailed set of protocols, and several benchmarks and analyses for estimating tolerated sequences using flexible backbone protein design implemented in the Rosetta molecular modeling software suite. The input to the method is at least one experimentally determined three-dimensional protein structure or high-quality model. The starting structure(s are expanded or refined into a conformational ensemble using Monte Carlo simulations consisting of backrub backbone and side chain moves in Rosetta. The method then uses a combination of simulated annealing and genetic algorithm optimization methods to enrich for low-energy sequences for the individual members of the ensemble. To emphasize certain functional requirements (e.g. forming a binding interface, interactions between and within parts of the structure (e.g. domains can be reweighted in the scoring function. Results from each backbone structure are merged together to create a single estimate for the tolerated sequence space. We provide an extensive description of the protocol and its parameters, all source code, example analysis scripts and three tests applying this method to finding sequences predicted to stabilize proteins or protein interfaces. The generality of this method makes many other applications possible, for example stabilizing interactions with small molecules, DNA, or RNA. Through the use of within-domain reweighting and/or multistate design, it may also be possible to use this method to find sequences that stabilize particular protein conformations or binding interactions over others.

  11. Variation of protein backbone amide resonance by electrostatic field

    CERN Document Server

    Sharley, John N

    2015-01-01

    Amide resonance is found to be sensitive to electrostatic field with component parallel or antiparallel the amide C-N bond. This effect is linear and without threshold in the biologically plausible electrostatic field range -0.005 to 0.005 au. Variation of amide resonance varies Resonance Assisted Hydrogen Bonding such as occurs in the hydrogen bonded chains of backbone amides of protein secondary structures such as beta sheet and non-polyproline helix such as alpha helix, varying the stability of the secondary structure. The electrostatic properties including permittivity of amino acid residue sidegroups influence the electrostatic field component parallel or antiparallel the C-N bond of each amide. The significance of this factor relative to other factors in protein folding depends on the magnitude of electrostatic field component parallel or antiparallel the C-N bond of each amide, and preliminary protein-scale calculations of the magnitude of these components suggest this factor warrants investigation in ...

  12. Bioactivities of fish protein hydrolysates from defatted salmon backbones

    Directory of Open Access Journals (Sweden)

    Rasa Slizyte

    2016-09-01

    Full Text Available Bioactivities of bulk fish protein hydrolysates (FPH from defatted salmon backbones obtained with eight different commercial enzymes and their combinations were tested. All FPH showed antioxidative activity in vitro. DPPH scavenging activity increased, while iron chelating ability decreased with increasing time of hydrolysis. All FPH showed ACE inhibiting effect which depended on type of enzyme and increased with time of hydrolysis. The highest effect was found for FPH produced with Trypsin. Bromelain + Papain hydrolysates reduced the uptake of radiolabelled glucose into CaCo-2 cells, a model of human enterocytes, indicating a potential antidiabetic effect of FPH. FPH obtained by Trypsin, Bromelain + Papain and Protamex showed the highest ACE inhibitory, cellular glucose transporter (GLUT/SGLT inhibitory and in vitro antioxidative activities, respectively. Correlation was observed between the measured bioactivities, degree of hydrolysis and molecular weight profiles, supporting prolonged hydrolysis to obtain high bioactivities.

  13. Backbone building from quadrilaterals: a fast and accurate algorithm for protein backbone reconstruction from alpha carbon coordinates.

    Science.gov (United States)

    Gront, Dominik; Kmiecik, Sebastian; Kolinski, Andrzej

    2007-07-15

    In this contribution, we present an algorithm for protein backbone reconstruction that comprises very high computational efficiency with high accuracy. Reconstruction of the main chain atomic coordinates from the alpha carbon trace is a common task in protein modeling, including de novo structure prediction, comparative modeling, and processing experimental data. The method employed in this work follows the main idea of some earlier approaches to the problem. The details and careful design of the present approach are new and lead to the algorithm that outperforms all commonly used earlier applications. BBQ (Backbone Building from Quadrilaterals) program has been extensively tested both on native structures as well as on near-native decoy models and compared with the different available existing methods. Obtained results provide a comprehensive benchmark of existing tools and evaluate their applicability to a large scale modeling using a reduced representation of protein conformational space. The BBQ package is available for downloading from our website at http://biocomp.chem.uw.edu.pl/services/BBQ/. This webpage also provides a user manual that describes BBQ functions in detail.

  14. Wetting of nonconserved residue-backbones: A feature indicative of aggregation associated regions of proteins.

    Science.gov (United States)

    Pradhan, Mohan R; Pal, Arumay; Hu, Zhongqiao; Kannan, Srinivasaraghavan; Chee Keong, Kwoh; Lane, David P; Verma, Chandra S

    2016-02-01

    Aggregation is an irreversible form of protein complexation and often toxic to cells. The process entails partial or major unfolding that is largely driven by hydration. We model the role of hydration in aggregation using "Dehydrons." "Dehydrons" are unsatisfied backbone hydrogen bonds in proteins that seek shielding from water molecules by associating with ligands or proteins. We find that the residues at aggregation interfaces have hydrated backbones, and in contrast to other forms of protein-protein interactions, are under less evolutionary pressure to be conserved. Combining evolutionary conservation of residues and extent of backbone hydration allows us to distinguish regions on proteins associated with aggregation (non-conserved dehydron-residues) from other interaction interfaces (conserved dehydron-residues). This novel feature can complement the existing strategies used to investigate protein aggregation/complexation.

  15. Pseudo 5D HN(C)N Experiment to Facilitate the Assignment of Backbone Resonances in Proteins Exhibiting High Backbone Shift Degeneracy

    CERN Document Server

    Kumar, Dinesh; Shukla, Vaibhav Kumar; Pandey, Himanshu; Arora, Ashish; Guleria, Anupam

    2014-01-01

    Assignment of protein backbone resonances is most routinely carried out using triple resonance three dimensional NMR experiments involving amide 1H and 15N resonances. However for intrinsically unstructured proteins, alpha-helical proteins or proteins containing several disordered fragments, the assignment becomes problematic because of high degree of backbone shift degeneracy. In this backdrop, a novel reduced dimensionality (RD) experiment -(5,3)D-hNCO-CANH- is presented to facilitate (and/or to validate) the sequential backbone resonance assignment in such proteins. The proposed 3D NMR experiment makes use of the modulated amide 15N chemical shifts (resulting from the joint sampling along both its indirect dimensions) to resolve the ambiguity involved in connecting the neighboring amide resonances (i.e. HiNi and Hi-1Ni-1) for overlapping amide NH peaks. The experiment -encoding 5D spectral information- leads to a conventional 3D spectrum with significantly reduced spectral crowding and complexity. The impr...

  16. Using Excel To Study The Relation Between Protein Dihedral Angle Omega And Backbone Length

    Science.gov (United States)

    Shew, Christopher; Evans, Samari; Tao, Xiuping

    How to involve the uninitiated undergraduate students in computational biophysics research? We made use of Microsoft Excel to carry out calculations of bond lengths, bond angles and dihedral angles of proteins. Specifically, we studied protein backbone dihedral angle omega by examining how its distribution varies with the length of the backbone length. It turns out Excel is a respectable tool for this task. An ordinary current-day desktop or laptop can handle the calculations for midsized proteins in just seconds. Care has to be taken to enter the formulas for the spreadsheet column after column to minimize the computing load. Supported in part by NSF Grant #1238795.

  17. APSY-NMR for protein backbone assignment in high-throughput structural biology

    Energy Technology Data Exchange (ETDEWEB)

    Dutta, Samit Kumar; Serrano, Pedro; Proudfoot, Andrew; Geralt, Michael [The Scripps Research Institute, Department of Integrative Structural and Computational Biology (United States); Pedrini, Bill [Paul Scherrer Institute (PSI), SwissFEL Project (Switzerland); Herrmann, Torsten [Université de Lyon, Institut des Sciences Analytiques, Centre de RMN à Très Hauts Champs, UMR 5280 CNRS, ENS Lyon, UCB Lyon 1 (France); Wüthrich, Kurt, E-mail: wuthrich@scripps.edu [The Scripps Research Institute, Department of Integrative Structural and Computational Biology (United States)

    2015-01-15

    A standard set of three APSY-NMR experiments has been used in daily practice to obtain polypeptide backbone NMR assignments in globular proteins with sizes up to about 150 residues, which had been identified as targets for structure determination by the Joint Center for Structural Genomics (JCSG) under the auspices of the Protein Structure Initiative (PSI). In a representative sample of 30 proteins, initial fully automated data analysis with the software UNIO-MATCH-2014 yielded complete or partial assignments for over 90 % of the residues. For most proteins the APSY data acquisition was completed in less than 30 h. The results of the automated procedure provided a basis for efficient interactive validation and extension to near-completion of the assignments by reference to the same 3D heteronuclear-resolved [{sup 1}H,{sup 1}H]-NOESY spectra that were subsequently used for the collection of conformational constraints. High-quality structures were obtained for all 30 proteins, using the J-UNIO protocol, which includes extensive automation of NMR structure determination.

  18. Backbone dynamics of the EIAV-Tat protein from {sup 15}N relaxation studies

    Energy Technology Data Exchange (ETDEWEB)

    Ejchart, A.; Herrmann, F.; Roesch, P.; Sticht, H.; Willbold, D. [Bayreuth Univ., Bayreuth (Germany)

    1994-12-31

    The work investigates the mobility of EIAV-Tat protein backbone by measuring the relaxation parameters of the {sup 15}N nitrogens. High degree of the flexibility, non-typical of rigid, well structured proteins was shown. 3 refs, 2 figs.

  19. Reduced Dimensionality (4,3)D-hnCOCANH Experiment: An Efficient Backbone Assignment tool for NMR studies of Proteins

    CERN Document Server

    Kumar, Dinesh

    2013-01-01

    Sequence specific resonance assignment and secondary structure determination of proteins form the basis for variety of structural and functional proteomics studies by NMR. In this context, an efficient standalone method for rapid assignment of backbone (1H, 15N, 13Ca and 13C') resonances and secondary structure determination of proteins has been presented here. Compared to currently available strategies used for the purpose, the method employs only a single reduced dimensionality (RD) experiment -(4,3)D-hnCOCANH and exploits the linear combinations of backbone (13Ca and 13C') chemical shifts to achieve a dispersion relatively better compared to those of individual chemical shifts (see the text) for efficient and rapid data analysis. Further, the experiment leads to the spectrum with direct distinction of self (intra-residue) and sequential (inter-residue) carbon correlation peaks; these appear opposite in signs and therefore can easily be discriminated without using an additional complementary experiment. On ...

  20. A Novel Method for Sampling Alpha-Helical Protein Backbones

    Science.gov (United States)

    Fain, Boris; Levitt, Michael

    2001-01-01

    We present a novel technique of sampling the configurations of helical proteins. Assuming knowledge of native secondary structure, we employ assembly rules gathered from a database of existing structures to enumerate the geometrically possible 3-D arrangements of the constituent helices. We produce a library of possible folds for 25 helical protein cores. In each case the method finds significant numbers of conformations close to the native structure. In addition we assign coordinates to all atoms for 4 of the 25 proteins. In the context of database driven exhaustive enumeration our method performs extremely well, yielding significant percentages of structures (0.02%--82%) within 6A of the native structure. The method's speed and efficiency make it a valuable contribution towards the goal of predicting protein structure.

  1. Solvent-induced differentiation of protein backbone hydrogen bonds in calmodulin.

    Science.gov (United States)

    Juranić, Nenad; Atanasova, Elena; Streiff, John H; Macura, Slobodan; Prendergast, Franklyn G

    2007-07-01

    In apo and holoCaM, almost half of the hydrogen bonds (H-bonds) at the protein backbone expected from the corresponding NMR or X-ray structures were not detected by h3JNC' couplings. The paucity of the h3JNC' couplings was considered in terms of dynamic features of these structures. We examined a set of seven proteins and found that protein-backbone H-bonds form two groups according to the h3JNC' couplings measured in solution. H-bonds that have h3JNC' couplings above the threshold of 0.2 Hz show distance/angle correlation among the H-bond geometrical parameters, and appear to be supported by the backbone dynamics in solution. The other H-bonds have no such correlation; they populate the water-exposed and flexible regions of proteins, including many of the CaM helices. The observed differentiation in a dynamical behavior of backbone H-bonds in apo and holoCaM appears to be related to protein functions.

  2. On the purported "backbone fluorescence" in protein three-dimensional fluorescence spectra

    DEFF Research Database (Denmark)

    Bortolotti, Annalisa; Wong, Yin How; Korsholm, Stine S.

    2016-01-01

    as any traditional protein emission spectrum. The many papers in reputable journals erroneously reporting this peak assignment, contradicting 5 decades of prior knowledge, have led to the creation of a new dogma, where many authors and reviewers now take the purported backbone fluorescence......In this study, several proteins (albumin, lysozyme, insulin) and model compounds (Trp, Tyr, homopolypeptides) were used to demonstrate the origin of the fluorescence observed upon their excitation at 220-230 nm. In the last 10 years we have observed a worrying increase in the number of articles...... claiming that this fluorescence originates from the protein backbone, contrary to the established knowledge that UV protein emission is due to aromatic amino acids only. Overall, our data clearly demonstrate that the observed emission upon excitation at 220-230 nm is due to the excitation of Tyr and/or Trp...

  3. Protein side-chain resonance assignment and NOE assignment using RDC-defined backbones without TOCSY data.

    Science.gov (United States)

    Zeng, Jianyang; Zhou, Pei; Donald, Bruce Randall

    2011-08-01

    One bottleneck in NMR structure determination lies in the laborious and time-consuming process of side-chain resonance and NOE assignments. Compared to the well-studied backbone resonance assignment problem, automated side-chain resonance and NOE assignments are relatively less explored. Most NOE assignment algorithms require nearly complete side-chain resonance assignments from a series of through-bond experiments such as HCCH-TOCSY or HCCCONH. Unfortunately, these TOCSY experiments perform poorly on large proteins. To overcome this deficiency, we present a novel algorithm, called NASCA: (NOE Assignment and Side-Chain Assignment), to automate both side-chain resonance and NOE assignments and to perform high-resolution protein structure determination in the absence of any explicit through-bond experiment to facilitate side-chain resonance assignment, such as HCCH-TOCSY. After casting the assignment problem into a Markov Random Field (MRF), NASCA: extends and applies combinatorial protein design algorithms to compute optimal assignments that best interpret the NMR data. The MRF captures the contact map information of the protein derived from NOESY spectra, exploits the backbone structural information determined by RDCs, and considers all possible side-chain rotamers. The complexity of the combinatorial search is reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is employed to find a set of optimal side-chain resonance assignments that best fit the NMR data. These side-chain resonance assignments are then used to resolve the NOE assignment ambiguity and compute high-resolution protein structures. Tests on five proteins show that NASCA: assigns resonances for more than 90% of side-chain protons, and achieves about 80% correct assignments. The final structures computed using the NOE distance restraints assigned by NASCA: have backbone

  4. Protein backbone dynamics revealed by quasi spectral density function analysis of amide N-15 nuclei.

    Science.gov (United States)

    Ishima, R; Nagayama, K

    1995-03-14

    Spectral density functions J(0), J(omega N), and J(omega H + omega N) of individual amide N-15 nuclei in proteins were approximated by a quasi spectral density function (QSDF). Using this function, the backbone dynamics were analyzed for seven protein systems on which data have been published. We defined J(0; omega N) as the difference between the J(0) and the J(omega N) values, which describes motions slower than 50 (or 60) MHz, and J(omega N; omega H+N) as the difference between the J(omega N) and the J(omega H + omega N) values, which describes motions slower than 450 (or 540) MHz. The QSDF analysis can easily extract the J(0; omega N) of protein backbones, which have often some relation to biologically relevant reactions. Flexible N-terminal regions in eglin c and glucose permease IIA and a loop region in eglin c showed smaller values of both the J(0; omega N) and the J(omega N; omega H+N) as compared with the other regions, indicating increases in motions faster than nanosecond. The values of the J(0; omega N) for the backbone of the FK506 binding protein showed a large variation in the apoprotein but fell in a very narrow range after the binding of FK506. Characteristic increase or decrease in the values of J(0) and J(omega N) was observed in two or three residues located between secondary structures.

  5. Unconventional N-H…N Hydrogen Bonds Involving Proline Backbone Nitrogen in Protein Structures.

    Science.gov (United States)

    Deepak, R N V Krishna; Sankararamakrishnan, Ramasubbu

    2016-05-10

    Contrary to DNA double-helical structures, hydrogen bonds (H-bonds) involving nitrogen as the acceptor are not common in protein structures. We systematically searched N-H…N H-bonds in two different sets of protein structures. Data set I consists of neutron diffraction and ultrahigh-resolution x-ray structures (0.9 Å resolution or better) and the hydrogen atom positions in these structures were determined experimentally. Data set II contains structures determined using x-ray diffraction (resolution ≤ 1.8 Å) and the positions of hydrogen atoms were generated using a computational method. We identified 114 and 14,347 potential N-H…N H-bonds from these two data sets, respectively, and 56-66% of these were of the Ni+1-Hi+1…Ni type, with Ni being the proline backbone nitrogen. To further understand the nature of such unusual contacts, we performed quantum chemical calculations on the model compound N-acetyl-L-proline-N-methylamide (Ace-Pro-NMe) with coordinates taken from the experimentally determined structures. A potential energy profile generated by varying the ψ dihedral angle in Ace-Pro-NMe indicates that the conformation with the N-H…N H-bond is the most stable. An analysis of H-bond-forming proline residues reveals that more than 30% of the proline carbonyl groups are also involved in n → π(∗) interactions with the carbonyl carbon of the preceding residue. Natural bond orbital analyses demonstrate that the strength of N-H…N H-bonds is less than half of that observed for a conventional H-bond. This study clearly establishes the H-bonding capability of proline nitrogen and its prevalence in protein structures. We found many proteins with multiple instances of H-bond-forming prolines. With more than 15% of all proline residues participating in N-H…N H-bonds, we suggest a new, to our knowledge, structural role for proline in providing stability to loops and capping regions of secondary structures in proteins.

  6. Prediction of backbone dihedral angles and protein secondary structure using support vector machines

    Directory of Open Access Journals (Sweden)

    Hirst Jonathan D

    2009-12-01

    Full Text Available Abstract Background The prediction of the secondary structure of a protein is a critical step in the prediction of its tertiary structure and, potentially, its function. Moreover, the backbone dihedral angles, highly correlated with secondary structures, provide crucial information about the local three-dimensional structure. Results We predict independently both the secondary structure and the backbone dihedral angles and combine the results in a loop to enhance each prediction reciprocally. Support vector machines, a state-of-the-art supervised classification technique, achieve secondary structure predictive accuracy of 80% on a non-redundant set of 513 proteins, significantly higher than other methods on the same dataset. The dihedral angle space is divided into a number of regions using two unsupervised clustering techniques in order to predict the region in which a new residue belongs. The performance of our method is comparable to, and in some cases more accurate than, other multi-class dihedral prediction methods. Conclusions We have created an accurate predictor of backbone dihedral angles and secondary structure. Our method, called DISSPred, is available online at http://comp.chem.nottingham.ac.uk/disspred/.

  7. Coupling Protein Side-Chain and Backbone Flexibility Improves the Re-design of Protein-Ligand Specificity.

    Directory of Open Access Journals (Sweden)

    Noah Ollikainen

    Full Text Available Interactions between small molecules and proteins play critical roles in regulating and facilitating diverse biological functions, yet our ability to accurately re-engineer the specificity of these interactions using computational approaches has been limited. One main difficulty, in addition to inaccuracies in energy functions, is the exquisite sensitivity of protein-ligand interactions to subtle conformational changes, coupled with the computational problem of sampling the large conformational search space of degrees of freedom of ligands, amino acid side chains, and the protein backbone. Here, we describe two benchmarks for evaluating the accuracy of computational approaches for re-engineering protein-ligand interactions: (i prediction of enzyme specificity altering mutations and (ii prediction of sequence tolerance in ligand binding sites. After finding that current state-of-the-art "fixed backbone" design methods perform poorly on these tests, we develop a new "coupled moves" design method in the program Rosetta that couples changes to protein sequence with alterations in both protein side-chain and protein backbone conformations, and allows for changes in ligand rigid-body and torsion degrees of freedom. We show significantly increased accuracy in both predicting ligand specificity altering mutations and binding site sequences. These methodological improvements should be useful for many applications of protein-ligand design. The approach also provides insights into the role of subtle conformational adjustments that enable functional changes not only in engineering applications but also in natural protein evolution.

  8. Microsecond molecular dynamics simulation shows effect of slow loop dynamics on backbone amide order parameters of proteins

    DEFF Research Database (Denmark)

    Maragakis, Paul; Lindorff-Larsen, Kresten; Eastwood, Michael P

    2008-01-01

    methods. However, apparent systematic discrepancies between order parameters extracted from simulations and experiments are common, particularly for elements of noncanonical secondary structure. In this paper, results from a 1.2 micros explicit solvent MD simulation of the protein ubiquitin are compared...... with previously determined backbone order parameters derived from NMR relaxation experiments [Tjandra, N.; Feller, S. E.; Pastor, R. W.; Bax, A. J. Am. Chem. Soc. 1995, 117, 12562-12566]. The simulation reveals fluctuations in three loop regions that occur on time scales comparable to or longer than...

  9. Automatic assignment of protein backbone resonances by direct spectrum inspection in targeted acquisition of NMR data.

    Science.gov (United States)

    Wong, Leo E; Masse, James E; Jaravine, Victor; Orekhov, Vladislav; Pervushin, Konstantin

    2008-10-01

    The necessity to acquire large multidimensional datasets, a basis for assignment of NMR resonances, results in long data acquisition times during which substantial degradation of a protein sample might occur. Here we propose a method applicable for such a protein for automatic assignment of backbone resonances by direct inspection of multidimensional NMR spectra. In order to establish an optimal balance between completeness of resonance assignment and losses of cross-peaks due to dynamic processes/degradation of protein, assignment of backbone resonances is set as a stirring criterion for dynamically controlled targeted nonlinear NMR data acquisition. The result is demonstrated with the 12 kDa (13)C,(15) N-labeled apo-form of heme chaperone protein CcmE, where hydrolytic cleavage of 29 C-terminal amino acids is detected. For this protein, 90 and 98% of manually assignable resonances are automatically assigned within 10 and 40 h of nonlinear sampling of five 3D NMR spectra, respectively, instead of 600 h needed to complete the full time domain grid. In addition, resonances stemming from degradation products are identified. This study indicates that automatic resonance assignment might serve as a guiding criterion for optimal run-time allocation of NMR resources in applications to proteins prone to degradation.

  10. Automatic assignment of protein backbone resonances by direct spectrum inspection in targeted acquisition of NMR data

    Energy Technology Data Exchange (ETDEWEB)

    Wong, Leo E. [Nanyang Technological University, School of Biological Sciences (Singapore); Masse, James E. [National Institutes of Health (United States); Jaravine, Victor [J. W. Goethe-University Frankfurt, Institute of Biophysical Chemistry (Germany); Orekhov, Vladislav [Gothenburg University, Swedish NMR Centre (Sweden); Pervushin, Konstantin [Nanyang Technological University, School of Biological Sciences (Singapore)], E-mail: kpervushin@ntu.edu.sg

    2008-10-15

    The necessity to acquire large multidimensional datasets, a basis for assignment of NMR resonances, results in long data acquisition times during which substantial degradation of a protein sample might occur. Here we propose a method applicable for such a protein for automatic assignment of backbone resonances by direct inspection of multidimensional NMR spectra. In order to establish an optimal balance between completeness of resonance assignment and losses of cross-peaks due to dynamic processes/degradation of protein, assignment of backbone resonances is set as a stirring criterion for dynamically controlled targeted nonlinear NMR data acquisition. The result is demonstrated with the 12 kDa {sup 13}C,{sup 15} N-labeled apo-form of heme chaperone protein CcmE, where hydrolytic cleavage of 29 C-terminal amino acids is detected. For this protein, 90 and 98% of manually assignable resonances are automatically assigned within 10 and 40 h of nonlinear sampling of five 3D NMR spectra, respectively, instead of 600 h needed to complete the full time domain grid. In addition, resonances stemming from degradation products are identified. This study indicates that automatic resonance assignment might serve as a guiding criterion for optimal run-time allocation of NMR resources in applications to proteins prone to degradation.

  11. RosettaRemodel: a generalized framework for flexible backbone protein design.

    Directory of Open Access Journals (Sweden)

    Po-Ssu Huang

    Full Text Available We describe RosettaRemodel, a generalized framework for flexible protein design that provides a versatile and convenient interface to the Rosetta modeling suite. RosettaRemodel employs a unified interface, called a blueprint, which allows detailed control over many aspects of flexible backbone protein design calculations. RosettaRemodel allows the construction and elaboration of customized protocols for a wide range of design problems ranging from loop insertion and deletion, disulfide engineering, domain assembly, loop remodeling, motif grafting, symmetrical units, to de novo structure modeling.

  12. Predicting disease-related proteins based on clique backbone in protein-protein interaction network.

    Science.gov (United States)

    Yang, Lei; Zhao, Xudong; Tang, Xianglong

    2014-01-01

    Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases.

  13. Structure and assembly of group B streptococcus pilus 2b backbone protein.

    Science.gov (United States)

    Cozzi, Roberta; Malito, Enrico; Lazzarin, Maddalena; Nuccitelli, Annalisa; Castagnetti, Andrea; Bottomley, Matthew J; Margarit, Immaculada; Maione, Domenico; Rinaudo, C Daniela

    2015-01-01

    Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.

  14. Structure and assembly of group B streptococcus pilus 2b backbone protein.

    Directory of Open Access Journals (Sweden)

    Roberta Cozzi

    Full Text Available Group B Streptococcus (GBS is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468 encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.

  15. Protein Side-Chain Resonance Assignment and NOE Assignment Using RDC-Defined Backbones without TOCSY Data3

    Science.gov (United States)

    Zeng, Jianyang; Zhou, Pei; Donald, Bruce Randall

    2011-01-01

    One bottleneck in NMR structure determination lies in the laborious and time-consuming process of side-chain resonance and NOE assignments. Compared to the well-studied backbone resonance assignment problem, automated side-chain resonance and NOE assignments are relatively less explored. Most NOE assignment algorithms require nearly complete side-chain resonance assignments from a series of through-bond experiments such as HCCH-TOCSY or HCCCONH. Unfortunately, these TOCSY experiments perform poorly on large proteins. To overcome this deficiency, we present a novel algorithm, called NASCA (NOE Assignment and Side-Chain Assignment), to automate both side-chain resonance and NOE assignments and to perform high-resolution protein structure determination in the absence of any explicit through-bond experiment to facilitate side-chain resonance assignment, such as HCCH-TOCSY. After casting the assignment problem into a Markov Random Field (MRF), NASCA extends and applies combinatorial protein design algorithms to compute optimal assignments that best interpret the NMR data. The MRF captures the contact map information of the protein derived from NOESY spectra, exploits the backbone structural information determined by RDCs, and considers all possible side-chain rotamers. The complexity of the combinatorial search is reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is employed to find a set of optimal side-chain resonance assignments that best fit the NMR data. These side-chain resonance assignments are then used to resolve the NOE assignment ambiguity and compute high-resolution protein structures. Tests on five proteins show that NASCA assigns resonances for more than 90% of side-chain protons, and achieves about 80% correct assignments. The final structures computed using the NOE distance restraints assigned by NASCA have backbone RMSD 0

  16. Backbone structure of Yersinia pestis Ail determined in micelles by NMR-restrained simulated annealing with implicit membrane solvation

    Energy Technology Data Exchange (ETDEWEB)

    Marassi, Francesca M., E-mail: fmarassi@sbmri.org; Ding, Yi [Sanford-Burnham Medical Research Institute (United States); Schwieters, Charles D. [National Institutes of Health, Division of Computational Bioscience, Center for Information Technology (United States); Tian, Ye; Yao, Yong [Sanford-Burnham Medical Research Institute (United States)

    2015-09-15

    The outer membrane protein Ail (attachment invasion locus) is a virulence factor of Yersinia pestis that mediates cell invasion, cell attachment and complement resistance. Here we describe its three-dimensional backbone structure determined in decyl-phosphocholine (DePC) micelles by NMR spectroscopy. The NMR structure was calculated using the membrane function of the implicit solvation potential, eefxPot, which we have developed to facilitate NMR structure calculations in a physically realistic environment. We show that the eefxPot force field guides the protein towards its native fold. The resulting structures provide information about the membrane-embedded global position of Ail, and have higher accuracy, higher precision and improved conformational properties, compared to the structures calculated with the standard repulsive potential.

  17. Enhanced biosynthetically directed fractional carbon-13 enrichment of proteins for backbone NMR assignments.

    Science.gov (United States)

    Wenrich, Broc R; Sonstrom, Reilly E; Gupta, Riju A; Rovnyak, David

    2015-11-01

    Routes to carbon-13 enrichment of bacterially expressed proteins include achieving uniform or positionally selective (e.g. ILV-Me, or (13)C', etc.) enrichment. We consider the potential for biosynthetically directed fractional enrichment (e.g. carbon-13 incorporation in the protein less than 100%) for performing routine n-(D)dimensional NMR spectroscopy of proteins. First, we demonstrate an approach to fractional isotope addition where the initial growth media containing natural abundance glucose is replenished at induction with a small amount (e.g. 10%(w/w)u-(13)C-glucose) of enriched nutrient. The approach considered here is to add 10% (e.g. 200mg for a 2g/L culture) u-(13)C-glucose at the induction time (OD600=0.8), resulting in a protein with enhanced (13)C incorporation that gives almost the same NMR signal levels as an exact 20% (13)C sample. Second, whereas fractional enrichment is used for obtaining stereospecific methyl assignments, we find that (13)C incorporation levels no greater than 20%(w/w) yield (13)C and (13)C-(13)C spin pair incorporation sufficient to conduct typical 3D-bioNMR backbone experiments on moderate instrumentation (600 MHz, RT probe). Typical 3D-bioNMR experiments of a fractionally enriched protein yield expected backbone connectivities, and did not show amino acid biases in this work, with one exception. When adding 10% u-(13)C glucose to expression media at induction, there is poor preservation of (13)Cα-(13)Cβ spin pairs in the amino acids ILV, leading to the absence of Cβ signals in HNCACB spectra for ILV, a potentially useful editing effect. Enhanced fractional carbon-13 enrichment provides lower-cost routes to high throughput protein NMR studies, and makes modern protein NMR more cost-accessible.

  18. On correlation between protein secondary structure, backbone bond angles, and side-chain orientations

    CERN Document Server

    Lundgren, Martin

    2012-01-01

    We investigate the fine structure of the sp3 hybridized covalent bond geometry that governs the tetrahedral architecture around the central C$_\\alpha$ carbon of a protein backbone, and for this we develop new visualization techniques to analyze high resolution X-ray structures in Protein Data Bank. We observe that there is a correlation between the deformations of the ideal tetrahedral symmetry and the local secondary structure of the protein. We propose a universal coarse grained energy function to describe the ensuing side-chain geometry in terms of the C$_\\beta$ carbon orientations. The energy function can model the side-chain geometry with a sub-atomic precision. As an example we construct the C$_\\alpha$-C$_\\beta$ structure of HP35 chicken villin headpiece. We obtain a configuration that deviates less than 0.4 \\.A in root-mean-square distance from the experimental X-ray structure.

  19. Subpicosecond protein backbone changes detected during the green-absorbing proteorhodopsin primary photoreaction.

    Science.gov (United States)

    Amsden, Jason J; Kralj, Joel M; Chieffo, Logan R; Wang, Xihua; Erramilli, Shyamsunder; Spudich, Elena N; Spudich, John L; Ziegler, Lawrence D; Rothschild, Kenneth J

    2007-10-11

    Recent studies demonstrate that photoactive proteins can react within several picoseconds to photon absorption by their chromophores. Faster subpicosecond protein responses have been suggested to occur in rhodopsin-like proteins where retinal photoisomerization may impulsively drive structural changes in nearby protein groups. Here, we test this possibility by investigating the earliest protein structural changes occurring in proteorhodopsin (PR) using ultrafast transient infrared (TIR) spectroscopy with approximately 200 fs time resolution combined with nonperturbing isotope labeling. PR is a recently discovered microbial rhodopsin similar to bacteriorhodopsin (BR) found in marine proteobacteria and functions as a proton pump. Vibrational bands in the retinal fingerprint (1175-1215 cm(-1)) and ethylenic stretching (1500-1570 cm(-1)) regions characteristic of all-trans to 13-cis chromophore isomerization and formation of a red-shifted photointermediate appear with a 500-700 fs time constant after photoexcitation. Bands characteristic of partial return to the ground state evolve with a 2.0-3.5 ps time constant. In addition, a negative band appears at 1548 cm(-1) with a time constant of 500-700 fs, which on the basis of total-15N and retinal C15D (retinal with a deuterium on carbon 15) isotope labeling is assigned to an amide II peptide backbone mode that shifts to near 1538 cm(-1) concomitantly with chromophore isomerization. Our results demonstrate that one or more peptide backbone groups in PR respond with a time constant of 500-700 fs, almost coincident with the light-driven retinylidene chromophore isomerization. The protein changes we observe on a subpicosecond time scale may be involved in storage of the absorbed photon energy subsequently utilized for proton transport.

  20. Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding study.

    Science.gov (United States)

    Hein, Christopher; Löhr, Frank; Schwarz, Daniel; Dötsch, Volker

    2017-05-01

    Selective labeling with stable isotopes has long been recognized as a valuable tool in protein NMR to alleviate signal overlap and sensitivity limitations. In this study, combinatorial (15) N-, (13) C(α) -, and (13) C'-selective labeling has been used during the backbone assignment of human cyclophilin D to explore binding of an inhibitor molecule. Using a cell-free expression system, a scheme that involves (15) N, 1-(13) C, 2-(13) C, fully (15) N/(13) C, and unlabeled amino acids was optimized to gain a maximum of assignment information from three samples. This scheme was combined with time-shared triple-resonance NMR experiments, which allows a fast and efficient backbone assignment by giving the unambiguous assignment of unique amino acid pairs in the protein, the identity of ambiguous pairs and information about all 19 non-proline amino acid types. It is therefore well suited for binding studies where de novo assignments of amide (1) H and (15) N resonances need to be obtained, even in cases where sensitivity is the limiting factor. © 2016 Wiley Periodicals, Inc.

  1. Limits on variations in protein backbone dynamics from precise measurements of scalar couplings.

    Science.gov (United States)

    Vögeli, Beat; Ying, Jinfa; Grishaev, Alexander; Bax, Ad

    2007-08-01

    3JHN,Halpha, 3JHN,Cbeta, and 3JHN,C' couplings, all related to the backbone torsion angle phi, were measured for the third immunoglobulin binding domain of protein G, or GB3. Measurements were carried out using both previously published methods and novel sequences based on the multiple-quantum principle, which limit attenuation of experimental couplings caused by finite lifetimes of the spin states of passive spins. High reproducibility between the multiple-quantum and conventional approaches confirms the accuracy of the measurements. With few exceptions, close agreement between 3JHN,Halpha, 3JHN,Cbeta, and 3JHN,C' and values predicted by their respective Karplus equations is observed. For the three types of couplings, up to 20% better agreement is obtained when fitting the experimental couplings to a dynamic ensemble NMR structure, which has a phi angle root-mean-square spread of 9 +/- 4 degrees and was previously calculated on the basis of a very extensive set of residual dipolar couplings, than for any single static NMR structure. Fits of 3J couplings to a 1.1-A X-ray structure, with hydrogens added in idealized positions, are 40-90% worse. Approximately half of the improvement when fitting to the NMR structures relates to the amide proton deviating from its idealized, in-peptide-plane position, indicating that the positioning of hydrogens relative to the backbone atoms is one of the factors limiting the accuracy at which the backbone torsion angle phi can be extracted from 3J couplings. Introducing an additional, residue-specific variable for the amplitude of phi angle fluctuations does not yield a statistically significant improvement when fitting to a set of dynamic Karplus curves, pointing to a homogeneous behavior of these amplitudes.

  2. Coupling Protein Side-Chain and Backbone Flexibility Improves the Re-design of Protein-Ligand Specificity

    Science.gov (United States)

    Ollikainen, Noah; de Jong, René M.; Kortemme, Tanja

    2015-01-01

    Interactions between small molecules and proteins play critical roles in regulating and facilitating diverse biological functions, yet our ability to accurately re-engineer the specificity of these interactions using computational approaches has been limited. One main difficulty, in addition to inaccuracies in energy functions, is the exquisite sensitivity of protein–ligand interactions to subtle conformational changes, coupled with the computational problem of sampling the large conformational search space of degrees of freedom of ligands, amino acid side chains, and the protein backbone. Here, we describe two benchmarks for evaluating the accuracy of computational approaches for re-engineering protein-ligand interactions: (i) prediction of enzyme specificity altering mutations and (ii) prediction of sequence tolerance in ligand binding sites. After finding that current state-of-the-art “fixed backbone” design methods perform poorly on these tests, we develop a new “coupled moves” design method in the program Rosetta that couples changes to protein sequence with alterations in both protein side-chain and protein backbone conformations, and allows for changes in ligand rigid-body and torsion degrees of freedom. We show significantly increased accuracy in both predicting ligand specificity altering mutations and binding site sequences. These methodological improvements should be useful for many applications of protein – ligand design. The approach also provides insights into the role of subtle conformational adjustments that enable functional changes not only in engineering applications but also in natural protein evolution. PMID:26397464

  3. Optimization of Protein Backbone Dihedral Angles by Means of Hamiltonian Reweighting.

    Science.gov (United States)

    Margreitter, Christian; Oostenbrink, Chris

    2016-09-26

    Molecular dynamics simulations depend critically on the accuracy of the underlying force fields in properly representing biomolecules. Hence, it is crucial to validate the force-field parameter sets in this respect. In the context of the GROMOS force field, this is usually achieved by comparing simulation data to experimental observables for small molecules. In this study, we develop new amino acid backbone dihedral angle potential energy parameters based on the widely used 54A7 parameter set by matching to experimental J values and secondary structure propensity scales. In order to find the most appropriate backbone parameters, close to 100 000 different combinations of parameters have been screened. However, since the sheer number of combinations considered prohibits actual molecular dynamics simulations for each of them, we instead predicted the values for every combination using Hamiltonian reweighting. While the original 54A7 parameter set fails to reproduce the experimental data, we are able to provide parameters that match significantly better. However, to ensure applicability in the context of larger peptides and full proteins, further studies have to be undertaken.

  4. Polarizable Simulations with Second order Interaction Model (POSSIM) force field: Developing parameters for alanine peptides and protein backbone

    Science.gov (United States)

    Ponomarev, Sergei Y.; Kaminski, George A.

    2011-01-01

    A previously introduced POSSIM (POlarizable Simulations with Second order Interaction Model) force field has been extended to include parameters for alanine peptides and protein backbones. New features were introduced into the fitting protocol, as compared to the previous generation of the polarizable force field for proteins. A reduced amount of quantum mechanical data was employed in fitting the electrostatic parameters. Transferability of the electrostatics between our recently developed NMA model and the protein backbone was confirmed. Binding energy and geometry for complexes of alanine dipeptide with a water molecule were estimated and found in a good agreement with high-level quantum mechanical results (for example, the intermolecular distances agreeing within ca. 0.06Å). Following the previously devised procedure, we calculated average errors in alanine di- and tetra-peptide conformational energies and backbone angles and found the agreement to be adequate (for example, the alanine tetrapeptide extended-globular conformational energy gap was calculated to be 3.09 kcal/mol quantim mechanically and 3.14 kcal/mol with the POSSIM force field). However, we have now also included simulation of a simple alpha-helix in both gas-phase and water as the ultimate test of the backbone conformational behavior. The resulting alanine and protein backbone force field is currently being employed in further development of the POSSIM fast polarizable force field for proteins. PMID:21743799

  5. Probing the role of backbone hydrogen bonds in protein-peptide interactions by amide-to-ester mutations

    DEFF Research Database (Denmark)

    Eildal, Jonas N N; Hultqvist, Greta; Balle, Thomas;

    2013-01-01

    -protein interactions, those of the PDZ domain family involve formation of intermolecular hydrogen bonds: C-termini or internal linear motifs of proteins bind as β-strands to form an extended antiparallel β-sheet with the PDZ domain. Whereas extensive work has focused on the importance of the amino acid side chains...... of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding...... to natural protein ligands. Amide-to-ester mutations of the three C-terminal amides of the peptide ligand severely affected the affinity with the PDZ domain, demonstrating that hydrogen bonds contribute significantly to ligand binding (apparent changes in binding energy, ΔΔG = 1.3 to >3.8 kcal mol(-1...

  6. Quantitative residue-specific protein backbone torsion angle dynamics from concerted measurement of 3J couplings.

    Science.gov (United States)

    Lee, Jung Ho; Li, Fang; Grishaev, Alexander; Bax, Ad

    2015-02-04

    Three-bond (3)J(C'C') and (3)J(HNHα) couplings in peptides and proteins are functions of the intervening backbone torsion angle ϕ. In well-ordered regions, (3)J(HNHα) is tightly correlated with (3)J(C'C'), but the presence of large ϕ angle fluctuations differentially affects the two types of couplings. Assuming the ϕ angles follow a Gaussian distribution, the width of this distribution can be extracted from (3)J(C'C') and (3)J(HNHα), as demonstrated for the folded proteins ubiquitin and GB3. In intrinsically disordered proteins, slow transverse relaxation permits measurement of (3)J(C'C') and (3)J(HNH) couplings at very high precision, and impact of factors other than the intervening torsion angle on (3)J will be minimal, making these couplings exceptionally valuable structural reporters. Analysis of α-synuclein yields rather homogeneous widths of 69 ± 6° for the ϕ angle distributions and (3)J(C'C') values that agree well with those of a recent maximum entropy analysis of chemical shifts, J couplings, and (1)H-(1)H NOEs. Data are consistent with a modest (≤30%) population of the polyproline II region.

  7. Rapid analysis of protein backbone resonance assignments using cryogenic probes, a distributed Linux-based computing architecture, and an integrated set of spectral analysis tools.

    Science.gov (United States)

    Monleón, Daniel; Colson, Kimberly; Moseley, Hunter N B; Anklin, Clemens; Oswald, Robert; Szyperski, Thomas; Montelione, Gaetano T

    2002-01-01

    Rapid data collection, spectral referencing, processing by time domain deconvolution, peak picking and editing, and assignment of NMR spectra are necessary components of any efficient integrated system for protein NMR structure analysis. We have developed a set of software tools designated AutoProc, AutoPeak, and AutoAssign, which function together with the data processing and peak-picking programs NMRPipe and Sparky, to provide an integrated software system for rapid analysis of protein backbone resonance assignments. In this paper we demonstrate that these tools, together with high-sensitivity triple resonance NMR cryoprobes for data collection and a Linux-based computer cluster architecture, can be combined to provide nearly complete backbone resonance assignments and secondary structures (based on chemical shift data) for a 59-residue protein in less than 30 hours of data collection and processing time. In this optimum case of a small protein providing excellent spectra, extensive backbone resonance assignments could also be obtained using less than 6 hours of data collection and processing time. These results demonstrate the feasibility of high throughput triple resonance NMR for determining resonance assignments and secondary structures of small proteins, and the potential for applying NMR in large scale structural proteomics projects.

  8. Structural dynamics of protein backbone {phi} angles: extended molecular dynamics simulations versus experimental {sup 3}J scalar couplings

    Energy Technology Data Exchange (ETDEWEB)

    Markwick, Phineus R. L. [CNRS/CEA/UJF, Protein Dynamics and Flexibility, Institut de Biologie Structurale Jean-Pierre Ebel UMR 5075 (France); Showalter, Scott A. [Florida State University, Department of Chemistry and Biochemistry, NHMFL (United States); Bouvignies, Guillaume [CNRS/CEA/UJF, Protein Dynamics and Flexibility, Institut de Biologie Structurale Jean-Pierre Ebel UMR 5075 (France); Brueschweiler, Rafael [Florida State University, Department of Chemistry and Biochemistry, NHMFL (United States)], E-mail: bruschweiler@magnet.fsu.edu; Blackledge, Martin [CNRS/CEA/UJF, Protein Dynamics and Flexibility, Institut de Biologie Structurale Jean-Pierre Ebel UMR 5075 (France)], E-mail: martin.blackledge@ibs.fr

    2009-09-15

    {sup 3}J scalar couplings report on the conformational averaging of backbone {phi} angles in peptides and proteins, and therefore represent a potentially powerful tool for studying the details of both structure and dynamics in solution. We have compared an extensive experimental dataset with J-couplings predicted from unrestrained molecular dynamics simulation using enhanced sampling available from accelerated molecular dynamics or using long timescale trajectories (200 ns). The dynamic fluctuations predicted to be present along the backbone, in agreement with residual dipolar coupling analysis, are compatible with the experimental {sup 3}J scalar couplings providing a slightly better reproduction of these experimental parameters than a high-resolution static structure.

  9. A Multi-Objective Approach for Protein Structure Prediction Based on an Energy Model and Backbone Angle Preferences.

    Science.gov (United States)

    Tsay, Jyh-Jong; Su, Shih-Chieh; Yu, Chin-Sheng

    2015-07-03

    Protein structure prediction (PSP) is concerned with the prediction of protein tertiary structure from primary structure and is a challenging calculation problem. After decades of research effort, numerous solutions have been proposed for optimisation methods based on energy models. However, further investigation and improvement is still needed to increase the accuracy and similarity of structures. This study presents a novel backbone angle preference factor, which is one of the factors inducing protein folding. The proposed multiobjective optimisation approach simultaneously considers energy models and backbone angle preferences to solve the ab initio PSP. To prove the effectiveness of the multiobjective optimisation approach based on the energy models and backbone angle preferences, 75 amino acid sequences with lengths ranging from 22 to 88 amino acids were selected from the CB513 data set to be the benchmarks. The data sets were highly dissimilar, therefore indicating that they are meaningful. The experimental results showed that the root-mean-square deviation (RMSD) of the multiobjective optimization approach based on energy model and backbone angle preferences was superior to those of typical energy models, indicating that the proposed approach can facilitate the ab initio PSP.

  10. A Multi-Objective Approach for Protein Structure Prediction Based on an Energy Model and Backbone Angle Preferences

    Directory of Open Access Journals (Sweden)

    Jyh-Jong Tsay

    2015-07-01

    Full Text Available Protein structure prediction (PSP is concerned with the prediction of protein tertiary structure from primary structure and is a challenging calculation problem. After decades of research effort, numerous solutions have been proposed for optimisation methods based on energy models. However, further investigation and improvement is still needed to increase the accuracy and similarity of structures. This study presents a novel backbone angle preference factor, which is one of the factors inducing protein folding. The proposed multiobjective optimisation approach simultaneously considers energy models and backbone angle preferences to solve the ab initio PSP. To prove the effectiveness of the multiobjective optimisation approach based on the energy models and backbone angle preferences, 75 amino acid sequences with lengths ranging from 22 to 88 amino acids were selected from the CB513 data set to be the benchmarks. The data sets were highly dissimilar, therefore indicating that they are meaningful. The experimental results showed that the root-mean-square deviation (RMSD of the multiobjective optimization approach based on energy model and backbone angle preferences was superior to those of typical energy models, indicating that the proposed approach can facilitate the ab initio PSP.

  11. n Silico Analysis of Envelope Dengue Virus-2 and Envelope Dengue Virus-3 Protein as the Backbone of Dengue Virus Tetravalent Vaccine by Using Homology Modeling Method

    Directory of Open Access Journals (Sweden)

    Rizky I. Taufik

    2009-01-01

    Full Text Available Problem statement: Dengue fever, which was caused by Dengue virus infection, had became a major public health problem in the tropic and subtropical countries. Dengue virus (DENV had four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4, based on their immunogenic in the human body. Preventive measure will be necessary to decrease the prevalence of dengue fever, by developing modern vaccine. Approach: This research was focused on in silico study of dengue virus vaccines, by using envelope (E protein of DENV-2 and DENV-3 as their backbones. T cell epitope prediction was determined by using MULTIPRED server and B cell epitope prediction was determined by using Conformational Epitope Prediction server (CEP. Homology modeling study of E DENV-3 protein as the vaccine backbone had produced six dengue vaccine peptides (HMM Vaccine 1-6. Moreover, homology modeling study of E DENV-2 protein as vaccine backbone had produced six dengue vaccine peptides (ANN vaccine 1-6. Results: The BLAST analysis of HMM and ANN vaccines had produced 93% and 91% identity, respectively. The Ramachandran Plot of both vaccines had shown less than 15% non glycine residue in the disallowed region, therefore it showed the solid stability of the proteins. The VAST analysis of E DENV-3 backbone vaccines had determined, that HMM4 and HMM6 had the highest structure similarity with native E DENV-3. HMM4 and HMM6 had the highest VAST score of 64.5. Moreover, the VAST analysis of E DENV-2 backbone vaccines had determined, that ANN1, ANN3, ANN4, ANN5 and ANN6 had the highest structure similarity with native E DENV-2. ANN1, ANN3, ANN4, ANN5 and ANN6 have the highest VAST score of 64.7. Conclusion/Recommendation: It could be inferred from this research that HMM4; HMM6; ANN1; ANN3; ANN4; ANN5; and ANN6 were the best in silico vaccine design, based on their similarity with native E DENV Proteins. This research could be applied for the wet

  12. Direct measurement of the correlated dynamics of the protein-backbone and proximal waters of hydration in mechanically strained elastin

    CERN Document Server

    Sun, Cheng; Huang, Jiaxin; Boutis, Gregory S

    2011-01-01

    We report on the direct measurement of the correlation times of the protein backbone carbons and proximal waters of hydration in mechanically strained elastin by nuclear magnetic resonance methods. The experimental data indicate a decrease in the correlation times of the carbonyl carbons as the strain on the biopolymer is increased. These observations are in good agreement with short 4ns molecular dynamics simulations of (VPGVG)3, a well studied mimetic peptide of elastin. The experimental results also indicate a reduction in the correlation time of proximal waters of hydration with increasing strain applied to the elastomer. A simple model is suggested that correlates the increase in the motion of proximal waters of hydration to the increase in frequency of libration of the protein backbone that develops with increasing strain. Together, the reduction in the protein entropy accompanied with the increase in entropy of the proximal waters of hydration with increasing strain, support the notion that the source ...

  13. The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain.

    Directory of Open Access Journals (Sweden)

    Søren W. Pedersen

    Full Text Available Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.

  14. Improving prediction of secondary structure, local backbone angles, and solvent accessible surface area of proteins by iterative deep learning.

    Science.gov (United States)

    Heffernan, Rhys; Paliwal, Kuldip; Lyons, James; Dehzangi, Abdollah; Sharma, Alok; Wang, Jihua; Sattar, Abdul; Yang, Yuedong; Zhou, Yaoqi

    2015-01-01

    Direct prediction of protein structure from sequence is a challenging problem. An effective approach is to break it up into independent sub-problems. These sub-problems such as prediction of protein secondary structure can then be solved independently. In a previous study, we found that an iterative use of predicted secondary structure and backbone torsion angles can further improve secondary structure and torsion angle prediction. In this study, we expand the iterative features to include solvent accessible surface area and backbone angles and dihedrals based on Cα atoms. By using a deep learning neural network in three iterations, we achieved 82% accuracy for secondary structure prediction, 0.76 for the correlation coefficient between predicted and actual solvent accessible surface area, 19° and 30° for mean absolute errors of backbone φ and ψ angles, respectively, and 8° and 32° for mean absolute errors of Cα-based θ and τ angles, respectively, for an independent test dataset of 1199 proteins. The accuracy of the method is slightly lower for 72 CASP 11 targets but much higher than those of model structures from current state-of-the-art techniques. This suggests the potentially beneficial use of these predicted properties for model assessment and ranking.

  15. Systematic determination of the mosaic structure of bacterial genomes: species backbone versus strain-specific loops

    Directory of Open Access Journals (Sweden)

    Gendrault-Jacquemard A

    2005-07-01

    Full Text Available Abstract Background Public databases now contain multitude of complete bacterial genomes, including several genomes of the same species. The available data offers new opportunities to address questions about bacterial genome evolution, a task that requires reliable fine comparison data of closely related genomes. Recent analyses have shown, using pairwise whole genome alignments, that it is possible to segment bacterial genomes into a common conserved backbone and strain-specific sequences called loops. Results Here, we generalize this approach and propose a strategy that allows systematic and non-biased genome segmentation based on multiple genome alignments. Segmentation analyses, as applied to 13 different bacterial species, confirmed the feasibility of our approach to discern the 'mosaic' organization of bacterial genomes. Segmentation results are available through a Web interface permitting functional analysis, extraction and visualization of the backbone/loops structure of documented genomes. To illustrate the potential of this approach, we performed a precise analysis of the mosaic organization of three E. coli strains and functional characterization of the loops. Conclusion The segmentation results including the backbone/loops structure of 13 bacterial species genomes are new and available for use by the scientific community at the URL: http://genome.jouy.inra.fr/mosaic.

  16. ENZYME-IIB(CELLOBIOSE) OF THE PHOSPHOENOL-PYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI - BACKBONE ASSIGNMENT AND SECONDARY STRUCTURE DETERMINED BY 3-DIMENSIONAL NMR-SPECTROSCOPY

    NARCIS (Netherlands)

    AB, E; SCHUURMANWOLTERS, GK; SAIER, MH; REIZER, J; JACUINOD, M; ROEPSTORFF, P; DIJKSTRA, K; SCHEEK, RM; ROBILLARD, GT

    1994-01-01

    The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBCellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple re

  17. Enzyme IIBcellobiose of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli : Backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy

    NARCIS (Netherlands)

    AB, Eiso; Schuurman-Wolters, Gea K.; Saier, Milton H.; Reizer, Jonathan; Jacuinod, Michel; Roepstorff, Peter; Dijkstra, Klaas; Scheek, Ruud M.; Robillard, George T.

    1994-01-01

    The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple re

  18. Circular permutation prediction reveals a viable backbone disconnection for split proteins: an approach in identifying a new functional split intein.

    Directory of Open Access Journals (Sweden)

    Yun-Tzai Lee

    Full Text Available Split-protein systems have emerged as a powerful tool for detecting biomolecular interactions and reporting biological reactions. However, reliable methods for identifying viable split sites are still unavailable. In this study, we demonstrated the feasibility that valid circular permutation (CP sites in proteins have the potential to act as split sites and that CP prediction can be used to search for internal permissive sites for creating new split proteins. Using a protein ligase, intein, as a model, CP predictor facilitated the creation of circular permutants in which backbone opening imposes the least detrimental effects on intein folding. We screened a series of predicted intein CPs and identified stable and native-fold CPs. When the valid CP sites were introduced as split sites, there was a reduction in folding enthalpy caused by the new backbone opening; however, the coincident loss in entropy was sufficient to be compensated, yielding a favorable free energy for self-association. Since split intein is exploited in protein semi-synthesis, we tested the related protein trans-splicing (PTS activities of the corresponding split inteins. Notably, a novel functional split intein composed of the N-terminal 36 residues combined with the remaining C-terminal fragment was identified. Its PTS activity was shown to be better than current reported two-piece intein with a short N-terminal segment. Thus, the incorporation of in silico CP prediction facilitated the design of split intein as well as circular permutants.

  19. The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

    Directory of Open Access Journals (Sweden)

    Tsinoremas NF

    2007-05-01

    Full Text Available Abstract Background The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues. Results We have developed an integrated software system for the automated analysis and representation of H/D exchange data that has been titled "The Deuterator". Novel approaches have been implemented that enable high throughput analysis, automated determination of deuterium incorporation, and deconvolution of overlapping peptides. This has been achieved by using methods involving iterative theoretical envelope fitting, and consideration of peak data within expected m/z ranges. Existing common file formats have been leveraged to allow compatibility with the output from the myriad of MS instrument platforms and peptide sequence database search engines. A web-based interface is used to integrate the components of The Deuterator that are able to analyze and present mass spectral data from instruments with varying resolving powers. The results, if necessary, can then be confirmed, adjusted, re-calculated and saved. Additional tools synchronize the curated calculation parameters with replicate time points, increasing throughput. Saved results can then

  20. Protein and peptide alkoxyl radicals can give rise to C-terminal decarboxylation and backbone cleavage

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    1996-01-01

    when the free amino acid does not, and that hydroperoxides can be formed on both the backbone (at alpha-carbon positions) and the side chain. Decomposition of alpha-carbon hydroperoxides by Fe(II)-EDTA gives initially an alkoxyl radical via a pseudo-Fenton reaction; these radicals fragment rapidly...... with k estimated as > or = 10(7) s(-1). With N-acetyl amino acids and dipeptides beta-scission of an alkoxyl radical at the C-terminal alpha-carbon results in C-terminal decarboxylation, with release of CO2.-; the corresponding amides undergo deamidation with release of .C(O)NH2. Cyclic dipeptides...... undergo analogous reactions with cleavage of the alpha-carbon to carbonyl-carbon bond and formation of .C(O)NHR radicals. With substrates with large aliphatic side chains, radicals from side-chain hydroperoxides are also observed. C-terminal decarboxylation and backbone fragmentation are also observed...

  1. Carbon and amide detect backbone assignment methods of a novel repeat protein from the staphylocoagulase in S. aureus.

    Science.gov (United States)

    Voehler, Markus; Ashoka, Maddur Appajaiah; Meiler, Jens; Bock, Paul E

    2017-08-17

    The C-terminal repeat domain of staphylocoagulase that is secreted by the S. aureus is believed to play an important role interacting with fibrinogen and promotes blood clotting. To study this interaction by NMR, full assignment of each amide residue in the HSQC spectrum was required. Despite of the short sequence of the repeat construct, the HSQC spectrum contained a substantial amount of overlapped and exchange broadened resonances, indicating little secondary or tertiary structure. This caused severe problems while using the conventional, amide based NMR method for the backbone assignment. With the growing interest in small apparently disordered proteins, these issues are being faced more frequently. An alternative strategy to improve the backbone assignment capability involved carbon direct detection methods. Circumventing the amide proton detection offers a larger signal dispersion and more uniform signal intensity. For peptides with higher concentrations and in combination with the cold carbon channels of new cryoprobes, higher fields, and sufficiently long relaxation times, the disadvantage of the lower sensitivity of the (13)C nucleus can be overcome. Another advantage of this method is the assignment of the proline backbone residues. Complete assignment with the carbon-detected strategy was achieved with a set of only two 3D, one 2D, and a HNCO measurement, which was necessary to translate the information to the HSQC spectrum.

  2. General order parameter based correlation analysis of protein backbone motions between experimental NMR relaxation measurements and molecular dynamics simulations

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qing; Shi, Chaowei [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); Yu, Lu [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 Science, Hefei, Anhui, 230031 (China); Zhang, Longhua [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); Xiong, Ying, E-mail: yxiong73@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); 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 Science, Hefei, Anhui, 230031 (China)

    2015-02-13

    Internal backbone dynamic motions are essential for different protein functions and occur on a wide range of time scales, from femtoseconds to seconds. Molecular dynamic (MD) simulations and nuclear magnetic resonance (NMR) spin relaxation measurements are valuable tools to gain access to fast (nanosecond) internal motions. However, there exist few reports on correlation analysis between MD and NMR relaxation data. Here, backbone relaxation measurements of {sup 15}N-labeled SH3 (Src homology 3) domain proteins in aqueous buffer were used to generate general order parameters (S{sup 2}) using a model-free approach. Simultaneously, 80 ns MD simulations of SH3 domain proteins in a defined hydrated box at neutral pH were conducted and the general order parameters (S{sup 2}) were derived from the MD trajectory. Correlation analysis using the Gromos force field indicated that S{sup 2} values from NMR relaxation measurements and MD simulations were significantly different. MD simulations were performed on models with different charge states for three histidine residues, and with different water models, which were SPC (simple point charge) water model and SPC/E (extended simple point charge) water model. S{sup 2} parameters from MD simulations with charges for all three histidines and with the SPC/E water model correlated well with S{sup 2} calculated from the experimental NMR relaxation measurements, in a site-specific manner. - Highlights: • Correlation analysis between NMR relaxation measurements and MD simulations. • General order parameter (S{sup 2}) as common reference between the two methods. • Different protein dynamics with different Histidine charge states in neutral pH. • Different protein dynamics with different water models.

  3. Comparison of solid-state dipolar couplings and solution relaxation data provides insight into protein backbone dynamics.

    Science.gov (United States)

    Chevelkov, Veniamin; Xue, Yi; Linser, Rasmus; Skrynnikov, Nikolai R; Reif, Bernd

    2010-04-14

    Analyses of solution (15)N relaxation data and solid-state (1)H(N)-(15)N dipolar couplings from a small globular protein, alpha-spectrin SH3 domain, produce a surprisingly similar pattern of order parameters. This result suggests that there is little or no ns-mus dynamics throughout most of the sequence and, in particular, in the structured portion of the backbone. At the same time, evidence of ns-mus motions is found in the flexible loops and termini. These findings, corroborated by the MD simulations of alpha-spectrin SH3 in a hydrated crystalline environment and in solution, are consistent with the picture of protein dynamics that has recently emerged from the solution studies employing residual dipolar couplings.

  4. Unraveling the complexity of protein backbone dynamics with combined (13)C and (15)N solid-state NMR relaxation measurements.

    Science.gov (United States)

    Lamley, Jonathan M; Lougher, Matthew J; Sass, Hans Juergen; Rogowski, Marco; Grzesiek, Stephan; Lewandowski, Józef R

    2015-09-14

    Typically, protein dynamics involve a complex hierarchy of motions occurring on different time scales between conformations separated by a range of different energy barriers. NMR relaxation can in principle provide a site-specific picture of both the time scales and amplitudes of these motions, but independent relaxation rates sensitive to fluctuations in different time scale ranges are required to obtain a faithful representation of the underlying dynamic complexity. This is especially pertinent for relaxation measurements in the solid state, which report on dynamics in a broader window of time scales by more than 3 orders of magnitudes compared to solution NMR relaxation. To aid in unraveling the intricacies of biomolecular dynamics we introduce (13)C spin-lattice relaxation in the rotating frame (R1ρ) as a probe of backbone nanosecond-microsecond motions in proteins in the solid state. We present measurements of (13)C'R1ρ rates in fully protonated crystalline protein GB1 at 600 and 850 MHz (1)H Larmor frequencies and compare them to (13)C'R1, (15)N R1 and R1ρ measured under the same conditions. The addition of carbon relaxation data to the model free analysis of nitrogen relaxation data leads to greatly improved characterization of time scales of protein backbone motions, minimizing the occurrence of fitting artifacts that may be present when (15)N data is used alone. We also discuss how internal motions characterized by different time scales contribute to (15)N and (13)C relaxation rates in the solid state and solution state, leading to fundamental differences between them, as well as phenomena such as underestimation of picosecond-range motions in the solid state and nanosecond-range motions in solution.

  5. NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition.

    Science.gov (United States)

    Mukherjee, Munia; Dutta, Kaushik; White, Mark A; Cowburn, David; Fox, Robert O

    2006-06-01

    Flaviviruses cause many human diseases, including dengue fever, yellow fever, West Nile viral encephalitis, and hemorrhagic fevers, and are transmitted to their vertebrate hosts by infected mosquitoes and ticks. Domain III of the envelope protein (E-D3) is considered to be the primary viral determinant involved in the virus-host-cell receptor interaction, and thus represents an excellent target for antiviral drug development. Langat (LGT) virus is a naturally attenuated BSL-2 TBE virus and is a model for the pathogenic BSL-3 and BSL-4 viruses in the serogroup. We have determined the solution structure of LGT-E-D3 using heteronuclear NMR spectroscopy. The backbone dynamics of LGT-E-D3 have been investigated using 15N relaxation measurements. A detailed analysis of the solution structure and dynamics of LGT-E-D3 suggests potential residues that could form a surface for molecular recognition, and thereby represent a target site for antiviral therapeutics design.

  6. A Network Flow Approach to Predict Protein Targets and Flavonoid Backbones to Treat Respiratory Syncytial Virus Infection

    Directory of Open Access Journals (Sweden)

    José Eduardo Vargas

    2015-01-01

    Full Text Available Background. Respiratory syncytial virus (RSV infection is the major cause of respiratory disease in lower respiratory tract in infants and young children. Attempts to develop effective vaccines or pharmacological treatments to inhibit RSV infection without undesired effects on human health have been unsuccessful. However, RSV infection has been reported to be affected by flavonoids. The mechanisms underlying viral inhibition induced by these compounds are largely unknown, making the development of new drugs difficult. Methods. To understand the mechanisms induced by flavonoids to inhibit RSV infection, a systems pharmacology-based study was performed using microarray data from primary culture of human bronchial cells infected by RSV, together with compound-proteomic interaction data available for Homo sapiens. Results. After an initial evaluation of 26 flavonoids, 5 compounds (resveratrol, quercetin, myricetin, apigenin, and tricetin were identified through topological analysis of a major chemical-protein (CP and protein-protein interacting (PPI network. In a nonclustered form, these flavonoids regulate directly the activity of two protein bottlenecks involved in inflammation and apoptosis. Conclusions. Our findings may potentially help uncovering mechanisms of action of early RSV infection and provide chemical backbones and their protein targets in the difficult quest to develop new effective drugs.

  7. Structural basis for the enhanced stability of protein model compounds and peptide backbone unit in ammonium ionic liquids.

    Science.gov (United States)

    Vasantha, T; Attri, Pankaj; Venkatesu, Pannuru; Devi, R S Rama

    2012-10-04

    Protein folding/unfolding is a fascinating study in the presence of cosolvents, which protect/disrupt the native structure of protein, respectively. The structure and stability of proteins and their functional groups may be modulated by the addition of cosolvents. Ionic liquids (ILs) are finding a vast array of applications as novel cosolvents for a wide variety of biochemical processes that include protein folding. Here, the systematic and quantitative apparent transfer free energies (ΔG'(tr)) of protein model compounds from water to ILs through solubility measurements as a function of IL concentration at 25 °C have been exploited to quantify and interpret biomolecular interactions between model compounds of glycine peptides (GPs) with ammonium based ILs. The investigated aqueous systems consist of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly(2)), triglycine (Gly(3)), tetraglycine (Gly(4)), and cyclic glycylglycine (c(GG)) in the presence of six ILs such as diethylammonium acetate (DEAA), diethylammonium hydrogen sulfate (DEAS), triethylammonium acetate (TEAA), triethylammonium hydrogen sulfate (TEAS), triethylammonium dihydrogen phosphate (TEAP), and trimethylammonium acetate (TMAA). We have observed positive values of ΔG'(tr) for GPs from water to ILs, indicating that interactions between ILs and GPs are unfavorable, which leads to stabilization of the structure of model protein compounds. Moreover, our experimental data ΔG'(tr) is used to obtain transfer free energies (Δg'(tr)) of the peptide backbone unit (or glycyl unit) (-CH(2)C═ONH-), which is the most numerous group in globular proteins, from water to IL solutions. To obtain the mechanism events of the ILs' role in enhancing the stability of the model compounds, we have further obtained m-values for GPs from solubility limits. These results explicitly elucidate that all alkyl ammonium ILs act as stabilizers for model compounds through the exclusion of ILs from model compounds of

  8. Solution Structure and Backbone Dynamics of Human Liver Fatty Acid Binding Protein: Fatty Acid Binding Revisited

    OpenAIRE

    Cai, Jun; Lücke, Christian; Chen, Zhongjing; Qiao, Ye; Klimtchuk, Elena; Hamilton, James A.

    2012-01-01

    Liver fatty acid binding protein (L-FABP), a cytosolic protein most abundant in liver, is associated with intracellular transport of fatty acids, nuclear signaling, and regulation of intracellular lipolysis. Among the members of the intracellular lipid binding protein family, L-FABP is of particular interest as it can i), bind two fatty acid molecules simultaneously and ii), accommodate a variety of bulkier physiological ligands such as bilirubin and fatty acyl CoA. To better understand the p...

  9. MERA: a webserver for evaluating backbone torsion angle distributions in dynamic and disordered proteins from NMR data

    Energy Technology Data Exchange (ETDEWEB)

    Mantsyzov, Alexey B. [M.V. Lomonosov Moscow State University, Faculty of Fundamental Medicine (Russian Federation); Shen, Yang; Lee, Jung Ho [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Hummer, Gerhard [Max Planck Institute of Biophysics (Germany); Bax, Ad, E-mail: bax@nih.gov [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    2015-09-15

    MERA (Maximum Entropy Ramachandran map Analysis from NMR data) is a new webserver that generates residue-by-residue Ramachandran map distributions for disordered proteins or disordered regions in proteins on the basis of experimental NMR parameters. As input data, the program currently utilizes up to 12 different parameters. These include three different types of short-range NOEs, three types of backbone chemical shifts ({sup 15}N, {sup 13}C{sup α}, and {sup 13}C′), six types of J couplings ({sup 3}J{sub HNHα}, {sup 3}J{sub C′C′}, {sup 3}J{sub C′Hα}, {sup 1}J{sub HαCα}, {sup 2}J{sub CαN} and {sup 1}J{sub CαN}), as well as the {sup 15}N-relaxation derived J(0) spectral density. The Ramachandran map distributions are reported in terms of populations of their 15° × 15° voxels, and an adjustable maximum entropy weight factor is available to ensure that the obtained distributions will not deviate more from a newly derived coil library distribution than required to account for the experimental data. MERA output includes the agreement between each input parameter and its distribution-derived value. As an application, we demonstrate performance of the program for several residues in the intrinsically disordered protein α-synuclein, as well as for several static and dynamic residues in the folded protein GB3.

  10. Backbone resonance assignments for G protein α(i3) subunit in the GDP-bound state.

    Science.gov (United States)

    Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

    2014-10-01

    Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gαi3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα(i3) in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα(i3)·GDP would be useful for the analyses of the dynamics of Gα(i3) and its interactions with various target molecules.

  11. Two Dimensional Electronic Correlation Spectroscopy of the npi* and pipi* Protein Backbone Transitions: A Simulation Study.

    Science.gov (United States)

    Li, Zhenyu; Abramavicius, Darius; Zhuang, Wei; Mukamel, Shaul

    2007-11-15

    The two dimensional (2D) photon echo spectrum of the amide ultraviolet (UV) bands of proteins are simulated. Two effective exciton Hamiltonian parameter sets developed by Woody and Hirst, which predict similar CD spectra, may be distinguished by their very different 2DUV spectra. These differences are enhanced in specific configurations of pulse polarizations which provide chirality-induced signals.

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

    KAUST Repository

    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.

  13. Determination of Equine Cytochrome c Backbone Amide Hydrogen/Deuterium Exchange Rates by Mass Spectrometry Using a Wider Time Window and Isotope Envelope

    Science.gov (United States)

    Hamuro, Yoshitomo

    2017-03-01

    A new strategy to analyze amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) data is proposed, utilizing a wider time window and isotope envelope analysis of each peptide. While most current scientific reports present HDX-MS data as a set of time-dependent deuteration levels of peptides, the ideal HDX-MS data presentation is a complete set of backbone amide hydrogen exchange rates. The ideal data set can provide single amide resolution, coverage of all exchange events, and the open/close ratio of each amide hydrogen in EX2 mechanism. Toward this goal, a typical HDX-MS protocol was modified in two aspects: measurement of a wider time window in HDX-MS experiments and deconvolution of isotope envelope of each peptide. Measurement of a wider time window enabled the observation of deuterium incorporation of most backbone amide hydrogens. Analysis of the isotope envelope instead of centroid value provides the deuterium distribution instead of the sum of deuteration levels in each peptide. A one-step, global-fitting algorithm optimized exchange rate and deuterium retention during the analysis of each amide hydrogen by fitting the deuterated isotope envelopes at all time points of all peptides in a region. Application of this strategy to cytochrome c yielded 97 out of 100 amide hydrogen exchange rates. A set of exchange rates determined by this approach is more appropriate for a patent or regulatory filing of a biopharmaceutical than a set of peptide deuteration levels obtained by a typical protocol. A wider time window of this method also eliminates false negatives in protein-ligand binding site identification.

  14. Pseudo-4D triple resonance experiments to resolve HN overlap in the backbone assignment of unfolded proteins.

    Science.gov (United States)

    Bagai, Ireena; Ragsdale, Stephen W; Zuiderweg, Erik R P

    2011-02-01

    The solution NMR resonance assignment of the protein backbone is most commonly carried out using triple resonance experiments that involve (15)N and (1)HN resonances. The assignment becomes problematic when there is resonance overlap of (15)N-(1)HN cross peaks. For such residues, one cannot unambiguously link the "left" side of the NH root to the "right" side, and the residues associated with such overlapping HN resonances remain often unassigned. Here we present a solution to this problem: a hybrid (4d,3d) reduced-dimensionality HN(CO)CA(CON)CA sequence. In this experiment, the Ca(i) resonance is modulated with the frequency of the Ca(i-1) resonance, which helps in resolving the ambiguity involved in connecting the Ca(i) and Ca(i-1) resonances for overlapping NH roots. The experiment has limited sensitivity, and is only suited for small or unfolded proteins. In a companion experiment, (4d,3d) reduced-dimensionality HNCO(N)CA, the Ca(i) resonance is modulated with the frequency of the CO(i-1) resonance, hence resolving the ambiguity existent in pairing up the Ca(i) and CO(i-1) resonances for overlapping NH roots.

  15. Quantitative analysis of backbone motion in proteins using MAS solid-state NMR spectroscopy.

    Science.gov (United States)

    Chevelkov, Veniamin; Fink, Uwe; Reif, Bernd

    2009-09-01

    We present a comprehensive analysis of protein dynamics for a micro-crystallin protein in the solid-state. Experimental data include (15)N T (1) relaxation times measured at two different magnetic fields as well as (1)H-(15)N dipole, (15)N CSA cross correlated relaxation rates which are sensitive to the spectral density function J(0) and are thus a measure of T (2) in the solid-state. In addition, global order parameters are included from a (1)H,(15)N dipolar recoupling experiment. The data are analyzed within the framework of the extended model-free Clore-Lipari-Szabo theory. We find slow motional correlation times in the range of 5 and 150 ns. Assuming a wobbling in a cone motion, the amplitude of motion of the respective amide moiety is on the order of 10 degrees for the half-opening angle of the cone in most of the cases. The experiments are demonstrated using a perdeuterated sample of the chicken alpha-spectrin SH3 domain.

  16. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yong-Geun [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of); Park, Chin-Ju [Gwangju Institute of Science and Technology, Division of Liberal Arts and Sciences and Department of Chemistry (Korea, Republic of); Kim, Hee-Eun; Seo, Yeo-Jin; Lee, Ae-Ree; Choi, Seo-Ree; Lee, Shim Sung; Lee, Joon-Hwa, E-mail: joonhwa@gnu.ac.kr [Gyeongsang National University, Department of Chemistry and Research Institute of Natural Science (Korea, Republic of)

    2015-02-15

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3{sub 10}-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

  17. Protein Nitrogen Determination

    Science.gov (United States)

    Nielsen, S. Suzanne

    The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.

  18. a Proposal for a General Method for Determining Semi-Experimental Equilibrium Structures of Carbon Atom Backbones

    Science.gov (United States)

    Craig, Norman C.

    2010-06-01

    Semi-experimental equilibrium structures are determined from ground state rotational constants derived from the analysis of rotational transitions in high-resolution spectra and from the quantum chemical calculation of spectroscopic alphas. In the full application of this method, spectra of numerous isotopic species must be investigated. Most of these isotopic species require specialized synthesis. We now propose focusing on the carbon atoms, for which microwave spectroscopy routinely yields spectra for polar molecules with 13C substitution in natural abundance. Needed spectroscopic alphas can be computed with Gaussian software. Application of the Kraitchman substitution relationships gives Cartesian coordinates for the carbon atoms and thence bond parameters for the carbon backbone. This method will be evaluated with ethylene, 1,1-difluoroethylene, 1,1-difluorocyclopropane, propene, and butadiene. The method will then be applied to cis-hexatriene and the two conformers of glycidol. R. D. Suenram, B. H. Pate, A. Lessari, J. L. Neill, S. Shipman, R. A. Holmes, M. C. Leyden, and N. C. Craig, J. Phys. Chem. A 113, 1864-1868 (2009). A. R. Conrad, N. H. Teumelsan, P. E. Wang, and M. J. Tubergen, J. Phys. Chem. A 114, 336-342 (2010).

  19. Using MUSIC and CC(CONH for Backbone Assignment of Two Medium-Sized Proteins Not Fully Accessible to Standard 3D NMR

    Directory of Open Access Journals (Sweden)

    Annette K. Brenner

    2014-06-01

    Full Text Available The backbone assignment of medium-sized proteins is rarely as straightforward as that of small proteins, and thus often requires creative solutions. Here, we describe the application of a combination of standard 3D heteronuclear methods with CC(CONH and a variety of MUltiplicity Selective In-phase Coherence transfer (MUSIC experiments. Both CC(CONH and MUSIC are, in theory, very powerful methods for the backbone assignment of proteins. Due to low sensitivity, their use has usually been linked to small proteins only. However, we found that combining CC(CONH and MUSIC experiments simplified the assignment of two challenging medium-sized proteins of 13 and 19.5 kDa, respectively. These methods are to some extent complementary to each other: CC(CONH acquired with a long isotropic mixing time can identify amino acids with large aliphatic side chains. Whereas the most sensitive MUSIC experiments identify amino acid types that cannot be detected by CC(CONH, comprising the residues with acid and amide groups, and aromatic rings in their side chains. Together these methods provide a means of identifying the majority of peaks in the 2D 15N HSQC spectrum which simplifies the backbone assignment work even for proteins, e.g., small kinases, whose standard spectra resulted in little spectral resolution and low signal intensities.

  20. Improving the prediction accuracy of residue solvent accessibility and real-value backbone torsion angles of proteins by guided-learning through a two-layer neural network.

    Science.gov (United States)

    Faraggi, Eshel; Xue, Bin; Zhou, Yaoqi

    2009-03-01

    This article attempts to increase the prediction accuracy of residue solvent accessibility and real-value backbone torsion angles of proteins through improved learning. Most methods developed for improving the backpropagation algorithm of artificial neural networks are limited to small neural networks. Here, we introduce a guided-learning method suitable for networks of any size. The method employs a part of the weights for guiding and the other part for training and optimization. We demonstrate this technique by predicting residue solvent accessibility and real-value backbone torsion angles of proteins. In this application, the guiding factor is designed to satisfy the intuitive condition that for most residues, the contribution of a residue to the structural properties of another residue is smaller for greater separation in the protein-sequence distance between the two residues. We show that the guided-learning method makes a 2-4% reduction in 10-fold cross-validated mean absolute errors (MAE) for predicting residue solvent accessibility and backbone torsion angles, regardless of the size of database, the number of hidden layers and the size of input windows. This together with introduction of two-layer neural network with a bipolar activation function leads to a new method that has a MAE of 0.11 for residue solvent accessibility, 36 degrees for psi, and 22 degrees for phi. The method is available as a Real-SPINE 3.0 server in http://sparks.informatics.iupui.edu.

  1. A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein, an intrinsically disordered protein.

    Science.gov (United States)

    Mantsyzov, Alexey B; Maltsev, Alexander S; Ying, Jinfa; Shen, Yang; Hummer, Gerhard; Bax, Ad

    2014-09-01

    α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential H(N) − H(α) and H(N) − H(N) NOEs, values for (3) JHNHα, (1) JHαCα, (2) JCαN, and (1) JCαN, as well as chemical shifts of (15)N, (13)C(α), and (13)C' nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20-30%) of the PPII region and a somewhat higher population of the potentially aggregation-prone β region (20-40%) than seen in the database. A generally lower population of the αR region (10-20%) is found. Analysis of (1)H − (1)H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein.

  2. A maximum entropy approach to the study of residue-specific backbone angle distributions in α-synuclein, an intrinsically disordered protein

    Science.gov (United States)

    Mantsyzov, Alexey B; Maltsev, Alexander S; Ying, Jinfa; Shen, Yang; Hummer, Gerhard; Bax, Ad

    2014-01-01

    α-Synuclein is an intrinsically disordered protein of 140 residues that switches to an α-helical conformation upon binding phospholipid membranes. We characterize its residue-specific backbone structure in free solution with a novel maximum entropy procedure that integrates an extensive set of NMR data. These data include intraresidue and sequential HN–Hα and HN–HN NOEs, values for 3JHNHα, 1JHαCα, 2JCαN, and 1JCαN, as well as chemical shifts of 15N, 13Cα, and 13C′ nuclei, which are sensitive to backbone torsion angles. Distributions of these torsion angles were identified that yield best agreement to the experimental data, while using an entropy term to minimize the deviation from statistical distributions seen in a large protein coil library. Results indicate that although at the individual residue level considerable deviations from the coil library distribution are seen, on average the fitted distributions agree fairly well with this library, yielding a moderate population (20–30%) of the PPII region and a somewhat higher population of the potentially aggregation-prone β region (20–40%) than seen in the database. A generally lower population of the αR region (10–20%) is found. Analysis of 1H–1H NOE data required consideration of the considerable backbone diffusion anisotropy of a disordered protein. PMID:24976112

  3. Structure determination of archaea-specific ribosomal protein L46a reveals a novel protein fold

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Yingang, E-mail: fengyg@qibebt.ac.cn [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Song, Xiaxia [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Lin, Jinzhong [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Xuan, Jinsong [Department of Biological Science and Engineering, School of Chemical and Biological Engineering, University of Science and Technology Beijing, Beijing 100083 (China); Cui, Qiu [Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101 (China); Wang, Jinfeng [National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2014-07-18

    Highlights: • The archaea-specific ribosomal protein L46a has no homology to known proteins. • Three dimensional structure and backbone dynamics of L46a were determined by NMR. • The structure of L46a represents a novel protein fold. • A potential rRNA-binding surface on L46a was identified. • The potential position of L46a on the ribosome was proposed. - Abstract: Three archaea-specific ribosomal proteins recently identified show no sequence homology with other known proteins. Here we determined the structure of L46a, the most conserved one among the three proteins, from Sulfolobus solfataricus P2 using NMR spectroscopy. The structure presents a twisted β-sheet formed by the N-terminal part and two helices at the C-terminus. The L46a structure has a positively charged surface which is conserved in the L46a protein family and is the potential rRNA-binding site. Searching homologous structures in Protein Data Bank revealed that the structure of L46a represents a novel protein fold. The backbone dynamics identified by NMR relaxation experiments reveal significant flexibility at the rRNA binding surface. The potential position of L46a on the ribosome was proposed by fitting the structure into a previous electron microscopy map of the ribosomal 50S subunit, which indicated that L46a contacts to domain I of 23S rRNA near a multifunctional ribosomal protein L7ae.

  4. Proton-detected MAS NMR experiments based on dipolar transfers for backbone assignment of highly deuterated proteins

    Science.gov (United States)

    Chevelkov, Veniamin; Habenstein, Birgit; Loquet, Antoine; Giller, Karin; Becker, Stefan; Lange, Adam

    2014-05-01

    Proton-detected solid-state NMR was applied to a highly deuterated insoluble, non-crystalline biological assembly, the Salmonella typhimurium type iii secretion system (T3SS) needle. Spectra of very high resolution and sensitivity were obtained at a low protonation level of 10-20% at exchangeable amide positions. We developed efficient experimental protocols for resonance assignment tailored for this system and the employed experimental conditions. Using exclusively dipolar-based interspin magnetization transfers, we recorded two sets of 3D spectra allowing for an almost complete backbone resonance assignment of the needle subunit PrgI. The additional information provided by the well-resolved proton dimension revealed the presence of two sets of resonances in the N-terminal helix of PrgI, while in previous studies employing 13C detection only a single set of resonances was observed.

  5. Testing Backbone.js

    CERN Document Server

    Roemer, Ryan

    2013-01-01

    This book is packed with the step by step tutorial and instructions in recipe format helping you setup test infrastructure and gradually advance your skills to plan, develop, and test your backbone applications.If you are a JavaScript developer looking for recipes to create and implement test support for your backbone application, then this book is ideal for you.

  6. Ribosomal proteins L11 and L10.(L12)4 and the antibiotic thiostrepton interact with overlapping regions of the 23 S rRNA backbone in the ribosomal GTPase centre

    DEFF Research Database (Denmark)

    Rosendahl, G; Douthwaite, S

    1993-01-01

    The Escherichia coli ribosomal protein (r-protein) L11 and its binding site on 23 S ribosomal RNA (rRNA) are associated with ribosomal hydrolysis of guanosine 5'-triphosphate (GTP). We have used hydroxyl radical footprinting to map the contacts between L11 and the backbone riboses in 23 S rRNA, a...

  7. Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis

    Science.gov (United States)

    Qi, Xingmei; Xiong, Sidong

    2017-01-01

    CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect. PMID:28148910

  8. Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon shermani.

    Directory of Open Access Journals (Sweden)

    Damien B Wilburn

    Full Text Available In response to pervasive sexual selection, protein sex pheromones often display rapid mutation and accelerated evolution of corresponding gene sequences. For proteins, the general dogma is that structure is maintained even as sequence or function may rapidly change. This phenomenon is well exemplified by the three-finger protein (TFP superfamily: a diverse class of vertebrate proteins co-opted for many biological functions - such as components of snake venoms, regulators of the complement system, and coordinators of amphibian limb regeneration. All of the >200 structurally characterized TFPs adopt the namesake "three-finger" topology. In male red-legged salamanders, the TFP pheromone Plethodontid Modulating Factor (PMF is a hypervariable protein such that, through extensive gene duplication and pervasive sexual selection, individual male salamanders express more than 30 unique isoforms. However, it remained unclear how this accelerated evolution affected the protein structure of PMF. Using LC/MS-MS and multidimensional NMR, we report the 3D structure of the most abundant PMF isoform, PMF-G. The high resolution structural ensemble revealed a highly modified TFP structure, including a unique disulfide bonding pattern and loss of secondary structure, that define a novel protein topology with greater backbone flexibility in the third peptide finger. Sequence comparison, models of molecular evolution, and homology modeling together support that this flexible third finger is the most rapidly evolving segment of PMF. Combined with PMF sequence hypervariability, this structural flexibility may enhance the plasticity of PMF as a chemical signal by permitting potentially thousands of structural conformers. We propose that the flexible third finger plays a critical role in PMF:receptor interactions. As female receptors co-evolve, this flexibility may allow PMF to still bind its receptor(s without the immediate need for complementary mutations. Consequently

  9. Structural insights into the evolution of a sexy protein: novel topology and restricted backbone flexibility in a hypervariable pheromone from the red-legged salamander, Plethodon shermani.

    Science.gov (United States)

    Wilburn, Damien B; Bowen, Kathleen E; Doty, Kari A; Arumugam, Sengodagounder; Lane, Andrew N; Feldhoff, Pamela W; Feldhoff, Richard C

    2014-01-01

    In response to pervasive sexual selection, protein sex pheromones often display rapid mutation and accelerated evolution of corresponding gene sequences. For proteins, the general dogma is that structure is maintained even as sequence or function may rapidly change. This phenomenon is well exemplified by the three-finger protein (TFP) superfamily: a diverse class of vertebrate proteins co-opted for many biological functions - such as components of snake venoms, regulators of the complement system, and coordinators of amphibian limb regeneration. All of the >200 structurally characterized TFPs adopt the namesake "three-finger" topology. In male red-legged salamanders, the TFP pheromone Plethodontid Modulating Factor (PMF) is a hypervariable protein such that, through extensive gene duplication and pervasive sexual selection, individual male salamanders express more than 30 unique isoforms. However, it remained unclear how this accelerated evolution affected the protein structure of PMF. Using LC/MS-MS and multidimensional NMR, we report the 3D structure of the most abundant PMF isoform, PMF-G. The high resolution structural ensemble revealed a highly modified TFP structure, including a unique disulfide bonding pattern and loss of secondary structure, that define a novel protein topology with greater backbone flexibility in the third peptide finger. Sequence comparison, models of molecular evolution, and homology modeling together support that this flexible third finger is the most rapidly evolving segment of PMF. Combined with PMF sequence hypervariability, this structural flexibility may enhance the plasticity of PMF as a chemical signal by permitting potentially thousands of structural conformers. We propose that the flexible third finger plays a critical role in PMF:receptor interactions. As female receptors co-evolve, this flexibility may allow PMF to still bind its receptor(s) without the immediate need for complementary mutations. Consequently, this unique

  10. Integral membrane protein structure determination using pseudocontact shifts

    Energy Technology Data Exchange (ETDEWEB)

    Crick, Duncan J.; Wang, Jue X. [University of Cambridge, Department of Biochemistry (United Kingdom); Graham, Bim; Swarbrick, James D. [Monash University, Monash Institute of Pharmaceutical Sciences (Australia); Mott, Helen R.; Nietlispach, Daniel, E-mail: dn206@cam.ac.uk [University of Cambridge, Department of Biochemistry (United Kingdom)

    2015-04-15

    Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.

  11. ``Pinning strategy": a novel approach for predicting the backbone structure in terms of protein blocks from sequence

    Indian Academy of Sciences (India)

    A G De Brevern; C Etchebest; C Benros; S Hazout

    2007-01-01

    The description of protein 3D structures can be performed through a library of 3D fragments, named a structural alphabet. Our structural alphabet is composed of 16 small protein fragments of 5 C in length, called protein blocks (PBs). It allows an efficient approximation of the 3D protein structures and a correct prediction of the local structure. The 72 most frequent series of 5 consecutive PBs, called structural words (SWs) are able to cover more than 90% of the 3D structures. PBs are highly conditioned by the presence of a limited number of transitions between them. In this study, we propose a new method called “pinning strategy” that used this specific feature to predict long protein fragments. Its goal is to define highly probable successions of PBs. It starts from the most probable SW and is then extended with overlapping SWs. Starting from an initial prediction rate of 34.4%, the use of the SWs instead of the PBs allows a gain of 4.5%. The pinning strategy simply applied to the SWs increases the prediction accuracy to 39.9%. In a second step, the sequence-structure relationship is optimized, the prediction accuracy reaches 43.6%.

  12. Intra-residue interactions in proteins: interplay between serine or cysteine side chains and backbone conformations, revealed by laser spectroscopy of isolated model peptides.

    Science.gov (United States)

    Alauddin, Mohammad; Biswal, Himansu S; Gloaguen, Eric; Mons, Michel

    2015-01-21

    Intra-residue interactions play an important role in proteins by influencing local folding of the backbone. Taking advantage of the capability of gas phase experiments to provide relevant information on the intrinsic H-bonding pattern of isolated peptide chains, the intra-residue interactions of serine and cysteine residues, i.e., OH/SH···OC(i) C6 and NH(i···)O/S C5 interactions in Ser/Cys residues, are probed by laser spectroscopy of isolated peptides. The strength of these local side chain-main chain interactions, elegantly documented from their IR spectral features for well-defined conformations of the main chain, demonstrates that a subtle competition exists between the two types of intra-residue bond: the C6 H-bond is the major interaction with Ser, in contrast to Cys where C5 interaction takes over. The restricted number of conformers observed in the gas phase experiment with Ser compared to Cys (where both extended and folded forms are observed) also suggests a significant mediation role of these intra-residue interactions on the competition between the several main chain folding patterns.

  13. Reduced Dimensionality tailored HN(C)N Pulse Sequences for Efficient Backbone Resonance Assignment of Proteins through Rapid Identification of Sequential HSQC peaks

    CERN Document Server

    Kumar, Dinesh

    2013-01-01

    Two novel reduced dimensionality (RD) experiments -(4,3)D-hNCOcaNH and (4,3)D-hNcoCANH- have been presented here to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. The experiments basically represent an improvisation of previously reported HN(C)N experiment [Panchal et. al., J. Biomol. NMR. (2002), 20 (2), 135-147] and exploit the simple reduced dimensionality NMR concept [Szyperski et. al. (2002), Proc. Natl. Acad. Sci. U.S.A. 99(12), 8009-8014] to achieve (a) higher dispersion and resolution along the co-evolved F1 dimension and (b) rapid identification of sequential HSQC peaks on its F2(15N)- F3(1H) planes. The current implementation is based on the fact that the linear combination of 15N and 13CO/13Ca chemical shifts offers relatively better dispersion and randomness compared to the individual chemical shifts; thus enables the assignment of crowded HSQC spectra by resolving the ambiguities generally encountered in HNCN based assignment protocol because of ...

  14. Study on Lyophilized Protective for Recombinant Anti - artherosclerosis Protein on the Basis of SAK Backbone%重组SRH蛋白冻干保护剂的研究

    Institute of Scientific and Technical Information of China (English)

    邹民吉; 徐涛; 刘深; 付文亮; 徐东刚

    2011-01-01

    Objective To select a lyophilized protective for recombinant anti - artherosclerosis protein on the basis of SAK backbone. Methods Mannitol, dextran 40, sucrose,and glycine in their different concentrations were selected to make up a single recipe or different complex recipes of their concentrations. After lyophilization, the thrombolytic activity and plasticity change should be examined, in the meantime, the lyophilized sample was rapidly thowed at 371 and the changes of thrombolytic activity and plasticity also should be examined. Results The protective effects on the protein of interest were not significantly different between glyceral and dextran 40 in their different concentrations. Both of them possessed better protective effect, however, that of the sucrose and glycine in different concentrations were less effective. The complex recipe of dextran 40, sucrose and glycine displayed the best protective effect on the protein of interest among the complex recipes, but the complex of gyceral, sucrose and glycine in the different concentrations had no obvious protective effect. Conclusion The dextran 40, sucrose and gycine in differet concentrations possess a significantly protective effect on the lyophilized process of the recombinant anti - artherosclerosis protein on the basis of SAK backbone.%目的 筛选并优化重组SRH蛋白的冻千保护剂,使其能够保持良好的生物学活性.方法 选择不同浓度的冻干保护剂,包括甘露醇、右旋糖酐40、蔗糖和甘氨酸等组成不同浓度的单一和复合配方,冷冻干燥后检测冻干样品的溶栓活性和成型的变化,同时,冻干样品经过37℃加速实验后,检测其溶栓活性和成型状况的改变,对比分析各组保护剂的效果.结果 不同浓度的甘露醇和右旋糖苷40对SRH蛋白的保护作用良好,没有明显差异,而不同浓度的蔗糖和甘氨酸保护效果较差.复方中以右旋糖苷40、蔗糖和甘胺酸的复合对目的蛋白的保

  15. Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded {sup 15}N/{sup 19}F-labeled unnatural amino acid

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Pan [National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China); School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026 (China); Xi, Zhaoyong; Wang, Hu [School of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026 (China); Shi, Chaowei [National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China); School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026 (China); Xiong, Ying, E-mail: yxiong73@ustc.edu.cn [School of Life Science, University of Science and Technology of China, Hefei, Anhui 230026 (China); Tian, Changlin, E-mail: cltian@ustc.edu.cn [National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026 (China)

    2010-11-19

    Research highlights: {yields} Chemical synthesis of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine. {yields} Site-specific incorporation of {sup 15}N/{sup 19}F-trifluomethyl phenylalanine to SH3. {yields} Site-specific backbone and side chain chemical shift and relaxation analysis. {yields} Different internal motions at different sites of SH3 domain upon ligand binding. -- Abstract: SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for {sup 15}N/{sup 19}F-trifluoromethyl-phenylalanine ({sup 15}N/{sup 19}F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ({sup 15}N){sup 1}H and side-chain {sup 19}F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.

  16. Efficient protocol for backbone and side-chain assignments of large, intrinsically disordered proteins: transient secondary structure analysis of 49.2 kDa microtubule associated protein 2c

    Energy Technology Data Exchange (ETDEWEB)

    Novacek, Jiri; Janda, Lubomir; Dopitova, Radka; Zidek, Lukas, E-mail: lzidek@chemi.muni.cz; Sklenar, Vladimir [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic)

    2013-08-15

    Microtubule-associated proteins (MAPs) are abundantly present in axons and dendrites, and have been shown to play crucial role during the neuronal morphogenesis. The period of main dendritic outgrowth and synaptogenesis coincides with high expression levels of one of MAPs, the MAP2c, in rats. The MAP2c is a 49.2 kDa intrinsically disordered protein. To achieve an atomic resolution characterization of such a large protein, we have developed a protocol based on the acquisition of two five-dimensional {sup 13}C-directly detected NMR experiments. Our previously published 5D CACONCACO experiment (Novacek et al. in J Biomol NMR 50(1):1-11, 2011) provides the sequential assignment of the backbone resonances, which is not interrupted by the presence of the proline residues in the amino acid sequence. A novel 5D HC(CC-TOCSY)CACON experiment facilitates the assignment of the aliphatic side chain resonances. To streamline the data analysis, we have developed a semi-automated procedure for signal assignments. The obtained data provides the first atomic resolution insight into the conformational state of MAP2c and constitutes a model for further functional studies of MAPs.

  17. Backbone dynamics of reduced plastocyanin from the cyanobacterium Anabaena variabilis: Regions involved in electron transfer have enhanced mobility

    DEFF Research Database (Denmark)

    Ma, L.X.; Hass, M.A.S.; Vierick, N.;

    2003-01-01

    The dynamics of the backbone of the electron-transfer protein plastocyanin from the cyanobacterium Anabaena variabilis were determined from the N-15 and C-13(alpha) R-1 and R-2) relaxation rates and steady-state [H-1]-N-15 and [H-1]-C-13 nuclear Overhauser effects (NOEs) using the model-free appr......The dynamics of the backbone of the electron-transfer protein plastocyanin from the cyanobacterium Anabaena variabilis were determined from the N-15 and C-13(alpha) R-1 and R-2) relaxation rates and steady-state [H-1]-N-15 and [H-1]-C-13 nuclear Overhauser effects (NOEs) using the model...

  18. Fractional {sup 13}C enrichment of isolated carbons using [1-{sup 13}C]- or [2-{sup 13}C]-glucose facilitates the accurate measurement of dynamics at backbone C{sup {alpha}} and side-chain methyl positions in proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lundstroem, Patrik [University of Toronto, Departments of Medical Genetics and Chemistry (Canada); Teilum, Kaare; Carstensen, Tommy [Lund University, Department of Biophysical Chemistry (Sweden); Bezsonova, Irina [University of Toronto, Department of Chemistry (Canada); Wiesner, Silke [University of Toronto, Department of Biochemistry (Canada); Hansen, D. Flemming [University of Toronto, Departments of Medical Genetics and Chemistry (Canada); Religa, Tomasz L. [Medical Research Council Centre for Protein Engineering (United Kingdom); Akke, Mikael [Lund University, Department of Biophysical Chemistry (Sweden); Kay, Lewis E. [University of Toronto, Departments of Medical Genetics and Chemistry (Canada)], E-mail: kay@pound.med.utoronto.ca

    2007-07-15

    A simple labeling approach is presented based on protein expression in [1-{sup 13}C]- or [2-{sup 13}C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C{sup {alpha}} sites, respectively. All of the methyl groups, with the exception of Thr and Ile({delta}1) are produced with isolated {sup 13}C spins (i.e., no {sup 13}C-{sup 13}C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-{alpha} sites are labeled without concomitant labeling at C{sup {beta}} positions for 17 of the common 20 amino acids and there are no cases for which {sup 13}C{sup {alpha}}-{sup 13}CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone {sup 15}N studies. The utility of the labeling is established by recording {sup 13}C R{sub 1{rho}} and CPMG-based experiments on a number of different protein systems.

  19. NET amyloidogenic backbone in human activated neutrophils.

    Science.gov (United States)

    Pulze, L; Bassani, B; Gini, E; D'Antona, P; Grimaldi, A; Luini, A; Marino, F; Noonan, D M; Tettamanti, G; Valvassori, R; de Eguileor, M

    2016-03-01

    Activated human neutrophils produce a fibrillar DNA network [neutrophil extracellular traps (NETs)] for entrapping and killing bacteria, fungi, protozoa and viruses. Our results suggest that the neutrophil extracellular traps show a resistant amyloidogenic backbone utilized for addressing reputed proteins and DNA against the non-self. The formation of amyloid fibrils in neutrophils is regulated by the imbalance of reactive oxygen species (ROS) in the cytoplasm. The intensity and source of the ROS signal is determinant for promoting stress-associated responses such as amyloidogenesis and closely related events: autophagy, exosome release, activation of the adrenocorticotrophin hormone/α-melanocyte-stimulating hormone (ACTH/α-MSH) loop and synthesis of specific cytokines. These interconnected responses in human activated neutrophils, that have been evaluated from a morphofunctional and quantitative viewpoint, represent primitive, but potent, innate defence mechanisms. In invertebrates, circulating phagocytic immune cells, when activated, show responses similar to those described previously for activated human neutrophils. Invertebrate cells within endoplasmic reticulum cisternae produce a fibrillar material which is then assembled into an amyloidogenic scaffold utilized to convey melanin close to the invader. These findings, in consideration to the critical role played by NET in the development of several pathologies, could explain the structural resistance of these scaffolds and could provide the basis for developing new diagnostic and therapeutic approaches in immunomediated diseases in which the innate branch of the immune system has a pivotal role.

  20. Protein fold determined by paramagnetic magic-angle spinning solid-state NMR spectroscopy

    Science.gov (United States)

    Sengupta, Ishita; Nadaud, Philippe S.; Helmus, Jonathan J.; Schwieters, Charles D.; Jaroniec, Christopher P.

    2012-01-01

    Biomacromolecules that are challenging for the usual structural techniques can be studied with atomic resolution by solid-state nuclear magnetic resonance. However, the paucity of >5 Å distance restraints, traditionally derived from measurements of magnetic dipole-dipole couplings between protein nuclei, is a major bottleneck that hampers such structure elucidation efforts. Here we describe a general approach that enables the rapid determination of global protein fold in the solid phase via measurements of nuclear paramagnetic relaxation enhancements (PREs) in several analogs of the protein of interest containing covalently-attached paramagnetic tags, without the use of conventional internuclear distance restraints. The method is demonstrated using six cysteine-EDTA-Cu2+ mutants of the 56-residue B1 immunoglobulin-binding domain of protein G, for which ~230 longitudinal backbone 15N PREs corresponding to ~10-20 Å distances were obtained. The mean protein fold determined in this manner agrees with the X-ray structure with a backbone atom root-mean-square deviation of 1.8 Å. PMID:22522262

  1. Extracting Backbones from Weighted Complex Networks with Incomplete Information

    Directory of Open Access Journals (Sweden)

    Liqiang Qian

    2015-01-01

    Full Text Available The backbone is the natural abstraction of a complex network, which can help people understand a networked system in a more simplified form. Traditional backbone extraction methods tend to include many outliers into the backbone. What is more, they often suffer from the computational inefficiency—the exhaustive search of all nodes or edges is often prohibitively expensive. In this paper, we propose a backbone extraction heuristic with incomplete information (BEHwII to find the backbone in a complex weighted network. First, a strict filtering rule is carefully designed to determine edges to be preserved or discarded. Second, we present a local search model to examine part of edges in an iterative way, which only relies on the local/incomplete knowledge rather than the global view of the network. Experimental results on four real-life networks demonstrate the advantage of BEHwII over the classic disparity filter method by either effectiveness or efficiency validity.

  2. Probabilistic Determination of Native State Ensembles of Proteins.

    Science.gov (United States)

    Olsson, Simon; Vögeli, Beat Rolf; Cavalli, Andrea; Boomsma, Wouter; Ferkinghoff-Borg, Jesper; Lindorff-Larsen, Kresten; Hamelryck, Thomas

    2014-08-12

    The motions of biological macromolecules are tightly coupled to their functions. However, while the study of fast motions has become increasingly feasible in recent years, the study of slower, biologically important motions remains difficult. Here, we present a method to construct native state ensembles of proteins by the combination of physical force fields and experimental data through modern statistical methodology. As an example, we use NMR residual dipolar couplings to determine a native state ensemble of the extensively studied third immunoglobulin binding domain of protein G (GB3). The ensemble accurately describes both local and nonlocal backbone fluctuations as judged by its reproduction of complementary experimental data. While it is difficult to assess precise time-scales of the observed motions, our results suggest that it is possible to construct realistic conformational ensembles of biomolecules very efficiently. The approach may allow for a dramatic reduction in the computational as well as experimental resources needed to obtain accurate conformational ensembles of biological macromolecules in a statistically sound manner.

  3. Constructing backbone network by using tinker algorithm

    Science.gov (United States)

    He, Zhiwei; Zhan, Meng; Wang, Jianxiong; Yao, Chenggui

    2017-01-01

    Revealing how a biological network is organized to realize its function is one of the main topics in systems biology. The functional backbone network, defined as the primary structure of the biological network, is of great importance in maintaining the main function of the biological network. We propose a new algorithm, the tinker algorithm, to determine this core structure and apply it in the cell-cycle system. With this algorithm, the backbone network of the cell-cycle network can be determined accurately and efficiently in various models such as the Boolean model, stochastic model, and ordinary differential equation model. Results show that our algorithm is more efficient than that used in the previous research. We hope this method can be put into practical use in relevant future studies.

  4. Design of HIV-1 protease inhibitors with pyrrolidinones and oxazolidinones as novel P1'-ligands to enhance backbone-binding interactions with protease: synthesis, biological evaluation, and protein-ligand X-ray studies

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Arun K.; Leshchenko-Yashchuk, Sofiya; Anderson, David D.; Baldridge, Abigail; Noetzel, Marcus; Miller, Heather B.; Tie, Yunfeng; Wang, Yuan-Fang; Koh, Yasuhiro; Weber, Irene T.; Mitsuya, Hiroaki; (GSU); (Purdue); (NCI); (Kumamoto University School of Medicine)

    2009-09-02

    Structure-based design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors are described. In an effort to enhance interactions with protease backbone atoms, we have incorporated stereochemically defined methyl-2-pyrrolidinone and methyl oxazolidinone as the P1{prime}-ligands. These ligands are designed to interact with Gly-27{prime} carbonyl and Arg-8 side chain in the S1{prime}-subsite of the HIV protease. We have investigated the potential of these ligands in combination with our previously developed bis-tetrahydrofuran (bis-THF) and cyclopentanyltetrahydrofuran (Cp-THF) as the P2-ligands. Inhibitor 19b with a (R)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. This inhibitor maintained near full potency against multi-PI-resistant clinical HIV-1 variants. A high resolution protein-ligand X-ray crystal structure of 19b-bound HIV-1 protease revealed that the P1{prime}-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1{prime} and S2 subsites of HIV-1 protease.

  5. Peptaibol antiamoebin I: spatial structure, backbone dynamics, interaction with bicelles and lipid-protein nanodiscs, and pore formation in context of barrel-stave model.

    Science.gov (United States)

    Shenkarev, Zakhar O; Paramonov, Alexander S; Lyukmanova, Ekaterina N; Gizatullina, Albina K; Zhuravleva, Anastasia V; Tagaev, Andrey A; Yakimenko, Zoya A; Telezhinskaya, Irina N; Kirpichnikov, Mikhail P; Ovchinnikova, Tatiana V; Arseniev, Alexander S

    2013-05-01

    Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. In comparison with other 16-amino acid-residue peptaibols, e.g., zervamicin IIB (Zrv-IIB), Aam-I possesses relatively weak biological and channel-forming activities. In MeOH solution, Aam-I demonstrates fast cooperative transitions between right-handed and left-handed helical conformation of the N-terminal (1-8) region. We studied Aam-I spatial structure and backbone dynamics in the membrane-mimicking environment (DMPC/DHPC bicelles)(1) ) by heteronuclear (1) H,(13) C,(15) N-NMR spectroscopy. Interaction with the bicelles stabilizes the Aam-I right-handed helical conformation retaining significant intramolecular mobility on the ms-μs time scale. Extensive ms-μs dynamics were also detected in the DPC and DHPC micelles and DOPG nanodiscs. In contrast, Zrv-IIB in the DPC micelles demonstrates appreciably lesser mobility on the μs-ms time scale. Titration with Mn(2+) and 16-doxylstearate paramagnetic probes revealed Aam-I binding to the bicelle surface with the N-terminus slightly immersed into hydrocarbon region. Fluctuations of the Aam-I helix between surface-bound and transmembrane (TM) state were observed in the nanodisc membranes formed from the short-chain (diC12 : 0) DLPC/DLPG lipids. All the obtained experimental data are in agreement with the barrel-stave model of TM pore formation, similarly to the mechanism proposed for Zrv-IIB and other peptaibols. The observed extensive intramolecular dynamics explains the relatively low activity of Aam-I.

  6. Simultaneous determination of protein structure and dynamics

    DEFF Research Database (Denmark)

    Lindorff-Larsen, Kresten; Best, Robert B.; DePristo, M. A.

    2005-01-01

    We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy-for obtaining experimental information at ...

  7. Protein Structure Determination Using Chemical Shifts

    DEFF Research Database (Denmark)

    Christensen, Anders Steen

    In this thesis, a protein structure determination using chemical shifts is presented. The method is implemented in the open source PHAISTOS protein simulation framework. The method combines sampling from a generative model with a coarse-grained force field and an energy function that includes...... chemical shifts. The method is benchmarked on folding simulations of five small proteins. In four cases the resulting structures are in excellent agreement with experimental data, the fifth case fail likely due to inaccuracies in the energy function. For the Chymotrypsin Inhibitor protein, a structure...

  8. Protein Structure Determination Using Chemical Shifts

    DEFF Research Database (Denmark)

    Christensen, Anders Steen

    In this thesis, a protein structure determination using chemical shifts is presented. The method is implemented in the open source PHAISTOS protein simulation framework. The method combines sampling from a generative model with a coarse-grained force field and an energy function that includes...... chemical shifts. The method is benchmarked on folding simulations of five small proteins. In four cases the resulting structures are in excellent agreement with experimental data, the fifth case fail likely due to inaccuracies in the energy function. For the Chymotrypsin Inhibitor protein, a structure...... is determined using only chemical shifts recorded and assigned through automated processes. The CARMSD to the experimental X-ray for this structure is 1.1. Å. Additionally, the method is combined with very sparse NOE-restraints and evolutionary distance restraints and tested on several protein structures >100...

  9. Backbone and sidechain 1H, 13C and 15N resonance assignments of the human brain-type fatty acid binding protein (FABP7) in its apo form and the holo forms binding to DHA, oleic acid, linoleic acid and elaidic acid

    DEFF Research Database (Denmark)

    Oeemig, Jesper S; Jørgensen, Mathilde L; Hansen, Mikka S

    2009-01-01

    In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid.......In this manuscript, we present the backbone and side chain assignments of human brain-type fatty acid binding protein, also known as FABP7, in its apo form and in four different holo forms, bound to DHA, oleic acid, linoleic acid and elaidic acid....

  10. Membrane protein structure determination in membrana.

    Science.gov (United States)

    Ding, Yi; Yao, Yong; Marassi, Francesca M

    2013-09-17

    The two principal components of biological membranes, the lipid bilayer and the proteins integrated within it, have coevolved for specific functions that mediate the interactions of cells with their environment. Molecular structures can provide very significant insights about protein function. In the case of membrane proteins, the physical and chemical properties of lipids and proteins are highly interdependent; therefore structure determination should include the membrane environment. Considering the membrane alongside the protein eliminates the possibility that crystal contacts or detergent molecules could distort protein structure, dynamics, and function and enables ligand binding studies to be performed in a natural setting. Solid-state NMR spectroscopy is compatible with three-dimensional structure determination of membrane proteins in phospholipid bilayer membranes under physiological conditions and has played an important role in elucidating the physical and chemical properties of biological membranes, providing key information about the structure and dynamics of the phospholipid components. Recently, developments in the recombinant expression of membrane proteins, sample preparation, pulse sequences for high-resolution spectroscopy, radio frequency probes, high-field magnets, and computational methods have enabled a number of membrane protein structures to be determined in lipid bilayer membranes. In this Account, we illustrate solid-state NMR methods with examples from two bacterial outer membrane proteins (OmpX and Ail) that form integral membrane β-barrels. The ability to measure orientation-dependent frequencies in the solid-state NMR spectra of membrane-embedded proteins provides the foundation for a powerful approach to structure determination based primarily on orientation restraints. Orientation restraints are particularly useful for NMR structural studies of membrane proteins because they provide information about both three-dimensional structure

  11. hNCOcanH pulse sequence and a robust protocol for rapid and unambiguous assignment of backbone ((1)H(N), (15)N and (13)C') resonances in (15)N/(13)C-labeled proteins.

    Science.gov (United States)

    Kumar, Dinesh; Hosur, Ramakrishna V

    2011-09-01

    A three-dimensional nuclear magnetic resonance (NMR) pulse sequence named as hNCOcanH has been described to aid rapid sequential assignment of backbone resonances in (15)N/(13)C-labeled proteins. The experiment has been derived by a simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147]; t2 evolution is used to frequency label (13)C' rather than (15)N (similar trick has also been used in the design of hNCAnH pulse sequence from hNcaNH [Frueh et al., JACS, 131 (2009) 12880-12881]). The modification results in a spectrum equivalent to HNCO, but in addition to inter-residue correlation peaks (i.e. Hi , Ci-1), the spectrum also contains additional intra-residue correlation peaks (i.e. Hi-1 , Ci-1) in the direct proton dimension which has maximum resolution. This is the main strength of the experiment and thus, even a small difference in amide (1) H chemical shifts (5-6 Hz) can be used for establishing a sequential connectivity. This experiment in combination with the HNN experiment described previously [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] leads to a more robust assignment protocol for backbone resonances ((1) H(N) , (15)N) than could be derived from the combination of HNN and HN(C)N experiments [Bhavesh et al., Biochemistry, 40 (2001) 14727-14735]. Further, this new protocol enables assignment of (13)C' resonances as well. We believe that the experiment and the protocol presented here will be of immense value for structural-and functional-proteomics research by NMR. Performance of this experiment has been demonstrated using (13)C/(15)N labeled ubiquitin.

  12. Future High Capacity Backbone Networks

    DEFF Research Database (Denmark)

    Wang, Jiayuan

    This thesis - Future High Capacity Backbone Networks - deals with the energy efficiency problems associated with the development of future optical networks. In the first half of the thesis, novel approaches for using multiple/single alternative energy sources for improving energy efficiency...... the context of the integrated control plane structure. Results show improvements of energy efficiency over three types of traffic, while still keeping acceptable QoS levels for high priority traffic....

  13. Simultaneous determination of protein structure and dynamics

    DEFF Research Database (Denmark)

    Lindorff-Larsen, Kresten; Best, Robert B.; DePristo, M. A.

    2005-01-01

    We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy-for obtaining experimental information...... at the atomic level about the structural and dynamical features of proteins-with the ability of molecular dynamics simulations to explore a wide range of protein conformations. We illustrate the method for human ubiquitin in solution and find that there is considerable conformational heterogeneity throughout...... the protein structure. The interior atoms of the protein are tightly packed in each individual conformation that contributes to the ensemble but their overall behaviour can be described as having a significant degree of liquid-like character. The protocol is completely general and should lead to significant...

  14. Adding diverse noncanonical backbones to rosetta: enabling peptidomimetic design.

    Directory of Open Access Journals (Sweden)

    Kevin Drew

    Full Text Available Peptidomimetics are classes of molecules that mimic structural and functional attributes of polypeptides. Peptidomimetic oligomers can frequently be synthesized using efficient solid phase synthesis procedures similar to peptide synthesis. Conformationally ordered peptidomimetic oligomers are finding broad applications for molecular recognition and for inhibiting protein-protein interactions. One critical limitation is the limited set of design tools for identifying oligomer sequences that can adopt desired conformations. Here, we present expansions to the ROSETTA platform that enable structure prediction and design of five non-peptidic oligomer scaffolds (noncanonical backbones, oligooxopiperazines, oligo-peptoids, [Formula: see text]-peptides, hydrogen bond surrogate helices and oligosaccharides. This work is complementary to prior additions to model noncanonical protein side chains in ROSETTA. The main purpose of our manuscript is to give a detailed description to current and future developers of how each of these noncanonical backbones was implemented. Furthermore, we provide a general outline for implementation of new backbone types not discussed here. To illustrate the utility of this approach, we describe the first tests of the ROSETTA molecular mechanics energy function in the context of oligooxopiperazines, using quantum mechanical calculations as comparison points, scanning through backbone and side chain torsion angles for a model peptidomimetic. Finally, as an example of a novel design application, we describe the automated design of an oligooxopiperazine that inhibits the p53-MDM2 protein-protein interaction. For the general biological and bioengineering community, several noncanonical backbones have been incorporated into web applications that allow users to freely and rapidly test the presented protocols (http://rosie.rosettacommons.org. This work helps address the peptidomimetic community's need for an automated and expandable

  15. Protein structure quality assessment based on the distance profiles of consecutive backbone Cα atoms [v3; ref status: indexed, http://f1000r.es/2kg

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    2013-12-01

    Full Text Available Predicting the three dimensional native state structure of a protein from its primary sequence is an unsolved grand challenge in molecular biology. Two main computational approaches have evolved to obtain the structure from the protein sequence - ab initio/de novo methods and template-based modeling - both of which typically generate multiple possible native state structures. Model quality assessment programs (MQAP validate these predicted structures in order to identify the correct native state structure. Here, we propose a MQAP for assessing the quality of protein structures based on the distances of consecutive Cα atoms. We hypothesize that the root-mean-square deviation of the distance of consecutive Cα (RDCC atoms from the ideal value of 3.8 Å, derived from a statistical analysis of high quality protein structures (top100H database, is minimized in native structures. Based on tests with the top100H set, we propose a RDCC cutoff value of 0.012 Å, above which a structure can be filtered out as a non-native structure. We applied the RDCC discriminator on decoy sets from the Decoys 'R' Us database to show that the native structures in all decoy sets tested have RDCC below the 0.012 Å cutoff. While most decoy sets were either indistinguishable using this discriminator or had very few violations, all the decoy structures in the fisa decoy set were discriminated by applying the RDCC criterion. This highlights the physical non-viability of the fisa decoy set, and possible issues in benchmarking other methods using this set. The source code and manual is made available at https://github.com/sanchak/mqap and permanently available on 10.5281/zenodo.7134.

  16. Deuterium isotope shifts for backbone ¹H, ¹⁵N and ¹³C nuclei in intrinsically disordered protein α-synuclein.

    Science.gov (United States)

    Maltsev, Alexander S; Ying, Jinfa; Bax, Ad

    2012-10-01

    Intrinsically disordered proteins (IDPs) are abundant in nature and characterization of their potential structural propensities remains a widely pursued but challenging task. Analysis of NMR secondary chemical shifts plays an important role in such studies, but the output of such analyses depends on the accuracy of reference random coil chemical shifts. Although uniform perdeuteration of IDPs can dramatically increase spectral resolution, a feature particularly important for the poorly dispersed IDP spectra, the impact of deuterium isotope shifts on random coil values has not yet been fully characterized. Very precise (2)H isotope shift measurements for (13)C(α), (13)C(β), (13)C', (15)N, and (1)H(N) have been obtained by using a mixed sample of protonated and uniformly perdeuterated α-synuclein, a protein with chemical shifts exceptionally close to random coil values. Decomposition of these isotope shifts into one-bond, two-bond and three-bond effects as well as intra- and sequential residue contributions shows that such an analysis, which ignores conformational dependence, is meaningful but does not fully describe the total isotope shift to within the precision of the measurements. Random coil (2)H isotope shifts provide an important starting point for analysis of such shifts in structural terms in folded proteins, where they are known to depend strongly on local geometry.

  17. Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.

    Science.gov (United States)

    Harris, Paul W R; Brimble, Margaret A

    2015-03-01

    The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilizing tail, was assembled in a convergent manner from four unprotected peptide α-thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc solid phase peptide synthesis (SPPS) respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic C-terminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein. © 2015 Wiley Periodicals, Inc.

  18. Six methods for determining urinary protein compared.

    Science.gov (United States)

    Dilena, B A; Penberthy, L A; Fraser, C G

    1983-03-01

    Inter-laboratory surveys have shown that "routine methods" of urinary protein determination are often unsatisfactory. Therefore, we compared six frequently used methods for determination of protein in urine with respect to linearity, within-batch and between-batch precision, comparative bias, and practicability. We assayed dilutions of human and bovine albumin and serum, and fresh and lyophilized human urine. We find that the AACC Selected Method has poor practicability and poor precision under routine conditions, but good linearity. The sulfosalicylic acid/biuret technique is also impracticable, requires a large sample, and is not linear at low concentrations of urine protein. The Coomassie Brilliant Blue technique has a narrow range of linearity and poor precision. The sulfosalicylic acid/sodium sulfate turbidimetric method is not precise and cannot be standardized with bovine materials. The Ponceau-S technique has good performance characteristics and practicability, and we recommend it for routine laboratory use.

  19. Solution structure and backbone dynamics of the pleckstrin homology domain of the human protein kinase B (PKB/Akt). Interaction with inositol phosphates.

    Science.gov (United States)

    Auguin, Daniel; Barthe, Philippe; Augé-Sénégas, Marie-Thérèse; Stern, Marc-Henri; Noguchi, Masayuki; Roumestand, Christian

    2004-02-01

    The programmed cell death occurs as part of normal mammalian development. The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 3'-OH kinase (PI3K)/Protein Kinase B (PKB, also named Akt) cascade. Several targets of the PI3K/PKB signaling pathway have been identified that may underlie the ability of this regulatory cascade to promote cell survival. PKB possesses a N-terminal Pleckstrin Homology (PH) domain that binds specifically and with high affinity to PtIns(3,4,5)P(3) and PtIns(3,4)P(2), the PI3K second messengers. PKB is then recruited to the plasma membrane by virtue of its interaction with 3'-OH phosphatidylinositides and activated. Recent evidence indicates that PKB is active in various types of human cancer; constitutive PKB signaling activation is believed to promote proliferation and increased cell survival, thereby contributing to cancer progression. Thus, it has been shown that induction of PKB activity is augmented by the TCL1/MTCP1 oncoproteins through a physical association requiring the PKB PH domain. Here we present the three-dimensional solution structure of the PH domain of the human protein PKB (isoform beta). PKBbeta-PH is an electrostatically polarized molecule that adopts the same fold and topology as other PH-domains, consisting of a beta-sandwich of seven strands capped on one top by an alpha-helix. The opposite face presents three variable loops that appear poorly defined in the NMR structure. Measurements of (15)N spin relaxation times and heteronuclear (15)N[(1)H]NOEs showed that this poor definition is due to intrinsic flexibility, involving complex motions on different time scales. Chemical shift mapping studies correctly defined the binding site of Ins(1,3,4,5)P(4) (the head group of PtIns(3,4,5)P(3)), as was previously proposed

  20. Cross-Correlated Relaxation of Dipolar Coupling and Chemical-Shift Anisotropy in Magic-Angle Spinning R1ρ NMR Measurements: Application to Protein Backbone Dynamics Measurements.

    Science.gov (United States)

    Kurauskas, Vilius; Weber, Emmanuelle; Hessel, Audrey; Ayala, Isabel; Marion, Dominique; Schanda, Paul

    2016-09-01

    Transverse relaxation rate measurements in magic-angle spinning solid-state nuclear magnetic resonance provide information about molecular motions occurring on nanosecond-to-millisecond (ns-ms) time scales. The measurement of heteronuclear ((13)C, (15)N) relaxation rate constants in the presence of a spin-lock radiofrequency field (R1ρ relaxation) provides access to such motions, and an increasing number of studies involving R1ρ relaxation in proteins have been reported. However, two factors that influence the observed relaxation rate constants have so far been neglected, namely, (1) the role of CSA/dipolar cross-correlated relaxation (CCR) and (2) the impact of fast proton spin flips (i.e., proton spin diffusion and relaxation). We show that CSA/D CCR in R1ρ experiments is measurable and that the CCR rate constant depends on ns-ms motions; it can thus provide insight into dynamics. We find that proton spin diffusion attenuates this CCR due to its decoupling effect on the doublet components. For measurements of dynamics, the use of R1ρ rate constants has practical advantages over the use of CCR rates, and this article reveals factors that have so far been disregarded and which are important for accurate measurements and interpretation.

  1. CASD-NMR 2: robust and accurate unsupervised analysis of raw NOESY spectra and protein structure determination with UNIO

    Energy Technology Data Exchange (ETDEWEB)

    Guerry, Paul; Duong, Viet Dung; Herrmann, Torsten, E-mail: torsten.herrmann@ens-lyon.fr [Université de Lyon (UMR 5280 CNRS, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1), Institut des Sciences Analytiques, Centre de RMN à très Hauts Champs (France)

    2015-08-15

    UNIO is a comprehensive software suite for protein NMR structure determination that enables full automation of all NMR data analysis steps involved—including signal identification in NMR spectra, sequence-specific backbone and side-chain resonance assignment, NOE assignment and structure calculation. Within the framework of the second round of the community-wide stringent blind NMR structure determination challenge (CASD-NMR 2), we participated in two categories of CASD-NMR 2, namely using either raw NMR spectra or unrefined NOE peak lists as input. A total of 15 resulting NMR structure bundles were submitted for 9 out of 10 blind protein targets. All submitted UNIO structures accurately coincided with the corresponding blind targets as documented by an average backbone root mean-square deviation to the reference proteins of only 1.2 Å. Also, the precision of the UNIO structure bundles was virtually identical to the ensemble of reference structures. By assessing the quality of all UNIO structures submitted to the two categories, we find throughout that only the UNIO–ATNOS/CANDID approach using raw NMR spectra consistently yielded structure bundles of high quality for direct deposition in the Protein Data Bank. In conclusion, the results obtained in CASD-NMR 2 are another vital proof for robust, accurate and unsupervised NMR data analysis by UNIO for real-world applications.

  2. Protein topology determines cysteine oxidation fate: the case of sulfenyl amide formation among protein families.

    Science.gov (United States)

    Defelipe, Lucas A; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A; Turjanski, Adrián G

    2015-03-01

    Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function.

  3. Protein Structure Determination by Assembling Super-Secondary Structure Motifs Using Pseudocontact Shifts.

    Science.gov (United States)

    Pilla, Kala Bharath; Otting, Gottfried; Huber, Thomas

    2017-03-07

    Computational and nuclear magnetic resonance hybrid approaches provide efficient tools for 3D structure determination of small proteins, but currently available algorithms struggle to perform with larger proteins. Here we demonstrate a new computational algorithm that assembles the 3D structure of a protein from its constituent super-secondary structural motifs (Smotifs) with the help of pseudocontact shift (PCS) restraints for backbone amide protons, where the PCSs are produced from different metal centers. The algorithm, DINGO-PCS (3D assembly of Individual Smotifs to Near-native Geometry as Orchestrated by PCSs), employs the PCSs to recognize, orient, and assemble the constituent Smotifs of the target protein without any other experimental data or computational force fields. Using a universal Smotif database, the DINGO-PCS algorithm exhaustively enumerates any given Smotif. We benchmarked the program against ten different protein targets ranging from 100 to 220 residues with different topologies. For nine of these targets, the method was able to identify near-native Smotifs.

  4. How biomass composition determines protein extractability

    NARCIS (Netherlands)

    Sari, Y.W.; Syafitri, U.; Sanders, J.P.M.; Bruins, M.E.

    2015-01-01

    Biomass consists of a complex mixture of different components, of which protein potentially has a high added value for biorefinery. In this study, protein extractability of different types of biomass, mostly by-products, was analyzed. Protein yield obtained from a three step extraction using alkali

  5. How biomass composition determines protein extractability

    NARCIS (Netherlands)

    Sari, Y.W.; Syafitri, U.; Sanders, J.P.M.; Bruins, M.E.

    2015-01-01

    Biomass consists of a complex mixture of different components, of which protein potentially has a high added value for biorefinery. In this study, protein extractability of different types of biomass, mostly by-products, was analyzed. Protein yield obtained from a three step extraction using alkali

  6. Exercise: The Backbone of Spine Treatment

    Medline Plus

    Full Text Available Exercise: The Backbone of Spine Treatment | View Video Back About Video Struggling with Low Back Pain? Many people are surprised to learn that carefully selected exercise can actually reduce back pain. Some exercises can ...

  7. Assays for Determination of Protein Concentration.

    Science.gov (United States)

    Olson, Bradley J S C

    2016-06-01

    Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also included is a rapid, inexpensive, and reliable BCA assay of total protein in SDS-PAGE sample buffer that is used for equal loading of SDS-PAGE gels. © 2016 by John Wiley & Sons, Inc.

  8. Determination of membrane protein glycation in diabetic tissue

    OpenAIRE

    Zhang, Eric Y.; Swaan, Peter W.

    1999-01-01

    Diabetes-associated hyperglycemia causes glycation of proteins at reactive amino groups, which can adversely affect protein function Although the effects of glycation on soluble proteins are well characterized, there is no information regarding membrane-associated proteins, mainly because of the lack of reproducible methods to determine protein glycation in vivo. The current study was conducted to establish such a method and to compare the glycation levels of membrane-associated proteins deri...

  9. Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions. Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Arun K.; Martyr, Cuthbert D.; Osswald, Heather L.; Sheri, Venkat Reddy; Kassekert, Luke A.; Chen, Shujing; Agniswamy, Johnson; Wang, Yuan-Fang; Hayashi, Hironori; Aoki, Manabu; Weber, Irene T.; Mitsuya, Hiroaki (GSU); (Kumamoto); (Purdue)

    2015-10-30

    Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand–binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolution X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand–binding site interactions are possibly responsible for their potent activity.

  10. Structural determination of intact proteins using mass spectrometry

    Science.gov (United States)

    Kruppa, Gary; Schoeniger, Joseph S.; Young, Malin M.

    2008-05-06

    The present invention relates to novel methods of determining the sequence and structure of proteins. Specifically, the present invention allows for the analysis of intact proteins within a mass spectrometer. Therefore, preparatory separations need not be performed prior to introducing a protein sample into the mass spectrometer. Also disclosed herein are new instrumental developments for enhancing the signal from the desired modified proteins, methods for producing controlled protein fragments in the mass spectrometer, eliminating complex microseparations, and protein preparatory chemical steps necessary for cross-linking based protein structure determination.Additionally, the preferred method of the present invention involves the determination of protein structures utilizing a top-down analysis of protein structures to search for covalent modifications. In the preferred method, intact proteins are ionized and fragmented within the mass spectrometer.

  11. Determination of protein allergenicity : studies in rats

    NARCIS (Netherlands)

    Penninks, A.H.; Knippels, L.M.J.

    2001-01-01

    For the safety evaluation of genetically engineered crops the potential allergenicity of the newly introduced protein(s) has become an important issue. There is, however, no universal and reliable test system for the evaluation of the allergenic potency of food products. The best known allergy

  12. Statistical analysis of protein kinase specificity determinants

    DEFF Research Database (Denmark)

    Kreegipuu, Andres; Blom, Nikolaj; Brunak, Søren;

    1998-01-01

    The site and sequence specificity of protein kinase, as well as the role of the secondary structure and surface accessibility of the phosphorylation sites on substrate proteins, was statistically analyzed. The experimental data were collected from the literature and are available on the World Wide...

  13. NMR study of non-structural proteins--part I: (1)H, (13)C, (15)N backbone and side-chain resonance assignment of macro domain from Mayaro virus (MAYV).

    Science.gov (United States)

    Melekis, Efstathios; Tsika, Aikaterini C; Lichière, Julie; Chasapis, Christos T; Margiolaki, Irene; Papageorgiou, Nicolas; Coutard, Bruno; Bentrop, Detlef; Spyroulias, Georgios A

    2015-04-01

    Macro domains are ADP-ribose-binding modules present in all eukaryotic organisms, bacteria and archaea. They are also found in non-structural proteins of several positive strand RNA viruses such as alphaviruses. Here, we report the high yield expression and preliminary structural analysis through solution NMR spectroscopy of the macro domain from New World Mayaro Alphavirus. The recombinant protein was well-folded and in a monomeric state. An almost complete sequence-specific assignment of its (1)H, (15)N and (13)C resonances was obtained and its secondary structure determined by TALOS+.

  14. Membrane topology of transmembrane proteins: determinants and experimental tools.

    Science.gov (United States)

    Lee, Hunsang; Kim, Hyun

    2014-10-17

    Membrane topology refers to the two-dimensional structural information of a membrane protein that indicates the number of transmembrane (TM) segments and the orientation of soluble domains relative to the plane of the membrane. Since membrane proteins are co-translationally translocated across and inserted into the membrane, the TM segments orient themselves properly in an early stage of membrane protein biogenesis. Each membrane protein must contain some topogenic signals, but the translocation components and the membrane environment also influence the membrane topology of proteins. We discuss the factors that affect membrane protein orientation and have listed available experimental tools that can be used in determining membrane protein topology.

  15. An exhaustive survey of regular peptide conformations using a new metric for backbone handedness (h

    Directory of Open Access Journals (Sweden)

    Ranjan V. Mannige

    2017-05-01

    Full Text Available The Ramachandran plot is important to structural biology as it describes a peptide backbone in the context of its dominant degrees of freedom—the backbone dihedral angles φ and ψ (Ramachandran, Ramakrishnan & Sasisekharan, 1963. Since its introduction, the Ramachandran plot has been a crucial tool to characterize protein backbone features. However, the conformation or twist of a backbone as a function of φ and ψ has not been completely described for both cis and trans backbones. Additionally, little intuitive understanding is available about a peptide’s conformation simply from knowing the φ and ψ values of a peptide (e.g., is the regular peptide defined by φ = ψ =  − 100°  left-handed or right-handed?. This report provides a new metric for backbone handedness (h based on interpreting a peptide backbone as a helix with axial displacement d and angular displacement θ, both of which are derived from a peptide backbone’s internal coordinates, especially dihedral angles φ, ψ and ω. In particular, h equals sin(θd∕|d|, with range [−1, 1] and negative (or positive values indicating left(or right-handedness. The metric h is used to characterize the handedness of every region of the Ramachandran plot for both cis (ω = 0° and trans (ω = 180° backbones, which provides the first exhaustive survey of twist handedness in Ramachandran (φ, ψ space. These maps fill in the ‘dead space’ within the Ramachandran plot, which are regions that are not commonly accessed by structured proteins, but which may be accessible to intrinsically disordered proteins, short peptide fragments, and protein mimics such as peptoids. Finally, building on the work of (Zacharias & Knapp, 2013, this report presents a new plot based on d and θ that serves as a universal and intuitive alternative to the Ramachandran plot. The universality arises from the fact that the co-inhabitants of such a plot include every possible peptide backbone

  16. Green Network Planning Model for Optical Backbones

    DEFF Research Database (Denmark)

    Gutierrez Lopez, Jose Manuel; Riaz, M. Tahir; Jensen, Michael

    2010-01-01

    Communication networks are becoming more essential for our daily lives and critically important for industry and governments. The intense growth in the backbone traffic implies an increment of the power demands of the transmission systems. This power usage might have a significant negative effect...... on the environment in general. In network planning there are existing planning models focused on QoS provisioning, investment minimization or combinations of both and other parameters. But there is a lack of a model for designing green optical backbones. This paper presents novel ideas to be able to define...

  17. Inferring the determinants of protein evolutionary rates in mammals.

    Science.gov (United States)

    Zou, Yang; Shao, Xiaojian; Dong, Dong

    2016-06-15

    Understanding the determinants of protein evolutionary rates is one of the most fundamental evolutionary questions. Previous studies have revealed that many biological variables are tightly associated with protein evolutionary rates in mammals. However, the dominant role of these biological variables and their combinatorial effects to evolutionary rates of mammalian proteins are still less understood. In this work, we derived a quantitative model to correlate protein evolutionary rates with the levels of these variables. The result showed that only a small number of variables are necessary to accurately predict protein evolutionary rates, among which miRNA regulation plays the most important role. Our result suggested that biological variables are extensively interrelated and suffer from hidden redundancies in determining protein evolutionary rates. Various variables should be considered in a natural ensemble to comprehensively assess the determinants of protein evolutionary rate.

  18. Electroanalytical determination of tungsten and molybdenum in proteins.

    Science.gov (United States)

    Hagedoorn, P L; van't Slot, P; van Leeuwen, H P; Hagen, W R

    2001-10-01

    Recent crystal structure determinations accelerated the progress in the biochemistry of tungsten-containing enzymes. In order to characterize these enzymes, a sensitive determination of this metal in protein-containing samples is necessary. An electroanalytical tungsten determination has successfully been adapted to determine the tungsten and molybdenum content in enzymes. The tungsten and molybdenum content can be measured simultaneously from 1 to 10 microg of purified protein with little or no sample handling. More crude protein samples require precipitation of interfering surface active material with 10% perchloric acid. This method affords the isolation of novel molybdenum- and tungsten-containing proteins via molybdenum and tungsten monitoring of column fractions, without using radioactive isotopes. A screening of soluble proteins from Pyrococcus furiosus for tungsten, using anion-exchange column chromatography to separate the proteins, has been performed. The three known tungsten-containing enzymes from P. furiosus were recovered with this screening.

  19. Determination of the protein quality of cooked Canadian pulses

    OpenAIRE

    Nosworthy, Matthew G.; Neufeld, Jason; Frohlich, Peter; Young, Gina; Malcolmson, Linda; House, James D

    2017-01-01

    Abstract A study to determine the protein digestibility?corrected amino acid score and protein efficiency ratio of nine different cooked Canadian pulse classes was conducted in support of the establishment of protein quality claims in Canada and the United States. Split green and yellow pea, whole green lentil, split red lentil, Kabuli chickpea, navy bean, pinto bean, light red kidney bean, and black bean were investigated. Protein digestibility?corrected amino acid score (PDCAAS) and the pro...

  20. Microsoft Operations Framework implementation for The Backbone

    NARCIS (Netherlands)

    Kienhuis, G.H.

    2007-01-01

    Doel The Backbone ontwerpt, implementeert en beheert IT infrastructuren voor bedrijven en instellingen. Beheer wordt proactief uitgevoerd met behulp van Microsoft Operation Manager (MOM) 2005. MOM is een applicatie die de status en gebeurtenissen van systemen zichtbaar maakt vanuit één locatie. Om

  1. A simple model for the band structure and D.C. conductivity of an infinite C dbond O···H bond N chain perpendicular to the protein backbone

    Science.gov (United States)

    Bende, Attila; Bogár, Ferenc; Ladik, János

    The1 Hartree-Fock crystal orbital (CO) method in its linear combination of atomic orbitals form was applied to determine the band structure of histone proteins taking 0.041e charge transfer per nucleotide base from the PO4- groups of poly(guanilic acid) to the arginine, and lysine side chains in histones (see text). Assuming that there are infinite COs, perpendicular to the main chain, formed by the amide groups of one segment of the protein chain bound together by H-bonds with the C dbond O groups of another segment of the chain, we have calculated the band structure. From this, we have determined the mobility using the deformation potential approximation. Multiplying this with the mobile electron concentration due to the charge transfer between the PO4- groups of DNA and the positive side chains in histones, we have obtained for the direct current (D.C.) electron conductivity sigmafib = 1.07 × 10-9 Omega-1 cm for a single fiber and after division by the cross-section of 9.10 × 10-16 cm2, sigmaspec = 1.18 × 106 Omega-1 cm-1 for the specific conductivity.

  2. Detection of tropomyosin and determination of proteins in crustacean oils.

    Science.gov (United States)

    Vang, Birthe; Mæhre, Hanne K; Jensen, Ida-J; Olsen, Ragnar L

    2013-11-01

    Tropomyosin is known to be the main allergen in crustaceans and the objective of this study was to investigate if this protein could be detected in commercial crustacean oils from Antarctic krill (Euphausia superba) and the zooplankton Calanus finmarchicus. We also examined the possibility of determining the protein content in the oils by direct amino acid analysis. Western blotting showed that a commercial antibody against shrimp tropomyosin cross-reacted with a protein of similar size in Antarctic krill and C. finmarchicus. The protein tentatively identified as tropomyosin, was also detected in krill oil products, but not in oils from C. finmarchicus. The acetone-heptane method used for extracting proteins in the oils is however not optimal. Other extraction methods should therefore be considered when investigating the presence of allergenic proteins in oils. Direct amino acid analysis on oils should be further explored as a method for determining the total amount of proteins present.

  3. J-UNIO protocol used for NMR structure determination of the 206-residue protein NP-346487.1 from Streptococcus pneumoniae TIGR4

    Energy Technology Data Exchange (ETDEWEB)

    Jaudzems, Kristaps [Latvian Institute of Organic Synthesis (Latvia); Pedrini, Bill [Paul Scherrer Institute (PSI), SwissFEL Project (Switzerland); Geralt, Michael; Serrano, Pedro; Wüthrich, Kurt, E-mail: wuthrich@scripps.edu [The Scripps Research Institute, Department of Integrative Structural and Computational Biology (United States)

    2015-01-15

    The NMR structure of the 206-residue protein NP-346487.1 was determined with the J-UNIO protocol, which includes extensive automation of the structure determination. With input from three APSY-NMR experiments, UNIO-MATCH automatically yielded 77 % of the backbone assignments, which were interactively validated and extended to 97 %. With an input of the near-complete backbone assignments and three 3D heteronuclear-resolved [{sup 1}H,{sup 1}H]-NOESY spectra, automated side chain assignment with UNIO-ATNOS/ASCAN resulted in 77 % of the expected assignments, which was extended interactively to about 90 %. Automated NOE assignment and structure calculation with UNIO-ATNOS/CANDID in combination with CYANA was used for the structure determination of this two-domain protein. The individual domains in the NMR structure coincide closely with the crystal structure, and the NMR studies further imply that the two domains undergo restricted hinge motions relative to each other in solution. NP-346487.1 is so far the largest polypeptide chain to which the J-UNIO structure determination protocol has successfully been applied.

  4. Selectivity determinants of GPCR-G-protein binding

    DEFF Research Database (Denmark)

    Flock, Tilman; Hauser, Alexander S; Lund, Nadia

    2017-01-01

    The selective coupling of G-protein-coupled receptors (GPCRs) to specific G proteins is critical to trigger the appropriate physiological response. However, the determinants of selective binding have remained elusive. Here we reveal the existence of a selectivity barcode (that is, patterns of ami...

  5. Determination of the protein quality of cooked Canadian pulses.

    Science.gov (United States)

    Nosworthy, Matthew G; Neufeld, Jason; Frohlich, Peter; Young, Gina; Malcolmson, Linda; House, James D

    2017-07-01

    A study to determine the protein digestibility-corrected amino acid score and protein efficiency ratio of nine different cooked Canadian pulse classes was conducted in support of the establishment of protein quality claims in Canada and the United States. Split green and yellow pea, whole green lentil, split red lentil, Kabuli chickpea, navy bean, pinto bean, light red kidney bean, and black bean were investigated. Protein digestibility-corrected amino acid score (PDCAAS) and the protein efficiency ratio (PER) were determined using the appropriate rodent models. All pulses had high digestibility values, >70%, with PDCAAS values greater than 0.5, thereby qualifying as a quality protein in the United States, but only navy beans qualified as a good source of protein. All pulses except whole green lentils, split red lentils, and split green peas would qualify as sources of protein with protein ratings between 20 and 30.4 in Canada. These findings support the use of pulses as protein sources in the regulatory context of both the United States and Canada.

  6. Cancer associated proteins in blood plasma: Determining normal variation.

    Science.gov (United States)

    Stenemo, Markus; Teleman, Johan; Sjöström, Martin; Grubb, Gabriel; Malmström, Erik; Malmström, Johan; Niméus, Emma

    2016-07-01

    Protein biomarkers have the potential to improve diagnosis, stratification of patients into treatment cohorts, follow disease progression and treatment response. One distinct group of potential biomarkers comprises proteins which have been linked to cancer, known as cancer associated proteins (CAPs). We determined the normal variation of 86 CAPs in 72 individual plasma samples collected from ten individuals using SRM mass spectrometry. Samples were collected weekly during 5 weeks from ten volunteers and over one day at nine fixed time points from three volunteers. We determined the degree of the normal variation depending on interpersonal variation, variation due to time of day, and variation over weeks and observed that the variation dependent on the time of day appeared to be the most important. Subdivision of the proteins resulted in two predominant protein groups containing 21 proteins with relatively high variation in all three factors (day, week and individual), and 22 proteins with relatively low variation in all factors. We present a strategy for prioritizing biomarker candidates for future studies based on stratification over their normal variation and have made all data publicly available. Our findings can be used to improve selection of biomarker candidates in future studies and to determine which proteins are most suitable depending on study design.

  7. Spectrophotometric and Refractometric Determination of Total Protein in Avian Plasma

    Directory of Open Access Journals (Sweden)

    Rodica Căpriță

    2013-10-01

    Full Text Available The aim of this study was to compare the total protein values obtained in heparin plasma of chickens by a spectrophotometric technique (biuret method, and the values obtained on the same day in the same samples by refractometry. The results obtained by refractometry (average value 2.638±0.153g% were higher than those obtained by the spectrophotometric method (average value 2.441±0.181g%. There was a low correlation (r = 0.6709 between the total protein values, determined with both methods. Protein is the major determinant of plasma refractive index, but glucose contributes too. The refractometric method is not recommended in chickens for the determination of total protein, because avian blood glucose concentration averages about twice than in mammalian blood.

  8. Probing the physical determinants of thermal expansion of folded proteins.

    Science.gov (United States)

    Dellarole, Mariano; Kobayashi, Kei; Rouget, Jean-Baptiste; Caro, José Alfredo; Roche, Julien; Islam, Mohammad M; Garcia-Moreno E, Bertrand; Kuroda, Yutaka; Royer, Catherine A

    2013-10-24

    The magnitude and sign of the volume change upon protein unfolding are strongly dependent on temperature. This temperature dependence reflects differences in the thermal expansivity of the folded and unfolded states. The factors that determine protein molar expansivities and the large differences in thermal expansivity for proteins of similar molar volume are not well understood. Model compound studies have suggested that a major contribution is made by differences in the molar volume of water molecules as they transfer from the protein surface to the bulk upon heating. The expansion of internal solvent-excluded voids upon heating is another possible contributing factor. Here, the contribution from hydration density to the molar thermal expansivity of a protein was examined by comparing bovine pancreatic trypsin inhibitor and variants with alanine substitutions at or near the protein-water interface. Variants of two of these proteins with an additional mutation that unfolded them under native conditions were also examined. A modest decrease in thermal expansivity was observed in both the folded and unfolded states for the alanine variants compared with the parent protein, revealing that large changes can be made to the external polarity of a protein without causing large ensuing changes in thermal expansivity. This modest effect is not surprising, given the small molar volume of the alanine residue. Contributions of the expansion of the internal void volume were probed by measuring the thermal expansion for cavity-containing variants of a highly stable form of staphylococcal nuclease. Significantly larger (2-3-fold) molar expansivities were found for these cavity-containing proteins relative to the reference protein. Taken together, these results suggest that a key determinant of the thermal expansivities of folded proteins lies in the expansion of internal solvent-excluded voids.

  9. Using light scattering to determine the stoichiometry of protein complexes.

    Science.gov (United States)

    Mogridge, Jeremy

    2015-01-01

    The stoichiometry of a protein complex can be calculated from an accurate measurement of the complex's molecular weight. Multiangle laser light scattering in combination with size exclusion chromatography and interferometric refractometry provides a powerful means for determining the molecular weights of proteins and protein complexes. In contrast to conventional size exclusion chromatography and analytical centrifugation, measurements do not rely on the use of molecular weight standards and are not affected by the shape of the proteins. The technique is based on the direct relationship between the amount of light scattered by a protein in solution, and the product of its concentration and molecular weight. A typical experimental configuration includes a size exclusion column to fractionate the sample, a light scattering detector to measure scattered light, and an interferometric refractometer to measure protein concentration. The determination of the molecular weight of an anthrax toxin complex will be used to illustrate how multiangle laser light scattering can be used to determine the stoichiometry of protein complexes.

  10. Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity

    Directory of Open Access Journals (Sweden)

    Marie-Laurence Tremblay

    2016-08-01

    Full Text Available Spider aciniform (wrapping silk is a remarkable fibrillar biomaterial with outstanding mechanical properties. It is a modular protein consisting, in Argiope trifasciata, of a core repetitive domain of 200 amino acid units (W units. In solution, the W units comprise a globular folded core, with five α-helices, and disordered tails that are linked to form a ~63-residue intrinsically disordered linker in concatemers. Herein, we present nuclear magnetic resonance (NMR spectroscopy-based 15N spin relaxation analysis, allowing characterization of backbone dynamics as a function of residue on the ps–ns timescale in the context of the single W unit (W1 and the two unit concatemer (W2. Unambiguous mapping of backbone dynamics throughout W2 was made possible by segmental NMR active isotope-enrichment through split intein-mediated trans-splicing. Spectral density mapping for W1 and W2 reveals a striking disparity in dynamics between the folded core and the disordered linker and tail regions. These data are also consistent with rotational diffusion behaviour where each globular domain tumbles almost independently of its neighbour. At a localized level, helix 5 exhibits elevated high frequency dynamics relative to the proximal helix 4, supporting a model of fibrillogenesis where this helix unfolds as part of the transition to a mixed α-helix/β-sheet fibre.

  11. Porous solid backbone impregnation for electrochemical energy conversion systems

    KAUST Repository

    Boulfrad, Samir

    2013-09-19

    An apparatus and method for impregnating a porous solid backbone. The apparatus may include a platform for holding a porous solid backbone, an ink jet nozzle configured to dispense a liquid solution onto the porous solid backbone, a positioning mechanism configured to position the ink jet nozzle proximate to a plurality of locations of the porous solid backbone, and a control unit configured to control the positioning mechanism to position the ink jet nozzle proximate to the plurality of locations and cause the ink jet nozzle to dispense the liquid solution onto the porous solid backbone.

  12. A segmental labeling strategy for unambiguous determination of domain-domain interactions of large multi-domain proteins

    Energy Technology Data Exchange (ETDEWEB)

    Chen Jianglei; Wang Jianjun, E-mail: jjwang@med.wayne.edu [Wayne State University, Department of Biochemistry and Molecular Biology, School of Medicine (United States)

    2011-08-15

    NMR structural determination of large multi-domain proteins is a challenging task due to significant spectral overlap with a particular difficulty in unambiguous identification of domain-domain interactions. Segmental labeling is a NMR strategy that allows for isotopically labeling one domain and leaves the other domain unlabeled. This significantly simplifies spectral overlaps and allows for quick identification of domain-domain interaction. Here, a novel segmental labeling strategy is presented for detection of inter-domain NOEs. To identify domain-domain interactions in human apolipoprotein E (apoE), a multi-domain, 299-residues {alpha}-helical protein, on-column expressed protein ligation was utilized to generate a segmental-labeled apoE samples in which the N-terminal (NT-) domain was {sup 2}H(99%)/{sup 15}N-labeled whereas the C-terminal (CT-) domain was either {sup 15}N- or {sup 15}N/{sup 13}C-labeled. 3-D {sup 15}N-edited NOESY spectra of these segmental-labeled apoE samples allow for direct observation of the inter-domain NOEs between the backbone amide protons of the NT-domain and the aliphatic protons of the CT-domain. This straightforward approach permits unambiguous identification of 78 inter-domain NOEs, enabling accurate definition of the relative positions of both the NT- and the CT-domains and determination of the NMR structure of apoE.

  13. A protein-dependent side-chain rotamer library.

    KAUST Repository

    Bhuyan, M.S.

    2011-12-14

    Protein side-chain packing problem has remained one of the key open problems in bioinformatics. The three main components of protein side-chain prediction methods are a rotamer library, an energy function and a search algorithm. Rotamer libraries summarize the existing knowledge of the experimentally determined structures quantitatively. Depending on how much contextual information is encoded, there are backbone-independent rotamer libraries and backbone-dependent rotamer libraries. Backbone-independent libraries only encode sequential information, whereas backbone-dependent libraries encode both sequential and locally structural information. However, side-chain conformations are determined by spatially local information, rather than sequentially local information. Since in the side-chain prediction problem, the backbone structure is given, spatially local information should ideally be encoded into the rotamer libraries. In this paper, we propose a new type of backbone-dependent rotamer library, which encodes structural information of all the spatially neighboring residues. We call it protein-dependent rotamer libraries. Given any rotamer library and a protein backbone structure, we first model the protein structure as a Markov random field. Then the marginal distributions are estimated by the inference algorithms, without doing global optimization or search. The rotamers from the given library are then re-ranked and associated with the updated probabilities. Experimental results demonstrate that the proposed protein-dependent libraries significantly outperform the widely used backbone-dependent libraries in terms of the side-chain prediction accuracy and the rotamer ranking ability. Furthermore, without global optimization/search, the side-chain prediction power of the protein-dependent library is still comparable to the global-search-based side-chain prediction methods.

  14. A protein-dependent side-chain rotamer library

    Directory of Open Access Journals (Sweden)

    Bhuyan Md Shariful

    2011-12-01

    Full Text Available Abstract Background Protein side-chain packing problem has remained one of the key open problems in bioinformatics. The three main components of protein side-chain prediction methods are a rotamer library, an energy function and a search algorithm. Rotamer libraries summarize the existing knowledge of the experimentally determined structures quantitatively. Depending on how much contextual information is encoded, there are backbone-independent rotamer libraries and backbone-dependent rotamer libraries. Backbone-independent libraries only encode sequential information, whereas backbone-dependent libraries encode both sequential and locally structural information. However, side-chain conformations are determined by spatially local information, rather than sequentially local information. Since in the side-chain prediction problem, the backbone structure is given, spatially local information should ideally be encoded into the rotamer libraries. Methods In this paper, we propose a new type of backbone-dependent rotamer library, which encodes structural information of all the spatially neighboring residues. We call it protein-dependent rotamer libraries. Given any rotamer library and a protein backbone structure, we first model the protein structure as a Markov random field. Then the marginal distributions are estimated by the inference algorithms, without doing global optimization or search. The rotamers from the given library are then re-ranked and associated with the updated probabilities. Results Experimental results demonstrate that the proposed protein-dependent libraries significantly outperform the widely used backbone-dependent libraries in terms of the side-chain prediction accuracy and the rotamer ranking ability. Furthermore, without global optimization/search, the side-chain prediction power of the protein-dependent library is still comparable to the global-search-based side-chain prediction methods.

  15. A protein-dependent side-chain rotamer library

    Science.gov (United States)

    2011-01-01

    Background Protein side-chain packing problem has remained one of the key open problems in bioinformatics. The three main components of protein side-chain prediction methods are a rotamer library, an energy function and a search algorithm. Rotamer libraries summarize the existing knowledge of the experimentally determined structures quantitatively. Depending on how much contextual information is encoded, there are backbone-independent rotamer libraries and backbone-dependent rotamer libraries. Backbone-independent libraries only encode sequential information, whereas backbone-dependent libraries encode both sequential and locally structural information. However, side-chain conformations are determined by spatially local information, rather than sequentially local information. Since in the side-chain prediction problem, the backbone structure is given, spatially local information should ideally be encoded into the rotamer libraries. Methods In this paper, we propose a new type of backbone-dependent rotamer library, which encodes structural information of all the spatially neighboring residues. We call it protein-dependent rotamer libraries. Given any rotamer library and a protein backbone structure, we first model the protein structure as a Markov random field. Then the marginal distributions are estimated by the inference algorithms, without doing global optimization or search. The rotamers from the given library are then re-ranked and associated with the updated probabilities. Results Experimental results demonstrate that the proposed protein-dependent libraries significantly outperform the widely used backbone-dependent libraries in terms of the side-chain prediction accuracy and the rotamer ranking ability. Furthermore, without global optimization/search, the side-chain prediction power of the protein-dependent library is still comparable to the global-search-based side-chain prediction methods. PMID:22373394

  16. Peptoid-Peptide hybrid backbone architectures

    DEFF Research Database (Denmark)

    Olsen, Christian Adam

    2010-01-01

    -amino acids (alpha/beta-peptides) have been investigated in some detail as well. The present Minireview is a survey of the literature concerning hybrid structures of alpha-amino acids and peptoids, including beta-peptoids (N-alkyl-beta-alanine oligomers), and is intended to give an overview of this area......Peptidomimetic oligomers and foldamers have received considerable attention for over a decade, with beta-peptides and the so-called peptoids (N-alkylglycine oligomers) representing prominent examples of such architectures. Lately, hybrid or mixed backbones consisting of both alpha- and beta...

  17. Instant Backbone.js application development

    CERN Document Server

    Hunter, Thomas

    2013-01-01

    Get to grips with a new technology, understand what it is and what it can do for you, and then get to work with the most important features and tasks. This book is a practical, step-by-step tutorial that will teach you to build Backbone.js applications quickly and efficiently.This book is targeted towards developers. It is assumed that you have at least a basic understanding of JavaScript and jQuery selectors. If you are interested in building dynamic Single Page Applications that interact heavily with a backend server, then this is the book for you.

  18. Antigenic determinants of alpha-like proteins of Streptococcus agalactiae.

    Science.gov (United States)

    Maeland, Johan A; Bevanger, Lars; Lyng, Randi Valsoe

    2004-11-01

    The majority of group B streptococcus (GBS) isolates express one or more of a family of surface-anchored proteins that vary by strain and that form ladder-like patterns on Western blotting due to large repeat units. These proteins, which are important as GBS serotype markers and as inducers of protective antibodies, include the alpha C (Calpha) and R4 proteins and the recently described alpha-like protein 2 (Alp2), encoded by alp2, and Alp3, encoded by alp3. In this study, we examined antigenic determinants possessed by Alp2 and Alp3 by testing of antibodies raised in rabbits, mainly by using enzyme-linked immunosorbent assays (ELISA) and an ELISA absorption test. The results showed that Alp2 and Alp3 shared an antigenic determinant, which may be a unique immunological marker of the Alp variants of GBS proteins. Alp2, in addition, possessed an antigenic determinant which showed specificity for Alp2 and a third determinant which showed serological cross-reactivity with Calpha. Alp3, in addition to the determinant common to Alp2 and Alp3, harbored an antigenic site which also was present in the R4 protein, whereas no Alp3-specific antigenic site was detected. These ELISA-based results were confirmed by Western blotting and a fluorescent-antibody test. The results are consistent with highly complex antigenic structures of the alpha-like proteins in a fashion which is in agreement with the recently described structural mosaicism of the alp2 and alp3 genes. The results are expected to influence GBS serotyping, immunoprotection studies, and GBS vaccine developments.

  19. Ultrasound-based protein determination in maize seeds.

    Science.gov (United States)

    Drochioiu, Gabi; Ciobanu, Catalina Ionica; Bancila, Sabina; Ion, Laura; Petre, Brindusa Alina; Andries, Claudia; Gradinaru, Robert Vasile; Murariu, Manuela

    2016-03-01

    The need for a simple and accurate method for protein estimation in alcoholic extracts led to the reexamination of the optimum conditions of a colorimetric assay based on the biuret reaction. Sonication time and the other experimental parameters were optimized after kinetics study on the extraction of either zein or total proteins. Zein extraction and purity were investigated by (1)H and (13)C NMR spectroscopy, SDS-PAGE electrophoresis, and UV-visible spectrophotometry (UV-vis). A zein assay was proposed, which involves the reaction of copper ions in copper phosphate powder with zein extracted in ethanolic solutions under strong alkaline environment. Furthermore, we extended this procedure to determine total proteins in maize samples simultaneously with their ultrasonic-assisted (US) extraction with an alkaline-alcoholic solution. Proteins in both types of extracts were well characterized by UV-vis spectroscopy. However, the 545 nm absorbance of the violet-colored supernatants which is proportional to the protein content was found to be the key parameter of the improved biuret-based protein assay. Comparison of values obtained by this procedure and by Micro-Kjeldahl method was in excellent agreement. A scaled-down procedure agreed well with the standard procedure. Enhanced accuracy and repeatability was found in protein determination in maize using the modified biuret method. The optimization of reagent concentrations and incubation times were studied as well.

  20. Quantitative Determination of Spatial Protein-protein Proximity in Fluorescence Confocal Microscopy

    CERN Document Server

    Wu, Yong; Ou, Jimmy; Li, Min; Toro, Ligia; Stefani, Enrico

    2009-01-01

    To quantify spatial protein-protein proximity (colocalization) in fluorescence microscopic images, cross-correlation and autocorrelation functions were decomposed into fast and slowly decaying components. The fast component results from clusters of proteins specifically labeled and the slow one from background/image heterogeneity. We show that the calculation of the protein-protein proximity index and the correlation coefficient are more reliably determined by extracting the fast-decaying component. This new method is illustrated by analyzing colocalization in both simulated and biological images.

  1. Optimizing an emperical scoring function for transmembrane protein structure determination.

    Energy Technology Data Exchange (ETDEWEB)

    Young, Malin M.; Sale, Kenneth L.; Gray, Genetha Anne; Kolda, Tamara Gibson

    2003-10-01

    We examine the problem of transmembrane protein structure determination. Like many other questions that arise in biological research, this problem cannot be addressed by traditional laboratory experimentation alone. An approach that integrates experiment and computation is required. We investigate a procedure which states the transmembrane protein structure determination problem as a bound constrained optimization problem using a special empirical scoring function, called Bundler, as the objective function. In this paper, we describe the optimization problem and some of its mathematical properties. We compare and contrast results obtained using two different derivative free optimization algorithms.

  2. Optimizing an emperical scoring function for transmembrane protein structure determination.

    Energy Technology Data Exchange (ETDEWEB)

    Young, Malin M.; Sale, Kenneth L.; Gray, Genetha Anne; Kolda, Tamara Gibson

    2003-10-01

    We examine the problem of transmembrane protein structure determination. Like many other questions that arise in biological research, this problem cannot be addressed by traditional laboratory experimentation alone. An approach that integrates experiment and computation is required. We investigate a procedure which states the transmembrane protein structure determination problem as a bound constrained optimization problem using a special empirical scoring function, called Bundler, as the objective function. In this paper, we describe the optimization problem and some of its mathematical properties. We compare and contrast results obtained using two different derivative free optimization algorithms.

  3. Peptide-functionalized semiconductor surfaces: strong surface electronic effects from minor alterations to backbone composition.

    Science.gov (United States)

    Matmor, Maayan; Lengyel, George A; Horne, W Seth; Ashkenasy, Nurit

    2017-02-22

    The use of non-canonical amino acids is a powerful way to control protein structure. Here, we show that subtle changes to backbone composition affect the ability of a dipeptide to modify solid surface electronic properties. The extreme sensitivity of the interactions to the peptide structure suggests potential applications in improving the performance of electronic devices.

  4. Use of refractometry for determination of psittacine plasma protein concentration.

    Science.gov (United States)

    Cray, Carolyn; Rodriguez, Marilyn; Arheart, Kristopher L

    2008-12-01

    Previous studies have demonstrated both poor and good correlation of total protein concentrations in various avian species using refractometry and biuret methodologies. The purpose of the current study was to compare these 2 techniques of total protein determination using plasma samples from several psittacine species and to determine the effect of cholesterol and other solutes on refractometry results. Total protein concentration in heparinized plasma samples without visible lipemia was analyzed by refractometry and an automated biuret method on a dry reagent analyzer (Ortho 250). Cholesterol, glucose, and uric acid concentrations were measured using the same analyzer. Results were compared using Deming regression analysis, Bland-Altman bias plots, and Spearman's rank correlation. Correlation coefficients (r) for total protein results by refractometry and biuret methods were 0.49 in African grey parrots (n=28), 0.77 in Amazon parrots (20), 0.57 in cockatiels (20), 0.73 in cockatoos (36), 0.86 in conures (20), and 0.93 in macaws (38) (Prefractometry in Amazon parrots, conures, and macaws (n=25 each, PRefractometry can be used to accurately measure total protein concentration in nonlipemic plasma samples from some psittacine species. Method and species-specific reference intervals should be used in the interpretation of total protein values.

  5. A simplified electrophoretic system for determining molecular weights of proteins.

    Science.gov (United States)

    Manwell, C

    1977-09-01

    Electrophoresis of 31 different proteins in commercially prepared polyacrylamide gradient gels, Gradipore, yields a linear relationship between a hypothetical limiting pore size (the reciprocal of a limiting gel concentration, GL) and the cube root of the mol.wt., over the range 13 500-9000 000. A regression analysis of these data reveals that 98.6% of all variability in 1/GL is explained by the molecular weight, and this degree of accuracy compares favourably with existing methods for the determination of molecular weight by retardation of mobility in polyacrylamide. This new procedure has the additional advantages that molecular-weight standards can be obtained from readily available body fluids or tissue extracts by localizing enzymes and other proteins by standard histochemical methods, and that the same electrophoretic system can be used in determining molecular weights as is used in routine surveys of populations for individual and species variation in protein heterogeneity.

  6. Extracting the information backbone in online system.

    Science.gov (United States)

    Zhang, Qian-Ming; Zeng, An; Shang, Ming-Sheng

    2013-01-01

    Information overload is a serious problem in modern society and many solutions such as recommender system have been proposed to filter out irrelevant information. In the literature, researchers have been mainly dedicated to improving the recommendation performance (accuracy and diversity) of the algorithms while they have overlooked the influence of topology of the online user-object bipartite networks. In this paper, we find that some information provided by the bipartite networks is not only redundant but also misleading. With such "less can be more" feature, we design some algorithms to improve the recommendation performance by eliminating some links from the original networks. Moreover, we propose a hybrid method combining the time-aware and topology-aware link removal algorithms to extract the backbone which contains the essential information for the recommender systems. From the practical point of view, our method can improve the performance and reduce the computational time of the recommendation system, thus improving both of their effectiveness and efficiency.

  7. Sofosbuvir as backbone of interferon free treatments.

    Science.gov (United States)

    Bourlière, Marc; Oules, Valèrie; Ansaldi, Christelle; Adhoute, Xavier; Castellani, Paul

    2014-12-15

    Sofosbuvir is the first-in-class NS5B nucleotide analogues to be launched for hepatitis C virus (HCV) treatment. Its viral potency, pangenotypic activity and high barrier to resistance make it the ideal candidate to become a backbone for several IFN-free regimens. Recent data demonstrated that sofosbuvir either with ribavirin alone or in combination with other direct-acting antivirals (DAAs) as daclatasvir, ledipasvir or simeprevir are able to cure HCV in at least 90% or over of patients. Treatment experienced genotype 3 population may remain the most difficult to treat population, but ongoing DAA combination studies will help to fill this gap. Safety profile of sofosbuvir or combination with other DAAs is good. Resistance to sofosbuvir did not appear as a significant issue. The rationale for using this class of drug and the available clinical data are reviewed.

  8. Extracting the information backbone in online system

    CERN Document Server

    Zhang, Qian-Ming; Shang, Ming-Sheng

    2013-01-01

    Information overload is a serious problem in modern society and many solutions such as recommender system have been proposed to filter out irrelevant information. In the literature, researchers mainly dedicated to improve the recommendation performance (accuracy and diversity) of the algorithms while overlooked the influence of topology of the online user-object bipartite networks. In this paper, we find that some information provided by the bipartite networks is not only redundant but also misleading. With such "less can be more" feature, we design some algorithms to improve the recommendation performance by eliminating some links from the original networks. Moreover, we propose a hybrid method combining the time-aware and topology-aware link removal algorithms to extract the backbone which contains the essential information for the recommender systems. From the practical point of view, our method can improve the performance and reduce the computational time of the recommendation system, thus improve both of...

  9. Contribution of Peptide Backbone to Anti-Citrullinated Peptide Antibody Reactivity.

    Directory of Open Access Journals (Sweden)

    Nicole Hartwig Trier

    Full Text Available Rheumatoid arthritis (RA is one of the most common autoimmune diseases, affecting approximately 1-2% of the world population. One of the characteristic features of RA is the presence of autoantibodies. Especially the highly specific anti-citrullinated peptide antibodies (ACPAs, which have been found in up to 70% of RA patients' sera, have received much attention. Several citrullinated proteins are associated with RA, suggesting that ACPAs may react with different sequence patterns, separating them from traditional antibodies, whose reactivity usually is specific towards a single target. As ACPAs have been suggested to be involved in the development of RA, knowledge about these antibodies may be crucial. In this study, we examined the influence of peptide backbone for ACPA reactivity in immunoassays. The antibodies were found to be reactive with a central Cit-Gly motif being essential for ACPA reactivity and to be cross-reactive between the selected citrullinated peptides. The remaining amino acids within the citrullinated peptides were found to be of less importance for antibody reactivity. Moreover, these findings indicated that the Cit-Gly motif in combination with peptide backbone is essential for antibody reactivity. Based on these findings it was speculated that any amino acid sequence, which brings the peptide into a properly folded structure for antibody recognition is sufficient for antibody reactivity. These findings are in accordance with the current hypothesis that structural homology rather than sequence homology are favored between citrullinated epitopes. These findings are important in relation to clarifying the etiology of RA and to determine the nature of ACPAs, e.g., why some Cit-Gly-containing sequences are not targeted by ACPAs.

  10. Determination of covalently bound hypusine and deoxyhypusine to protein using submilligram of protein samples by HPLC.

    Science.gov (United States)

    Beppu, T; Shirahata, A; Samejima, K

    1996-01-01

    A sensitive and reliable method for the determination of hypusine and deoxyhypusine in eIF-5A protein, an initiation factor of protein synthesis, was developed. An advantage of this method is the use of N epsilon-(5-aminopentyl)lysine, an analogue of deoxyhypusine, as an internal standard. The application made it possible to determine hypusine in less than a mg of protein samples from cultured HTC cells and rat organs. After acid hydrolysis of protein samples to which had been added the internal standard, the hydrolysates were fractionated by carboxymethyl cellulose column chromatography. Also, diamine fractions containing a few pmol of hypusine and deoxyhypusine were successfully analyzed by a reversed phase HPLC with a fluorescence detection of o-phthalaldehyde. The method was applied for the determination of hypusine and deoxyhypusine in drug-treated HTC cells and normal rat organs. The results from HTC cells were discussed based on the known effects of each drug on hypusine biosynthesis.

  11. Probabilistic Determination of Native State Ensembles of Proteins

    DEFF Research Database (Denmark)

    Olsson, Simon; Vögeli, Beat Rolf; Cavalli, Andrea

    2014-01-01

    The motions of biological macromolecules are tightly coupled to their functions. However, while the study of fast motions has become increasingly feasible in recent years, the study of slower, biologically important motions remains difficult. Here, we present a method to construct native state...... ensembles of proteins by the combination of physical force fields and experimental data through modern statistical methodology. As an example, we use NMR residual dipolar couplings to determine a native state ensemble of the extensively studied third immunoglobulin binding domain of protein G (GB3...

  12. High-resolution protein structure determination by serial femtosecond crystallography.

    Science.gov (United States)

    Boutet, Sébastien; Lomb, Lukas; Williams, Garth J; Barends, Thomas R M; Aquila, Andrew; Doak, R Bruce; Weierstall, Uwe; DePonte, Daniel P; Steinbrener, Jan; Shoeman, Robert L; Messerschmidt, Marc; Barty, Anton; White, Thomas A; Kassemeyer, Stephan; Kirian, Richard A; Seibert, M Marvin; Montanez, Paul A; Kenney, Chris; Herbst, Ryan; Hart, Philip; Pines, Jack; Haller, Gunther; Gruner, Sol M; Philipp, Hugh T; Tate, Mark W; Hromalik, Marianne; Koerner, Lucas J; van Bakel, Niels; Morse, John; Ghonsalves, Wilfred; Arnlund, David; Bogan, Michael J; Caleman, Carl; Fromme, Raimund; Hampton, Christina Y; Hunter, Mark S; Johansson, Linda C; Katona, Gergely; Kupitz, Christopher; Liang, Mengning; Martin, Andrew V; Nass, Karol; Redecke, Lars; Stellato, Francesco; Timneanu, Nicusor; Wang, Dingjie; Zatsepin, Nadia A; Schafer, Donald; Defever, James; Neutze, Richard; Fromme, Petra; Spence, John C H; Chapman, Henry N; Schlichting, Ilme

    2012-07-20

    Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

  13. Spectrophotometric and Refractometric Determination of Total Protein in Avian Plasma

    OpenAIRE

    2013-01-01

    The aim of this study was to compare the total protein values obtained in heparin plasma of chickens by a spectrophotometric technique (biuret method), and the values obtained on the same day in the same samples by refractometry. The results obtained by refractometry (average value 2.638±0.153g%) were higher than those obtained by the spectrophotometric method (average value 2.441±0.181g%). There was a low correlation (r = 0.6709) between the total protein values, determined with both methods...

  14. Evaluation of impact of backbone outages in IP networks

    Science.gov (United States)

    Kogan, Yaakov; Choudhury, Gagan L.; Tarapore, Percy

    2004-09-01

    Nationwide IP networks typically include nodes in major cities and the following elements: customer equipment, access routers, backbone routers, peering routers, access links connecting customer equipment to access routers, access routers to backbone routers, and backbone links interconnecting backbone routers. The part of this network consisting of backbone routers and related interconnecting links is referred to as the "backbone". We develop a new approach for accurately computing the Availability measure of IP networks by directly simulating each type of backbone outage event and its impact on traffic loss. We use this approach to quantify availability improvement as a result of introducing various technological changes in the network such as IGP tuning, high availability router architecture, MPLS-TE and Fast Reroute. A situation, where operational backbone links do not have enough spare capacity to carry additional traffic during the outage time, is referred to as bandwidth loss. We concentrate on one unidirectional backbone link and derive asymptotic approximations for the expected bandwidth loss in the framework of generalized Erlang and Engset models when the total number of resource units and request arrival rates are proportionally large. Simulation results demonstrate good accuracy of the approximations.

  15. Backbone analysis and algorithm design for the quadratic assignment problem

    Institute of Scientific and Technical Information of China (English)

    JIANG He; ZHANG XianChao; CHEN GuoLiang; LI MingChu

    2008-01-01

    As the hot line in NP-hard problems research in recent years, backbone analysis is crucial for phase transition, hardness, and algorithm design. Whereas theoretical analysis of backbone and its applications in algorithm design are still at a begin-ning state yet, this paper took the quadratic assignment problem (QAP) as a case study and proved by theoretical analysis that it is NP-hard to find the backbone, l.e., no algorithm exists to obtain the backbone of a QAP in polynomial time. Results of this paper showed that it is reasonable to acquire approximate backbone by inter-section of local optimal solutions. Furthermore, with the method of constructing biased instances, this paper proposed a new meta-heuristic - biased instance based approximate backbone (BI-AB), whose basic idea is as follows: firstly, con-struct a new biased instance for every QAP instance (the optimal solution of the new instance is also optimal for the original one); secondly, the approximate backbone is obtained by intersection of multiple local optimal solutions computed by some existing algorithm; finally, search for the optimal solutions in the reduced space by fixing the approximate backbone. Work of the paper enhanced the re-search area of theoretical analysis of backbone. The meta-heuristic proposed in this paper provided a new way for general algorithm design of NP-hard problems as well.

  16. Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

    Energy Technology Data Exchange (ETDEWEB)

    Driscoll, P.C.; Gronenborn, A.M.; Beress, L.; Clore, G.M. (National Institutes of Health, Bethesda, MD (USA))

    1989-03-07

    The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 {phi} backbone and 21 {sub {chi}1} side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 {plus minus} 0.12 {angstrom} for the backbone atoms and 0.90 {plus minus} 0.17 {angstrom} for all atoms. The core of the protein is formed by a triple-stranded antiparallel {beta}-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel {beta}-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel {beta}-sheet are connected by a long exposed loop. A number of side-chain interactions are discussed in light of the structure.

  17. Factor H-related proteins determine complement-activating surfaces.

    Science.gov (United States)

    Józsi, Mihály; Tortajada, Agustin; Uzonyi, Barbara; Goicoechea de Jorge, Elena; Rodríguez de Córdoba, Santiago

    2015-06-01

    Complement factor H-related proteins (FHRs) are strongly associated with different diseases involving complement dysregulation, which suggests a major role for these proteins regulating complement activation. Because FHRs are evolutionarily and structurally related to complement inhibitor factor H (FH), the initial assumption was that the FHRs are also negative complement regulators. Whereas weak complement inhibiting activities were originally reported for these molecules, recent developments indicate that FHRs may enhance complement activation, with important implications for the role of these proteins in health and disease. We review these findings here, and propose that FHRs represent a complex set of surface recognition molecules that, by competing with FH, provide improved discrimination of self and non-self surfaces and play a central role in determining appropriate activation of the complement pathway.

  18. Hydrogen bond networks determine emergent mechanical and thermodynamic properties across a protein family

    Directory of Open Access Journals (Sweden)

    Dallakyan Sargis

    2008-08-01

    Full Text Available Abstract Background Gram-negative bacteria use periplasmic-binding proteins (bPBP to transport nutrients through the periplasm. Despite immense diversity within the recognized substrates, all members of the family share a common fold that includes two domains that are separated by a conserved hinge. The hinge allows the protein to cycle between open (apo and closed (ligated conformations. Conformational changes within the proteins depend on a complex interplay of mechanical and thermodynamic response, which is manifested as an increase in thermal stability and decrease of flexibility upon ligand binding. Results We use a distance constraint model (DCM to quantify the give and take between thermodynamic stability and mechanical flexibility across the bPBP family. Quantitative stability/flexibility relationships (QSFR are readily evaluated because the DCM links mechanical and thermodynamic properties. We have previously demonstrated that QSFR is moderately conserved across a mesophilic/thermophilic RNase H pair, whereas the observed variance indicated that different enthalpy-entropy mechanisms allow similar mechanical response at their respective melting temperatures. Our predictions of heat capacity and free energy show marked diversity across the bPBP family. While backbone flexibility metrics are mostly conserved, cooperativity correlation (long-range couplings also demonstrate considerable amount of variation. Upon ligand removal, heat capacity, melting point, and mechanical rigidity are, as expected, lowered. Nevertheless, significant differences are found in molecular cooperativity correlations that can be explained by the detailed nature of the hydrogen bond network. Conclusion Non-trivial mechanical and thermodynamic variation across the family is explained by differences within the underlying H-bond networks. The mechanism is simple; variation within the H-bond networks result in altered mechanical linkage properties that directly affect

  19. INFLUENCE OF BACKBONE RIGIDITY ON THE LIQUID CRYSTALLINITY OF MESOGEN-CONTAINING POLYACETYLENES

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    Two acetylene polymers containing cyanobiphenyl-based mesogens,poly{10-[((4'-cyano-4-biphenylyl)oxy)carbonyl]-1-decyne} (PA8CN), which has a relatively flexible polyalkyne backbone, and poly {[4-(((12-((4'-cyano-4-biphenylyl)oxy)dodecyl)oxy)carbonyl) phenyl]-acetylene} (PB12CN), which has a fairly rigid poly(phenylacetylene)backbone, were synthesized using respectively WCl6 and [Rh(nbd)Cl]2 as the catalysts.PA8CN exhibits enantiotropic interdigitated smectic A phase (SAd) over a temperature range as wide as ca. 100℃, whereas PB12CN is non-mesomorphic, demonstrating that the backbone rigidity plays an important role in determining the liquid crystallinity of the polyacetylenes.

  20. Backbones of evolutionary history test biodiversity theory for microbes.

    Science.gov (United States)

    O'Dwyer, James P; Kembel, Steven W; Sharpton, Thomas J

    2015-07-07

    Identifying the ecological and evolutionary mechanisms that determine biological diversity is a central question in ecology. In microbial ecology, phylogenetic diversity is an increasingly common and relevant means of quantifying community diversity, particularly given the challenges in defining unambiguous species units from environmental sequence data. We explore patterns of phylogenetic diversity across multiple bacterial communities drawn from different habitats and compare these data to evolutionary trees generated using theoretical models of biodiversity. We have two central findings. First, although on finer scales the empirical trees are highly idiosyncratic, on coarse scales the backbone of these trees is simple and robust, consistent across habitats, and displays bursts of diversification dotted throughout. Second, we find that these data demonstrate a clear departure from the predictions of standard neutral theories of biodiversity and that an alternative family of generalized models provides a qualitatively better description. Together, these results lay the groundwork for a theoretical framework to connect ecological mechanisms to observed phylogenetic patterns in microbial communities.

  1. Determination of in vivo protein synthesis in human palatine tonsil.

    Science.gov (United States)

    Januszkiewicz, Anna; Klaude, Maria; Loré, Karin; Andersson, Jan; Ringdén, Olle; Rooyackers, Olav; Wernerman, Jan

    2005-02-01

    The palatine tonsils are constantly exposed to ingested or inhaled antigens which, in turn, lead to a permanent activation of tonsillar immune cells, even in a basic physiological state. The aim of the present study was to investigate if the immunological activation of the human palatine tonsil is reflected by a high metabolic activity, as determined by in vivo measurement of protein synthesis. The protein synthesis rate of the tonsil was also compared with that of the circulating T-lymphocytes, the total blood mononuclear cells and the whole population of blood leucocytes. Phenotypic characterization of immune-competent cells in tonsil tissue and blood was performed by flow cytometry. Pinch tonsil biopsies were taken after induction of anaesthesia in healthy adult patients (n=12) scheduled for ear surgery, uvulopalatopharyngoplasty or nose surgery. Protein synthesis was quantitatively determined during a 90-min period by a flooding-dose technique. The in vivo protein synthesis rate in the palatine tonsils was 22.8+/-5.7%/24 h (mean+/-S.D.), whereas protein synthesis in the circulating T-lymphocytes was 10.7+/-3.4%/24 h, in mononuclear cells was 10.8+/-2.8%/24 h and in leucocytes was 3.2+/-1.2%/24 h. CD3+ lymphocytes were the most abundant cell population in the tonsil. The in vivo protein synthesis rate in human tonsils was higher compared with the circulating immune cells. This high metabolic rate may reflect the permanent immunological activity present in human tonsils, although cell phenotypes and activity markers do not explain the differences.

  2. Determining the Topology of Membrane-Bound Proteins Using PEGylation.

    Science.gov (United States)

    Howe, Vicky; Brown, Andrew J

    2017-01-01

    Biochemical methods can help elucidate the membrane topology of hydrophobic membrane proteins where X-ray crystallography is difficult or impractical, providing important structural data. Here, we describe the method of PEGylation, which uses a cysteine-reactive molecule, maleimide polyethylene glycol (mPEG), to determine the cytosolic accessibility of introduced cysteine residues. This accessibility is visualized using Western blotting to detect a band shift that indicates cysteine labeling by mPEG. Using scanning cysteine mutagenesis, followed by PEGylation, one can map the accessibility of the introduced cysteines, hence inferring the membrane topology of the protein.We used PEGylation to determine the membrane topology of the sterol regulatory domain of a cholesterol synthesis enzyme, squalene monooxygenase, identifying that it is anchored to the membrane via a re-entrant loop.

  3. Backbone Assignment of the MALT1 Paracaspase by Solution NMR.

    Science.gov (United States)

    Unnerståle, Sofia; Nowakowski, Michal; Baraznenok, Vera; Stenberg, Gun; Lindberg, Jimmy; Mayzel, Maxim; Orekhov, Vladislav; Agback, Tatiana

    2016-01-01

    Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a unique paracaspase protein whose protease activity mediates oncogenic NF-κB signalling in activated B cell-like diffuse large B cell lymphomas (ABC-DLBCLs). ABC-DLBCLs are aggressive lymphomas with high resistance to current chemotherapies. Low survival rate among patients emphasizes the urgent need for alternative treatment options. The characterization of the MALT1 will be an essential tool for developing new target-directed drugs against MALT1 dependent disorders. As the first step in the atomic-level NMR studies of the system, here we report, the (15)N/(13)C/(1)H backbone assignment of the apo form of the MALT1 paracaspase region together with the third immunoglobulin-like (Ig3) domain, 44 kDa, by high resolution NMR. In addition, the non-uniform sampling (NUS) based targeted acquisition procedure is evaluated as a mean of decreasing acquisition and analysis time for larger proteins.

  4. Backbone Assignment of the MALT1 Paracaspase by Solution NMR.

    Directory of Open Access Journals (Sweden)

    Sofia Unnerståle

    Full Text Available Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1 is a unique paracaspase protein whose protease activity mediates oncogenic NF-κB signalling in activated B cell-like diffuse large B cell lymphomas (ABC-DLBCLs. ABC-DLBCLs are aggressive lymphomas with high resistance to current chemotherapies. Low survival rate among patients emphasizes the urgent need for alternative treatment options. The characterization of the MALT1 will be an essential tool for developing new target-directed drugs against MALT1 dependent disorders. As the first step in the atomic-level NMR studies of the system, here we report, the (15N/(13C/(1H backbone assignment of the apo form of the MALT1 paracaspase region together with the third immunoglobulin-like (Ig3 domain, 44 kDa, by high resolution NMR. In addition, the non-uniform sampling (NUS based targeted acquisition procedure is evaluated as a mean of decreasing acquisition and analysis time for larger proteins.

  5. NMRFAM-SDF: a protein structure determination framework

    Energy Technology Data Exchange (ETDEWEB)

    Dashti, Hesam; Lee, Woonghee; Tonelli, Marco; Cornilescu, Claudia C.; Cornilescu, Gabriel; Assadi-Porter, Fariba M.; Westler, William M.; Eghbalnia, Hamid R.; Markley, John L., E-mail: markley@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison, Biochemistry Department (United States)

    2015-08-15

    The computationally demanding nature of automated NMR structure determination necessitates a delicate balancing of factors that include the time complexity of data collection, the computational complexity of chemical shift assignments, and selection of proper optimization steps. During the past two decades the computational and algorithmic aspects of several discrete steps of the process have been addressed. Although no single comprehensive solution has emerged, the incorporation of a validation protocol has gained recognition as a necessary step for a robust automated approach. The need for validation becomes even more pronounced in cases of proteins with higher structural complexity, where potentially larger errors generated at each step can propagate and accumulate in the process of structure calculation, thereby significantly degrading the efficacy of any software framework. This paper introduces a complete framework for protein structure determination with NMR—from data acquisition to the structure determination. The aim is twofold: to simplify the structure determination process for non-NMR experts whenever feasible, while maintaining flexibility by providing a set of modules that validate each step, and to enable the assessment of error propagations. This framework, called NMRFAM-SDF (NMRFAM-Structure Determination Framework), and its various components are available for download from the NMRFAM website ( http://nmrfam.wisc.edu/software.htm http://nmrfam.wisc.edu/software.htm )

  6. Protein oxidation implicated as the primary determinant of bacterial radioresistance.

    Directory of Open Access Journals (Sweden)

    Michael J Daly

    2007-04-01

    Full Text Available In the hierarchy of cellular targets damaged by ionizing radiation (IR, classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.

  7. Protein Nitrogen Determination by Kjeldahl Digestion and Ion Chromatography.

    Science.gov (United States)

    Wang, Hsiaoling; Pampati, Nagarani; McCormick, William M; Bhattacharyya, Lokesh

    2016-06-01

    We report development and validation of a simple, rapid, and accurate method for the quantitation of protein nitrogen, which combines Kjeldahl digestion and ion chromatography with suppressed conductivity detection and requires nanomolar amount of nitrogen in samples (≥10 μg protein). The mechanism of suppressed conductivity detection does not permit analysis of samples containing copper (present in Kjeldahl digestion solution) and aluminum (present in many vaccines as adjuvants) due to precipitation of their hydroxides within the suppressor. We overcame this problem by including 10 μM oxalic acid in Kjeldahl digests and in the eluent (30 mM methanesulfonic acid). The chromatography is performed using an IonPac CS-16 cation exchange column by isocratic elution. The method reduces the digestion time to less than 1 h and eliminates the distillation and titration steps of the Kjeldahl method, thereby reducing the analysis time significantly and improving precision and accuracy. To determine protein nitrogen in samples containing non-protein nitrogen, proteins are precipitated by a mixture of deoxycholate and trichloroacetic acid and the precipitates are analyzed after dissolving in KOH. The method is particularly useful for biological samples that are limited and can also be applied to food, environmental, and other materials.

  8. Determination of Protein Content by NIR Spectroscopy in Protein Powder Mix Products.

    Science.gov (United States)

    Ingle, Prashant D; Christian, Roney; Purohit, Piyush; Zarraga, Veronica; Handley, Erica; Freel, Keith; Abdo, Saleem

    2016-01-01

    Protein is a principal component in commonly used dietary supplements and health food products. The analysis of these products, within the consumer package form, is of critical importance for the purpose of ensuring quality and supporting label claims. A rapid test method was developed using near-infrared (NIR) spectroscopy as a compliment to current protein determination by the Dumas combustion method. The NIR method was found to be a rapid, low-cost, and green (no use of chemicals and reagents) complimentary technique. The protein powder samples analyzed in this study were in the range of 22-90% protein. The samples were prepared as mixtures of soy protein, whey protein, and silicon dioxide ingredients, which are common in commercially sold protein powder drink-mix products in the market. A NIR regression model was developed with 17 samples within the constituent range and was validated with 20 independent samples of known protein levels (85-88%). The results show that the NIR method is capable of predicting the protein content with a bias of ±2% and a maximum bias of 3% between NIR and the external Dumas method.

  9. Hydrogen-deuterium exchange mass spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam.

    Science.gov (United States)

    Hamuro, Yoshitomo; Molnar, Kathleen S; Coales, Stephen J; OuYang, Bo; Simorellis, Alana K; Pochapsky, Thomas C

    2008-02-01

    Backbone dynamics of the camphor monoxygenase cytochrome P450(cam) (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor- and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively). However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology.

  10. [Biochemical methods for the determination of a clinical protein catabolism].

    Science.gov (United States)

    Roth, E; Funovics, J; Schulz, F; Karner, J

    1980-12-01

    1. 20 patients before surgery received enteral nutrition for three days (12 g nitrogen, 1800 Kcal). Nitrogen and urea excretions in urine during the second and third day were determined. Eleven patients had a negative nitrogen balance (-2,7 and -2,4 g/day). In these patients urea production rates were 21,1 and 20,1 g/day. An urea production rate exceeding 15 g urea/day is probable an indication for a protein catabolism. The reason for this catabolic state seems to be a decreased protein utilisation (49 and 47 percent) as the result of a metabolic stress situation. This metabolic stress was determined according the stress index (Bistrian). The patients were in a stress situation comparable to postoperative stress (+3,7 and +3,9). The determination of urea production rate and catabolic index seems a suitable tool for defining a catabolic state. 2. 3-met-histidine excretion in urine were measured in seven patients postoperatively. In different periods saline or aminoacids solutions (5% alanine) were infused. During alanine administration protein (+49%)--and 3-met-histidine excretions (+50%) increased. It is not possible to state a catabolic situation out of the 3-met-histidine excretion, because an increased excretion may result from a stimulated protein synthesis in muscle tissue or from an increased muscleprotein wasting. 3. Free amino acid pools in plasma and muscle tissue were analysed in patients with severe illness of liver and pancreas. The free amino acid pattern differed from healthy volunteers. In patients with liver disease significantly increased concentrations of phenylalanine, tyrosine and methionine were found. In patients with acute pancreatitis highly abnormal pattern of intracellular amino acids occurred with decreased concentrations of glutamine, cysteine, histidine, lysine, arginine and ornithine. The highly significant decreased concentrations of glutamine (p less than 0,01) indicate a catabolic situation of these patients. A quantification of the

  11. NHC Backbone Configuration in Ruthenium-Catalyzed Olefin Metathesis

    Directory of Open Access Journals (Sweden)

    Veronica Paradiso

    2016-01-01

    Full Text Available The catalytic properties of olefin metathesis ruthenium complexes bearing N-heterocyclic carbene ligands with stereogenic centers on the backbone are described. Differences in catalytic behavior depending on the backbone configurations of symmetrical and unsymmetrical NHCs are discussed. In addition, an overview on asymmetric olefin metathesis promoted by chiral catalysts bearing C2-symmetric and C1-symmetric NHCs is provided.

  12. NHC Backbone Configuration in Ruthenium-Catalyzed Olefin Metathesis.

    Science.gov (United States)

    Paradiso, Veronica; Costabile, Chiara; Grisi, Fabia

    2016-01-20

    The catalytic properties of olefin metathesis ruthenium complexes bearing N-heterocyclic carbene ligands with stereogenic centers on the backbone are described. Differences in catalytic behavior depending on the backbone configurations of symmetrical and unsymmetrical NHCs are discussed. In addition, an overview on asymmetric olefin metathesis promoted by chiral catalysts bearing C₂-symmetric and C₁-symmetric NHCs is provided.

  13. Extracting the information backbone in online system.

    Directory of Open Access Journals (Sweden)

    Qian-Ming Zhang

    Full Text Available Information overload is a serious problem in modern society and many solutions such as recommender system have been proposed to filter out irrelevant information. In the literature, researchers have been mainly dedicated to improving the recommendation performance (accuracy and diversity of the algorithms while they have overlooked the influence of topology of the online user-object bipartite networks. In this paper, we find that some information provided by the bipartite networks is not only redundant but also misleading. With such "less can be more" feature, we design some algorithms to improve the recommendation performance by eliminating some links from the original networks. Moreover, we propose a hybrid method combining the time-aware and topology-aware link removal algorithms to extract the backbone which contains the essential information for the recommender systems. From the practical point of view, our method can improve the performance and reduce the computational time of the recommendation system, thus improving both of their effectiveness and efficiency.

  14. The Backbone of the Climate Networks

    Science.gov (United States)

    Zou, Y.; Donges, J. F.; Marwan, N.; Kurths, J.

    2009-12-01

    We propose a method to reconstruct and analyze a complex network from data generated by a spatio-temporal dynamical system, relying on the nonlinear mutual information of time series analysis and betweenness centrality of complex network theory. We show, that this approach reveals a rich internal structure in complex climate networks constructed from reanalysis and model surface air temperature data. Our novel method uncovers peculiar wave-like structures of high energy flow, that we relate to global surface ocean currents. This points to a major role of the oceanic surface circulation in coupling and stabilizing the global temperature field in the long term mean (140 years for the model run and 60 years for reanalysis data). We find that these results cannot be obtained using classical linear methods of multivariate data analysis. Furthermore, we introduce significance tests to quantify the robustness of measured network properties to uncertainties. References: [1] J.F. Donges, Y. Zou, N. Marwan, and J. Kurths. Complex networks in climate dynamics -- -- Comparing linear and nonlinear network construction methods. European Physical Journal -- Special Topics, 174, 157-179, 2009. [2] J.F. Donges, Y. Zou, N. Marwan, and J. Kurths. Backbone of the climate network. Europhysics Letters, in press, 2009.

  15. Extracting the Information Backbone in Online System

    Science.gov (United States)

    Zhang, Qian-Ming; Zeng, An; Shang, Ming-Sheng

    2013-01-01

    Information overload is a serious problem in modern society and many solutions such as recommender system have been proposed to filter out irrelevant information. In the literature, researchers have been mainly dedicated to improving the recommendation performance (accuracy and diversity) of the algorithms while they have overlooked the influence of topology of the online user-object bipartite networks. In this paper, we find that some information provided by the bipartite networks is not only redundant but also misleading. With such “less can be more” feature, we design some algorithms to improve the recommendation performance by eliminating some links from the original networks. Moreover, we propose a hybrid method combining the time-aware and topology-aware link removal algorithms to extract the backbone which contains the essential information for the recommender systems. From the practical point of view, our method can improve the performance and reduce the computational time of the recommendation system, thus improving both of their effectiveness and efficiency. PMID:23690946

  16. High Speed Fibre Optic Backbone LAN

    Science.gov (United States)

    Tanimoto, Masaaki; Hara, Shingo; Kajita, Yuji; Kashu, Fumitoshi; Ikeuchi, Masaru; Hagihara, Satoshi; Tsuzuki, Shinji

    1987-09-01

    Our firm has developed the SUMINET-4100 series, a fibre optic local area network (LAN), to serve the communications system trunk line needs for facilities, such as steel refineries, automobile plants and university campuses, that require large transmission capacity, and for the backbone networks used in intelligent building systems. The SUMINET-4100 series is already in service in various fields of application. Of the networks available in this series, the SUMINET-4150 has a trunk line speed of 128 Mbps and the multiplexer used for time division multiplexing (TDM) was enabled by designing an ECL-TTL gate array (3000 gates) based custom LSI. The synchronous, full-duplex V.24 and V.3.5 interfaces (SUMINET-2100) are provided for use with general purpose lines. And the IBM token ring network, the SUMINET-3200, designed for heterogeneous PCs and the Ethernet can all be connected to sub loops. Further, the IBM 3270 TCA and 5080 CADAM can be connected in the local mode. Interfaces are also provided for the NTT high-speed digital service, the digital PBX systems, and the Video CODEC system. The built-in loop monitor (LM) and network supervisory processor (NSP) provide management of loop utilization and send loop status signals to the host CPU's network configuration and control facility (NCCF). These built-in functions allow both the computer system and LAN to be managed from a single source at the host. This paper outlines features of the SUMINET-4150 and provides an example of its installation.

  17. Iron-regulated surface determinant (Isd) proteins of Staphylococcus lugdunensis.

    Science.gov (United States)

    Zapotoczna, Marta; Heilbronner, Simon; Speziale, Pietro; Foster, Timothy J

    2012-12-01

    Staphylococcus lugdunensis is the only coagulase-negative Staphylococcus species with a locus encoding iron-regulated surface determinant (Isd) proteins. In Staphylococcus aureus, the Isd proteins capture heme from hemoglobin and transfer it across the wall to a membrane-bound transporter, which delivers it into the cytoplasm, where heme oxygenases release iron. The Isd proteins of S. lugdunensis are expressed under iron-restricted conditions. We propose that S. lugdunensis IsdB and IsdC proteins perform the same functions as those of S. aureus. S. lugdunensis IsdB is the only hemoglobin receptor within the isd locus. It specifically binds human hemoglobin with a dissociation constant (K(d)) of 23 nM and transfers heme on IsdC. IsdB expression promotes bacterial growth in an iron-limited medium containing human hemoglobin but not mouse hemoglobin. This correlates with weak binding of IsdB to mouse hemoglobin in vitro. Unlike IsdB and IsdC, the proteins IsdJ and IsdK are not sorted to the cell wall in S. lugdunensis. In contrast, IsdJ expressed in S. aureus and Lactococcus lactis is anchored to peptidoglycan, suggesting that S. lugdunensis sortases may differ in signal recognition or could be defective. IsdJ and IsdK are present in the culture supernatant, suggesting that they could acquire heme from the external milieu. The IsdA protein of S. aureus protects bacteria from bactericidal lipids due to its hydrophilic C-terminal domain. IsdJ has a similar region and protected S. aureus and L. lactis as efficiently as IsdA but, possibly due to its location, was less effective in its natural host.

  18. CHD3 proteins and polycomb group proteins antagonistically determine cell identity in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Ernst Aichinger

    2009-08-01

    Full Text Available Dynamic regulation of chromatin structure is of fundamental importance for modulating genomic activities in higher eukaryotes. The opposing activities of Polycomb group (PcG and trithorax group (trxG proteins are part of a chromatin-based cellular memory system ensuring the correct expression of specific transcriptional programs at defined developmental stages. The default silencing activity of PcG proteins is counteracted by trxG proteins that activate PcG target genes and prevent PcG mediated silencing activities. Therefore, the timely expression and regulation of PcG proteins and counteracting trxG proteins is likely to be of fundamental importance for establishing cell identity. Here, we report that the chromodomain/helicase/DNA-binding domain CHD3 proteins PICKLE (PKL and PICKLE RELATED2 (PKR2 have trxG-like functions in plants and are required for the expression of many genes that are repressed by PcG proteins. The pkl mutant could partly suppress the leaf and flower phenotype of the PcG mutant curly leaf, supporting the idea that CHD3 proteins and PcG proteins antagonistically determine cell identity in plants. The direct targets of PKL in roots include the PcG genes SWINGER and EMBRYONIC FLOWER2 that encode subunits of Polycomb repressive complexes responsible for trimethylating histone H3 at lysine 27 (H3K27me3. Similar to mutants lacking PcG proteins, lack of PKL and PKR2 caused reduced H3K27me3 levels and, therefore, increased expression of a set of PcG protein target genes in roots. Thus, PKL and PKR2 are directly required for activation of PcG protein target genes and in roots are also indirectly required for repression of PcG protein target genes. Reduced PcG protein activity can lead to cell de-differentiation and callus-like tissue formation in pkl pkr2 mutants. Thus, in contrast to mammals, where PcG proteins are required to maintain pluripotency and to prevent cell differentiation, in plants PcG proteins are required to promote

  19. CHD3 proteins and polycomb group proteins antagonistically determine cell identity in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Ernst Aichinger

    2009-08-01

    Full Text Available Dynamic regulation of chromatin structure is of fundamental importance for modulating genomic activities in higher eukaryotes. The opposing activities of Polycomb group (PcG and trithorax group (trxG proteins are part of a chromatin-based cellular memory system ensuring the correct expression of specific transcriptional programs at defined developmental stages. The default silencing activity of PcG proteins is counteracted by trxG proteins that activate PcG target genes and prevent PcG mediated silencing activities. Therefore, the timely expression and regulation of PcG proteins and counteracting trxG proteins is likely to be of fundamental importance for establishing cell identity. Here, we report that the chromodomain/helicase/DNA-binding domain CHD3 proteins PICKLE (PKL and PICKLE RELATED2 (PKR2 have trxG-like functions in plants and are required for the expression of many genes that are repressed by PcG proteins. The pkl mutant could partly suppress the leaf and flower phenotype of the PcG mutant curly leaf, supporting the idea that CHD3 proteins and PcG proteins antagonistically determine cell identity in plants. The direct targets of PKL in roots include the PcG genes SWINGER and EMBRYONIC FLOWER2 that encode subunits of Polycomb repressive complexes responsible for trimethylating histone H3 at lysine 27 (H3K27me3. Similar to mutants lacking PcG proteins, lack of PKL and PKR2 caused reduced H3K27me3 levels and, therefore, increased expression of a set of PcG protein target genes in roots. Thus, PKL and PKR2 are directly required for activation of PcG protein target genes and in roots are also indirectly required for repression of PcG protein target genes. Reduced PcG protein activity can lead to cell de-differentiation and callus-like tissue formation in pkl pkr2 mutants. Thus, in contrast to mammals, where PcG proteins are required to maintain pluripotency and to prevent cell differentiation, in plants PcG proteins are required to promote

  20. CHD3 proteins and polycomb group proteins antagonistically determine cell identity in Arabidopsis.

    Science.gov (United States)

    Aichinger, Ernst; Villar, Corina B R; Farrona, Sara; Reyes, José C; Hennig, Lars; Köhler, Claudia

    2009-08-01

    Dynamic regulation of chromatin structure is of fundamental importance for modulating genomic activities in higher eukaryotes. The opposing activities of Polycomb group (PcG) and trithorax group (trxG) proteins are part of a chromatin-based cellular memory system ensuring the correct expression of specific transcriptional programs at defined developmental stages. The default silencing activity of PcG proteins is counteracted by trxG proteins that activate PcG target genes and prevent PcG mediated silencing activities. Therefore, the timely expression and regulation of PcG proteins and counteracting trxG proteins is likely to be of fundamental importance for establishing cell identity. Here, we report that the chromodomain/helicase/DNA-binding domain CHD3 proteins PICKLE (PKL) and PICKLE RELATED2 (PKR2) have trxG-like functions in plants and are required for the expression of many genes that are repressed by PcG proteins. The pkl mutant could partly suppress the leaf and flower phenotype of the PcG mutant curly leaf, supporting the idea that CHD3 proteins and PcG proteins antagonistically determine cell identity in plants. The direct targets of PKL in roots include the PcG genes SWINGER and EMBRYONIC FLOWER2 that encode subunits of Polycomb repressive complexes responsible for trimethylating histone H3 at lysine 27 (H3K27me3). Similar to mutants lacking PcG proteins, lack of PKL and PKR2 caused reduced H3K27me3 levels and, therefore, increased expression of a set of PcG protein target genes in roots. Thus, PKL and PKR2 are directly required for activation of PcG protein target genes and in roots are also indirectly required for repression of PcG protein target genes. Reduced PcG protein activity can lead to cell de-differentiation and callus-like tissue formation in pkl pkr2 mutants. Thus, in contrast to mammals, where PcG proteins are required to maintain pluripotency and to prevent cell differentiation, in plants PcG proteins are required to promote cell

  1. High Performance Infiltrated Backbones for Cathode-Supported SOFC's

    DEFF Research Database (Denmark)

    Gil, Vanesa; Kammer Hansen, Kent

    2014-01-01

    The concept of using highly ionic conducting backbones with subsequent infiltration of electronically conducting particles has widely been used to develop alternative anode-supported SOFC's. In this work, the idea was to develop infiltrated backbones as an alternative design based on cathode......-supported SOFC. The cathodes are obtained by infiltrating LSM into a sintered either thick (300 μm) yttria stabilized zirconia (YSZ) backbone or a thin YSZ backbone (10-15 μm) integrated onto a thick (300 μm) porous strontium substituted lanthanum manganite (LSM) and YSZ composite. Fabrication challenges...... printed symmetrical cells. Samples with LSM/YSZ composite and YSZ backbones made with graphite+PMMA as pore formers exhibited comparable Rp values to the screen printed LSM/YSZ cathode. This route was chosen as the best to fabricate the cathode supported cells. SEM micrograph of a cathode supported cell...

  2. Clusterin: an IR-inducible protein determining life and death

    Energy Technology Data Exchange (ETDEWEB)

    DAVID A. BOOTHMAN, Ph.D.

    2006-07-11

    and accumulation of nCLU signals cell death; and (3) sCLU is cytoprotective via blockage of IR-induced TGF-ß signaling, which causes growth inhibition and cell death by apoptosis. These hypotheses will be examined in the following three Aims: Specific Aim #1: Determine the regulatory elements and transcription factors regulating CLU mRNA induction by low dose IR, and repression by the p53 tumor suppressor protein. Specific Aim #2: Determine the functions of sCLU compared to nCLU using clusterin-deficient human or mouse cell lines. Specific Aim #3: Characterize and quantitate sCLU from low dose or low dose-rate IR-exposed human cancer cells, and examine potential bystander effects of the protein.

  3. Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR.

    Science.gov (United States)

    Knight, Michael J; Pell, Andrew J; Bertini, Ivano; Felli, Isabella C; Gonnelli, Leonardo; Pierattelli, Roberta; Herrmann, Torsten; Emsley, Lyndon; Pintacuda, Guido

    2012-07-10

    We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with (1)H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of (15)N and (13)C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu(+) (diamagnetic) or Cu(2+) (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to (1)H-(1)H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.

  4. Solution Structure Determination of Proteins by Solution NMR: Application to a Envelope Protein, LAP2

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ Recent advances in multidimensional NMR to obtain resonance assignments, interproton distance and torsion angle restraints, and restraints that characterize long range order, coupled with new methods of structure refinement, have permitted solution structures of proteins to be rapidly and quickly determined.

  5. Backbone cyclization of a recombinant cystine-knot peptide by engineered Sortase A.

    Science.gov (United States)

    Stanger, Karen; Maurer, Till; Kaluarachchi, Harini; Coons, Mary; Franke, Yvonne; Hannoush, Rami N

    2014-11-28

    Cyclotides belong to the family of cyclic cystine-knot peptides and have shown promise as scaffolds for protein engineering and pharmacological modulation of cellular protein activity. Cyclotides are characterized by a cystine-knotted topology and a head-to-tail cyclic polypeptide backbone. While they are primarily produced in plants, cyclotides have also been obtained by chemical synthesis. However, there is still a need for methods to generate cyclotides in high yields to near homogeneity. Here, we report a biomimetic approach which utilizes an engineered version of the enzyme Sortase A to catalyze amide backbone cyclization of the recombinant cyclotide MCoTI-II, thereby allowing the efficient production of active homogenous species in high yields. Our results provide proof of concept for using engineered Sortase A to produce cyclic MCoTI-II and should be generally applicable to generating other cyclic cystine-knot peptides.

  6. Using Protein-Confined Proximity To Determine Chemical Reactivity.

    Science.gov (United States)

    Kobayashi, Tomonori; Hoppmann, Christian; Yang, Bing; Wang, Lei

    2016-11-16

    Chemical reactivity is essential for functional modification of biomolecules with small molecules and the development of covalent drugs. The reactivity between a chemical functional group of a small molecule and that of a large biomolecule cannot be reliably predicted from the reactivity of the corresponding functional groups separately installed on two small molecules, because the proximity effect on reactivity resulting from the binding of the small molecule to the biomolecule is challenging to achieve by mixing two small molecules. Here we present a new strategy to determine the chemical reactivity of two functional groups in the context of close proximity afforded by proteins. The functional groups to be tested were separately installed at the interface of two interacting proteins in the format of amino acid side chains via the expansion of the genetic code. Reaction of the two functional groups resulted in covalent cross-linking of interacting proteins, readily detectable by gel electrophoresis. Using this strategy, we evolved new synthetases to genetically encode N(ε)-fluoroacetyllysine (FAcK), an isosteric fluorine analogue of acetyllysine. We demonstrated that fluoroacetamide installed on FAcK, previously thought inert to biological functional groups, actually reacted with the thiol group of cysteine when in proximity. This strategy should be valuable for accurately evaluating chemical reactivity of small molecules toward large biomolecules, which will help avoid undesired side reactions of drugs and expand the repertoire of functional groups to covalently target biomolecules.

  7. Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples.

    Science.gov (United States)

    Lange, Oliver F; Rossi, Paolo; Sgourakis, Nikolaos G; Song, Yifan; Lee, Hsiau-Wei; Aramini, James M; Ertekin, Asli; Xiao, Rong; Acton, Thomas B; Montelione, Gaetano T; Baker, David

    2012-07-03

    We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the "best effort" structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.

  8. High Performance Infiltrated Backbones for Cathode-Supported SOFC's

    DEFF Research Database (Denmark)

    Gil, Vanesa; Kammer Hansen, Kent

    2014-01-01

    A four-step infiltration method has been developed to infiltrate La0.75Sr0.25MnO3+δ (LSM25) nanoparticles into porous structures (YSZ or LSM-YSZ backbones). The pore size distribution in the backbones is obtained either by using PMMA and/or graphites as pore formers or by leaching treatment of sa...... of samples with Ni remained in the YSZ structure at high temperatures. All impregnated backbones, presented Rs comparable to a standard screen printed cathode, which proves that LSM nanoparticles forms a pathway for electron conduction....

  9. Signal transduction meets systems biology: deciphering specificity determinants for protein-protein interactions

    Science.gov (United States)

    Bourret, Robert B.

    2008-01-01

    Two recent papers [Gao et al. Mol. Microbiol. 69, 1358 (2008); Skerker et al. Cell 133, 1043 (2008)] describe investigations into the specificity of protein-protein interactions that occur during signal transduction by two-component regulatory systems. This MicroCommentary summarizes and provides context for the reported findings. The results offer insights into molecular determinants that provide specificity to maintain signal separation and thus prevent deleterious crosstalk between pathways, as well as the potential extent and nature of interactions that may combine signals to achieve beneficial cross regulation among pathways. The methods employed are suitable for application to other systems. PMID:18694439

  10. Backbone resonance assignments of the micro-RNA precursor binding region of human TRBP.

    Science.gov (United States)

    Benoit, Matthieu P M H; Plevin, Michael J

    2013-10-01

    TAR-RNA binding protein (TRBP) is a multidomain human protein involved in micro-RNA (miRNA) biogenesis. TRBP is a component of both the Dicer complex, which processes precursor miRNAs, and the RNA-induced silencing complex-loading complex. In addition, TRBP is implicated in the human immunodeficiency virus replication cycle and interferon-protein kinase R activity. TRBP contains 3 double-stranded RNA binding domains the first two of which have been shown to interact with miRNA precursors. Here we present the backbone resonance assignments and secondary structure of residues 19-228 of human TRBP2.

  11. A backbone lever-arm effect enhances polymer mechanochemistry

    Science.gov (United States)

    Klukovich, Hope M.; Kouznetsova, Tatiana B.; Kean, Zachary S.; Lenhardt, Jeremy M.; Craig, Stephen L.

    2013-02-01

    Mechanical forces along a polymer backbone can be used to bring about remarkable reactivity in embedded mechanically active functional groups, but little attention has been paid to how a given polymer backbone delivers that force to the reactant. Here, single-molecule force spectroscopy was used to directly quantify and compare the forces associated with the ring opening of gem-dibromo and gem-dichlorocyclopropanes affixed along the backbone of cis-polynorbornene and cis-polybutadiene. The critical force for isomerization drops by about one-third in the polynorbornene scaffold relative to polybutadiene. The root of the effect lies in more efficient chemomechanical coupling through the polynorbornene backbone, which acts as a phenomenological lever with greater mechanical advantage than polybutadiene. The experimental results are supported computationally and provide the foundation for a new strategy by which to engineer mechanochemical reactivity.

  12. LOAD AWARE ADAPTIVE BACKBONE SYNTHESIS IN WIRELESS MESH NETWORKS

    Institute of Scientific and Technical Information of China (English)

    Yuan Yuan; Zheng Baoyu

    2009-01-01

    Wireless Mesh Networks (WMNs) are envisioned to support the wired backbone with a wireless Backbone Networks (BNet) for providing internet connectivity to large-scale areas.With a wide range of internet-oriented applications with different Quality of Service (QoS) requirement,the large-scale WMNs should have good scalability and large bandwidth.In this paper,a Load Aware Adaptive Backbone Synthesis (LAABS) algorithm is proposed to automatically balance the traffic flow in the WMNs.The BNet will dynamically split into smaller size or merge into bigger one according to statistic load information of Backbone Nodes (BNs).Simulation results show LAABS generates moderate BNet size and converges quickly,thus providing scalable and stable BNet to facilitate traffic flow.

  13. Towards a natural classification and backbone tree for Sordariomycete

    Digital Repository Service at National Institute of Oceanography (India)

    Maharachchikumbura, S.S.N.; Hyde, K.D.; Jones, E.B.G.; McKenzie, E.H.C.; Huang, S.-K.; Abdel-Wahab, M.A.; Daranagama, D.A.; Dayarathne, M.; D'souza, M.J.; Goonasekara, I.D.; Hongsanan, S.; Jayawardena, R.S.; Kirk, P.M.; Konta, S.; Liu, J.-K.; Liu, Z.-Y.; Norphanphoun, C.; Pang, K.-L.; Perera, R.H.; Senanayake, I.C.; Shang, Q.; Shenoy, B.D.; Xiao, Y.; Bahkali, A.H.; Kang, J.; Somrothipol, S.; Suetrong, S.; Wen, T.; Xu, J.

    , lichenized or lichenicolous taxa The class includes freshwater, marine and terrestrial taxa and has a worldwide distribution This paper provides an updated outline of the Sordariomycetes and a backbone tree incorporating asexual and sexual genera in the class...

  14. "Parallel factor analysis of multi-excitation ultraviolet resonance Raman spectra for protein secondary structure determination".

    Science.gov (United States)

    Oshokoya, Olayinka O; JiJi, Renee D

    2015-09-10

    Protein secondary structural analysis is important for understanding the relationship between protein structure and function, or more importantly how changes in structure relate to loss of function. The structurally sensitive protein vibrational modes (amide I, II, III and S) in deep-ultraviolet resonance Raman (DUVRR) spectra resulting from the backbone C-O and N-H vibrations make DUVRR a potentially powerful tool for studying secondary structure changes. Experimental studies reveal that the position and intensity of the four amide modes in DUVRR spectra of proteins are largely correlated with the varying fractions of α-helix, β-sheet and disordered structural content of proteins. Employing multivariate calibration methods and DUVRR spectra of globular proteins with varying structural compositions, the secondary structure of a protein with unknown structure can be predicted. A disadvantage of multivariate calibration methods is the requirement of known concentration or spectral profiles. Second-order curve resolution methods, such as parallel factor analysis (PARAFAC), do not have such a requirement due to the "second-order advantage." An exceptional feature of DUVRR spectroscopy is that DUVRR spectra are linearly dependent on both excitation wavelength and secondary structure composition. Thus, higher order data can be created by combining protein DUVRR spectra of several proteins collected at multiple excitation wavelengths to give multi-excitation ultraviolet resonance Raman data (ME-UVRR). PARAFAC has been used to analyze ME-UVRR data of nine proteins to resolve the pure spectral, excitation and compositional profiles. A three factor model with non-negativity constraints produced three unique factors that were correlated with the relative abundance of helical, β-sheet and poly-proline II dihedral angles. This is the first empirical evidence that the typically resolved "disordered" spectrum represents the better defined poly-proline II type structure.

  15. Backbone topology, access, and the commercial Internet, 1997 - 2000

    OpenAIRE

    Morton E O'Kelly; Grubesic, Tony H.

    2002-01-01

    As the Internet grows in popularity, telecommunications infrastructure in the United States continues to increase in capacity and geographic reach to meet market demand. Important components of this infrastructure include the commercial fiber-optic backbones used to transport digital information between locations. The spatial organization of commercial Internet backbones reflects an increasingly competitive privatized market for service provision, in which certain locations are more accessibl...

  16. On Backbone Structure for a Future Multipurpose Network

    DEFF Research Database (Denmark)

    Gutierrez Lopez, Jose Manuel; Cuevas, Ruben; Riaz, M. Tahir

    2008-01-01

    Telecommunications are evolving towards the unification of services and infrastructures. This unification must be achieved at the highest hierarchical level for a complete synergy of services. Therefore, one of the requirements is a multipurpose backbone network capable of supporting all the curr......Telecommunications are evolving towards the unification of services and infrastructures. This unification must be achieved at the highest hierarchical level for a complete synergy of services. Therefore, one of the requirements is a multipurpose backbone network capable of supporting all...

  17. Determination of human muscle protein fractional synthesis rate

    DEFF Research Database (Denmark)

    Bornø, Andreas; Hulston, Carl J; van Hall, Gerrit

    2014-01-01

    In the present study, different MS methods for the determination of human muscle protein fractional synthesis rate (FSR) using [ring-(13)C6 ]phenylalanine as a tracer were evaluated. Because the turnover rate of human skeletal muscle is slow, only minute quantities of the stable isotopically......-MS/MS) and GC-tandem MS (GC-MS/MS) have made these techniques an option for human muscle FSR measurements. Human muscle biopsies were freeze dried, cleaned, and hydrolyzed, and the amino acids derivatized using either N-acetyl-n-propyl, phenylisothiocyanate, or N.......89 ± 0.01, P muscle FSR, (2) LC-MS/MS comes quite close and is a good alternative when tissue quantities are too small for GC-C-IRMS, and (3) If GC-MS/MS is to be used, then the HFBA derivative should be used instead...

  18. Topologia dos backbones de internet no Brasil / Internet backbone topology in Brazil

    Directory of Open Access Journals (Sweden)

    Marcelo Paiva da Motta

    2012-04-01

    Full Text Available Este artigo visa reafirmar o papel do espaço no estudo das Novas Tecnologias de Informação e Comunicação (NTICs. Examinamos a topologia dos backbones de internet no Brasil usando as ferramentas matemáticas da teoria dos grafos. Através do cálculo de índices de centralidade (proximidade e intermediação, bem como de outras técnicas quantitativas, as redes físicas que compõem a internet são relacionadas à rede urbana preexistente, mostrando que em suas características gerais o funcionamento não subverte a geografia econômica do país, a despeito do ideário antigeográfico suscitado por parte da literatura sobre os impactos da tecnologia.

  19. Triazine-Based Sequence-Defined Polymers with Side-Chain Diversity and Backbone-Backbone Interaction Motifs.

    Science.gov (United States)

    Grate, Jay W; Mo, Kai-For; Daily, Michael D

    2016-03-14

    Sequence control in polymers, well-known in nature, encodes structure and functionality. Here we introduce a new architecture, based on the nucleophilic aromatic substitution chemistry of cyanuric chloride, that creates a new class of sequence-defined polymers dubbed TZPs. Proof of concept is demonstrated with two synthesized hexamers, having neutral and ionizable side chains. Molecular dynamics simulations show backbone-backbone interactions, including H-bonding motifs and pi-pi interactions. This architecture is arguably biomimetic while differing from sequence-defined polymers having peptide bonds. The synthetic methodology supports the structural diversity of side chains known in peptides, as well as backbone-backbone hydrogen-bonding motifs, and will thus enable new macromolecules and materials with useful functions.

  20. Prediction of Factors Determining Changes in Stability in Protein Mutants

    OpenAIRE

    Parthiban, Vijayarangakannan

    2006-01-01

    Analysing the factors behind protein stability is a key research topic in molecular biology and has direct implications on protein structure prediction and protein-protein docking solutions. Protein stability upon point mutations were analysed using a distance dependant pair potential representing mainly through-space interactions and torsion angle potential representing neighbouring effects as a basic statistical mechanical setup for the analysis. The synergetic effect of accessible surface ...

  1. NMR backbone dynamics of VEK-30 bound to the human plasminogen kringle 2 domain.

    Science.gov (United States)

    Wang, Min; Prorok, Mary; Castellino, Francis J

    2010-07-07

    To gain insights into the mechanisms for the tight and highly specific interaction of the kringle 2 domain of human plasminogen (K2(Pg)) with a 30-residue internal peptide (VEK-30) from a group A streptococcal M-like protein, the dynamic properties of free and bound K2(Pg) and VEK-30 were investigated using backbone amide (15)N-NMR relaxation measurements. Dynamic parameters, namely the generalized order parameter, S(2), the local correlation time, tau(e), and the conformational exchange contribution, R(ex), were obtained for this complex by Lipari-Szabo model-free analysis. The results show that VEK-30 displays distinctly different dynamic behavior as a consequence of binding to K2(Pg), manifest by decreased backbone flexibility, particularly at the binding region of the peptide. In contrast, the backbone dynamics parameters of K2(Pg) displayed similar patterns in the free and bound forms, but, nonetheless, showed interesting differences. Based on our previous structure-function studies of this interaction, we also made comparisons of the VEK-30/K2(Pg) dynamics results from different kringle modules complexed with small lysine analogs. The differences in dynamics observed for kringles with different ligands provide what we believe to be new insights into the interactions responsible for protein-ligand recognition and a better understanding of the differences in binding affinity and binding specificity of kringle domains with various ligands.

  2. Determination of protein and solvent volumes in protein crystals from contrast variation data

    Energy Technology Data Exchange (ETDEWEB)

    Badger, J. [Brandeis Univ., Waltham, MA (United States)

    1994-12-31

    By varying the relative values of protein and solvent scattering densities in a crystal, it is possible to obtain information on the shape and dimensions of protein molecular envelopes. Neutron diffraction methods are ideally suited to these contrast variation experiments because H/D exchange leads to large differential changes in the protein and solvent scattering densities and is structurally non-perturbing. Low resolution structure factors have been measured from cubic insulin crystals with differing H/D contents. Structure factors calculated from a simple binary density model, in which uniform scattering densities represent the protein and solvent volumes in the crystals, were compared with these data. The contrast variation differences in the sets of measured structure factors were found to be accurately fitted by this simple model. Trial applications to two problems in crystal structure determination illustrate how this fact may be exploited. (1) A translation function that employs contrast variation data gave a sharp minimum within 1-9{Angstrom} of the correctly positioned insulin molecule and is relatively insensitive to errors in the atomic model. (2) An ab initio phasing method for the contrast variation data, based on analyzing histograms of the density distributions in trial maps, was found to recover the correct molecular envelope.

  3. Cost-effectiveness analysis of dolutegravir plus backbone compared with raltegravir plus backbone, darunavir+ritonavir plus backbone and efavirenz/tenofovir/emtricitabine in treatment naïve and experienced HIV-positive patients

    Directory of Open Access Journals (Sweden)

    Restelli U

    2017-06-01

    Full Text Available Umberto Restelli,1,2 Giuliano Rizzardini,3,4 Andrea Antinori,5 Adriano Lazzarin,6 Marzia Bonfanti,1 Paolo Bonfanti,7 Davide Croce1,2 1Centre for Research on Health Economics, Social and Health Care Management, LIUC – Università Cattaneo, Castellanza, Varese, Italy; 2School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; 3First and Second Divisions of Infectious Diseases, “Luigi Sacco” Hospital, Milan, Italy; 4School of Clinical Medicine, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa; 5National Institute for Infectious Diseases “L Spallanzani”, Rome, 6Department of Infectious Diseases, San Raffaele Scientific Institute, 7Department of Infectious and Tropical Diseases, A Manzoni Hospital, Lecco, Italy Background: In January 2014, the European Medicines Agency issued a marketing authorization for dolutegravir (DTG, a second-generation integrase strand transfer inhibitor for HIV treatment. The study aimed at determining the incremental cost-effectiveness ratio (ICER of the use of DTG+backbone compared with raltegravir (RAL+backbone, darunavir (DRV+ritonavir(r+backbone and efavirenz/tenofovir/emtricitabine (EFV/TDF/FTC in HIV-positive treatment-naïve patients and compared with RAL+backbone in treatment-experienced patients, from the Italian National Health Service’s point of view.Materials and methods: A published Monte Carlo Individual Simulation Model (ARAMIS-DTG model was used to perform the analysis. Patients pass through mutually exclusive health states (defined in terms of diagnosis of HIV with or without opportunistic infections [OIs] and cardiovascular disease [CVD] and successive lines of therapy. The model considers costs (2014 and quality of life per monthly cycle in a lifetime horizon. Costs and quality-adjusted life years (QALYs are dependent on OI, CVD, AIDS events, adverse events and antiretroviral therapies.Results: In

  4. Rigidity of transmembrane proteins determines their cluster shape

    CERN Document Server

    Jafarinia, Hamidreza; Jalali, Mir Abbas

    2015-01-01

    Protein aggregation in cell membrane is vital for majority of biological functions. Recent experimental results suggest that transmembrane domains of proteins such as $\\alpha$-helices and $\\beta$-sheets have different structural rigidity. We use molecular dynamics simulation of a coarse-grained model of protein-embedded lipid membranes to investigate the mechanisms of protein clustering. For a variety of protein concentrations, our simulations in thermal equilibrium conditions reveal that the structural rigidity of transmembrane domains dramatically affects interactions and changes the shape of the cluster. We have observed stable large aggregates even in the absence of hydrophobic mismatch which has been previously proposed as the mechanism of protein aggregation. According to our results, semi-flexible proteins aggregate to form two-dimensional clusters while rigid proteins, by contrast, form one-dimensional string-like structures. By assuming two probable scenarios for the formation of a two-dimensional tr...

  5. Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy.

    Science.gov (United States)

    Mote, Kaustubh R; Gopinath, T; Veglia, Gianluigi

    2013-10-01

    The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD ~0.44 Å, a tilt angle of 24° ± 1°, and an azimuthal angle of 55° ± 6°. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR.

  6. Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mote, Kaustubh R. [University of Minnesota, Department of Chemistry (United States); Gopinath, T. [University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics (United States); Veglia, Gianluigi, E-mail: vegli001@umn.edu [University of Minnesota, Department of Chemistry (United States)

    2013-10-15

    The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD {approx}0.44 A, a tilt angle of 24 Degree-Sign {+-} 1 Degree-Sign , and an azimuthal angle of 55 Degree-Sign {+-} 6 Degree-Sign . This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR.

  7. Redox-controlled backbone dynamics of human cytochrome c revealed by {sup 15}N NMR relaxation measurements

    Energy Technology Data Exchange (ETDEWEB)

    Sakamoto, Koichi [Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810 (Japan); Kamiya, Masakatsu [Graduate School of Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Uchida, Takeshi [Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810 (Japan); Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810 (Japan); Kawano, Keiichi [Graduate School of Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Ishimori, Koichiro, E-mail: koichiro@sci.hokudai.ac.jp [Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810 (Japan); Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810 (Japan)

    2010-07-23

    Research highlights: {yields} The dynamic parameters for the backbone dynamics in Cyt c were determined. {yields} The backbone mobility of Cyt c is highly restricted due to the covalently bound heme. {yields} The backbone mobility of Cyt c is more restricted upon the oxidation of the heme. {yields} The redox-dependent dynamics are shown in the backbone of Cyt c. {yields} The backbone dynamics of Cyt c would regulate the electron transfer from Cyt c. -- Abstract: Redox-controlled backbone dynamics in cytochrome c (Cyt c) were revealed by 2D {sup 15}N NMR relaxation experiments. {sup 15}N T{sub 1} and T{sub 2} values and {sup 1}H-{sup 15}N NOEs of uniformly {sup 15}N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S{sup 2}), the effective correlation time for internal motion ({tau}{sub e}), the {sup 15}N exchange broadening contributions (R{sub ex}) for each residue, and the overall correlation time ({tau}{sub m}) were estimated by model-free dynamics formalism. These dynamic parameters clearly showed that the backbone dynamics of Cyt c are highly restricted due to the covalently bound heme that functions as the stable hydrophobic core. Upon oxidation of the heme iron in Cyt c, the average S{sup 2} value was increased from 0.88 {+-} 0.01 to 0.92 {+-} 0.01, demonstrating that the mobility of the backbone is further restricted in the oxidized form. Such increases in the S{sup 2} values were more prominent in the loop regions, including amino acid residues near the thioether bonds to the heme moiety and positively charged region around Lys87. Both of the regions are supposed to form the interaction site for cytochrome c oxidase (CcO) and the electron pathway from Cyt c to CcO. The redox-dependent mobility of the backbone in the interaction site for the electron transfer to CcO suggests an electron transfer mechanism regulated by the backbone dynamics in the Cyt c-CcO system.

  8. Peptide Backbone Sampling Convergence with the Adaptive Biasing Force Algorithm

    Science.gov (United States)

    Faller, Christina E.; Reilly, Kyle A.; Hills, Ronald D.; Guvench, Olgun

    2013-01-01

    Complete Boltzmann sampling of reaction coordinates in biomolecular systems continues to be a challenge for unbiased molecular dynamics simulations. A growing number of methods have been developed for applying biases to biomolecular systems to enhance sampling while enabling recovery of the unbiased (Boltzmann) distribution of states. The Adaptive Biasing Force (ABF) algorithm is one such method, and works by canceling out the average force along the desired reaction coordinate(s) using an estimate of this force progressively accumulated during the simulation. Upon completion of the simulation, the potential of mean force, and therefore Boltzmann distribution of states, is obtained by integrating this average force. In an effort to characterize the expected performance in applications such as protein loop sampling, ABF was applied to the full ranges of the Ramachandran ϕ/ψ backbone dihedral reaction coordinates for dipeptides of the 20 amino acids using all-atom explicit-water molecular dynamics simulations. Approximately half of the dipeptides exhibited robust and rapid convergence of the potential of mean force as a function of ϕ/ψ in triplicate 50-ns simulations, while the remainder exhibited varying degrees of less complete convergence. The greatest difficulties in achieving converged ABF sampling were seen in the branched-sidechain amino acids threonine and valine, as well as the special case of proline. Proline dipeptide sampling was further complicated by trans-to-cis peptide bond isomerization not observed in unbiased control molecular dynamics simulations. Overall, the ABF method was found to be a robust means of sampling the entire ϕ/ψ reaction coordinate for the 20 amino acids, including high free-energy regions typically inaccessible in standard molecular dynamics simulations. PMID:23215032

  9. Using relaxation dispersion NMR spectroscopy to determine structures of excited, invisible protein states

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, D. Flemming; Vallurupalli, Pramodh; Kay, Lewis E. [University of Toronto, Departments of Molecular Genetics, Biochemistry and Chemistry (Canada)], E-mail: kay@pound.med.utoronto.ca

    2008-07-15

    Currently the main focus of structural biology is the determination of static three-dimensional representations of biomolecules that for the most part correspond to low energy (ground state) conformations. However, it is becoming increasingly well recognized that higher energy structures often play important roles in function as well. Because these conformers are populated to only low levels and are often only transiently formed their study is not amenable to many of the tools of structural biology. In this perspective we discuss the role of CPMG-based relaxation dispersion NMR spectroscopy in characterizing these low populated, invisible states. It is shown that robust methods for measuring both backbone chemical shifts and residual anisotropic interactions in the excited state are in place and that these data provide valuable restraints for structural studies of invisible conformers.

  10. Protein structure validation and refinement using amide proton chemical shifts derived from quantum mechanics

    DEFF Research Database (Denmark)

    Christensen, Anders Steen; Linnet, Troels Emtekær; Borg, Mikael;

    2013-01-01

    We present the ProCS method for the rapid and accurate prediction of protein backbone amide proton chemical shifts - sensitive probes of the geometry of key hydrogen bonds that determine protein structure. ProCS is parameterized against quantum mechanical (QM) calculations and reproduces high level...

  11. The Formation of Protein Structure

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren

    1996-01-01

    Dynamically induced curvature owing to long-range excitations along the backbones of protein molecules with non-linear elastic properties may control the folding of proteins.......Dynamically induced curvature owing to long-range excitations along the backbones of protein molecules with non-linear elastic properties may control the folding of proteins....

  12. Pseudocontact Shift-Driven Iterative Resampling for 3D Structure Determinations of Large Proteins.

    Science.gov (United States)

    Pilla, Kala Bharath; Otting, Gottfried; Huber, Thomas

    2016-01-29

    Pseudocontact shifts (PCSs) induced by paramagnetic lanthanides produce pronounced effects in nuclear magnetic resonance spectra, which are easily measured and which deliver valuable long-range structure restraints. Even sparse PCS data greatly enhance the success rate of 3D (3-dimensional) structure predictions of proteins by the modeling program Rosetta. The present work extends this approach to 3D structures of larger proteins, comprising more than 200 residues, which are difficult to model by Rosetta without additional experimental restraints. The new algorithm improves the fragment assembly method of Rosetta by utilizing PCSs generated from paramagnetic lanthanide ions attached at four different sites as the only experimental restraints. The sparse PCS data are utilized at multiple stages, to identify native-like local structures, to rank the best structural models and to rebuild the fragment libraries. The fragment libraries are refined iteratively until convergence. The PCS-driven iterative resampling algorithm is strictly data dependent and shown to generate accurate models for a benchmark set of eight different proteins, ranging from 100 to 220 residues, using solely PCSs of backbone amide protons.

  13. PANADA: protein association network annotation, determination and analysis.

    Directory of Open Access Journals (Sweden)

    Alberto J M Martin

    Full Text Available Increasingly large numbers of proteins require methods for functional annotation. This is typically based on pairwise inference from the homology of either protein sequence or structure. Recently, similarity networks have been presented to leverage both the ability to visualize relationships between proteins and assess the transferability of functional inference. Here we present PANADA, a novel toolkit for the visualization and analysis of protein similarity networks in Cytoscape. Networks can be constructed based on pairwise sequence or structural alignments either on a set of proteins or, alternatively, by database search from a single sequence. The Panada web server, executable for download and examples and extensive help files are available at URL: http://protein.bio.unipd.it/panada/.

  14. Determination of dideoxyosone precursors of AGEs in human lens proteins.

    Science.gov (United States)

    Linetsky, Mikhail; Kaid Johar, S R; Meltretter, Jasmin; Padmanabha, Smitha; Parmar, Trilok; Vasavada, Abhay R; Pischetsrieder, Monika; Nagaraj, Ram H

    2011-10-01

    Dideoxyosones (DDOs) are intermediates in the synthesis of advanced glycation endproducts (AGEs), such as pentosidine and glucosepane. Although the formation of pentosidine and glucosepane in the human lens has been firmly established, the formation of DDOs has not been demonstrated. The aim of this study was to develop a reliable method to detect DDOs in lens proteins. A specific DDO trapping agent, biotinyl-diaminobenzene (3,4-diamino-N-(3-[5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoyl]aminopropyl)benzamide) (BDAB) was added during in vitro protein glycation or during protein extraction from human lenses. In vitro glycated human lens protein showed strong reaction in monomeric and polymeric crosslinked proteins by Western blot and ELISA. Glycation of BSA in the presence of BDAB resulted in covalent binding of BDAB to the protein and inhibited pentosidine formation. Mass spectrometric analysis of lysozyme glycated in the presence of BDAB showed the presence of quinoxalines at lysine residues at positions K1, K33, K96, and K116. The ELISA results indicated that cataractous lens proteins contain significantly higher levels of DDO than non-cataractous lenses (101.9±67.8 vs. 31.7±19.5AU/mg protein, p<0.0001). This study provides first direct evidence of DDO presence in human tissue proteins and establishes that AGE crosslink synthesis in the human lens occurs via DDO intermediates. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. In vitro Determination of Extracellular Proteins from Xylella fastidiosa

    Science.gov (United States)

    Mendes, Juliano S.; Santiago, André S.; Toledo, Marcelo A. S.; Horta, Maria A. C.; de Souza, Alessandra A.; Tasic, Ljubica; de Souza, Anete P.

    2016-01-01

    The phytopathogen Xylella fastidiosa causes economic losses in important agricultural crops. Xylem vessel occlusion caused by biofilm formation is the major mechanism underlying the pathogenicity of distinct strains of X. fastidiosa. Here, we provide a detailed in vitro characterization of the extracellular proteins of X. fastidiosa. Based on the results, we performed a comparison with a strain J1a12, which cannot induce citrus variegated chlorosis symptoms when inoculated into citrus plants. We then extend this approach to analyze the extracellular proteins of X. fastidiosa in media supplemented with calcium. We verified increases in extracellular proteins concomitant with the days of growth and, consequently, biofilm development (3–30 days). Outer membrane vesicles carrying toxins were identified beginning at 10 days of growth in the 9a5c strain. In addition, a decrease in extracellular proteins in media supplemented with calcium was observed in both strains. Using mass spectrometry, 71 different proteins were identified during 30 days of X. fastidiosa biofilm development, including proteases, quorum-sensing proteins, biofilm formation proteins, hypothetical proteins, phage-related proteins, chaperones, toxins, antitoxins, and extracellular vesicle membrane components. PMID:28082960

  16. Aromatic Copolyamides with Anthrazoline Units in the Backbone: Synthesis, Characterization, Pervaporation Application

    Directory of Open Access Journals (Sweden)

    Galina A. Polotskaya

    2016-10-01

    Full Text Available Copolyamides with anthrazoline units in the backbone (coPA were synthesized and dense nonporous films were prepared by solvent evaporation. Glass transition temperature, density, and fractional free volume were determined for the dense nonporous films composed of polyamide and two of its copolymers containing 20 and 30 mol % anthrazoline units in the backbone. Transport properties of the polymer films were estimated by sorption and pervaporation tests toward methanol, toluene, and their mixtures. An increase in anthrazoline fragments content leads to an increasing degree of methanol sorption but to a decreasing degree of toluene sorption. Pervaporation of a methanol–toluene mixture was studied over a wide range of feed concentration (10–90 wt % methanol. Maximal separation factor was observed for coPA-20 containing 20 mol % fragments with anthrazoline units; maximal total flux was observed for coPA-30 with the highest fractional free volume.

  17. Energy Efficient Low-Cost Virtual Backbone Construction for Optimal Routing in Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    K. Mohaideen Pitchai

    2014-01-01

    Full Text Available Many prominent applications in wireless sensor networks which require collected information have to be routed to end nodes in an efficient manner. In general, weighted connected dominating Sets (WCDS based routing is a promising approach for enhancing the routing efficiency in sensor networks. Backbone has been used extensively in routing. Here an efficient WCDS algorithm for constructing a virtual backbone with low total cost, hop spanning ratio, and minimum number of dominators is proposed. We report a systematic approach, which has three phases. Initial phase considers the issues of revoking a partial CDS tree from a complete CDS tree. Secondary and final phases make the design of the complete algorithm by considering the determination of dominators using an iteration process. Our findings reveal better performance than the existing algorithms in terms of total cost, hop spanning ratio, and number of dominators.

  18. APPECT: An Approximate Backbone-Based Clustering Algorithm for Tags

    DEFF Research Database (Denmark)

    Zong, Yu; Xu, Guandong; Jin, Pin

    2011-01-01

    algorithm for Tags (APPECT). The main steps of APPECT are: (1) we execute the K-means algorithm on a tag similarity matrix for M times and collect a set of tag clustering results Z={C1,C2,…,Cm}; (2) we form the approximate backbone of Z by executing a greedy search; (3) we fix the approximate backbone...... resulting from the severe difficulty of ambiguity, redundancy and less semantic nature of tags. Clustering method is a useful tool to address the aforementioned difficulties. Most of the researches on tag clustering are directly using traditional clustering algorithms such as K-means or Hierarchical...

  19. APPECT: An Approximate Backbone-Based Clustering Algorithm for Tags

    DEFF Research Database (Denmark)

    Zong, Yu; Xu, Guandong; Jin, Pin

    2011-01-01

    algorithm for Tags (APPECT). The main steps of APPECT are: (1) we execute the K-means algorithm on a tag similarity matrix for M times and collect a set of tag clustering results Z={C1,C2,…,Cm}; (2) we form the approximate backbone of Z by executing a greedy search; (3) we fix the approximate backbone...... resulting from the severe difficulty of ambiguity, redundancy and less semantic nature of tags. Clustering method is a useful tool to address the aforementioned difficulties. Most of the researches on tag clustering are directly using traditional clustering algorithms such as K-means or Hierarchical...

  20. Torsion stiffness of a protein pair determined by magnetic particles

    NARCIS (Netherlands)

    Janssen, X.J.A.; Van Noorloos, J.M.; Jacob, A.; Van IJzendoorn, L.J.; De Jong, A.M.; Prins, M.W.J.

    2012-01-01

    We demonstrate the ability to measure torsion stiffness of a proteincomplex by applying a controlled torque on a magnetic particle. Asa model system we use protein G bound to an IgG antibody. The protein pair is held between a magnetic particle and a polystyrene substrate. The angular orientation of

  1. HIV-1 phenotypic reverse transcriptase inhibitor drug resistance test interpretation is not dependent on the subtype of the virus backbone.

    Directory of Open Access Journals (Sweden)

    Michelle Bronze

    Full Text Available To date, the majority of HIV-1 phenotypic resistance testing has been performed with subtype B virus backbones (e.g. HXB2. However, the relevance of using this backbone to determine resistance in non-subtype B HIV-1 viruses still needs to be assessed. From 114 HIV-1 subtype C clinical samples (36 ARV-naïve, 78 ARV-exposed, pol amplicons were produced and analyzed for phenotypic resistance using both a subtype B- and C-backbone in which the pol fragment was deleted. Phenotypic resistance was assessed in resulting recombinant virus stocks (RVS for a series of antiretroviral drugs (ARV's and expressed as fold change (FC, yielding 1660 FC comparisons. These Antivirogram® derived FC values were categorized as having resistant or sensitive susceptibility based on biological cut-off values (BCOs. The concordance between resistance calls obtained for the same clinical sample but derived from two different backbones (i.e. B and C accounted for 86.1% (1429/1660 of the FC comparisons. However, when taking the assay variability into account, 95.8% (1590/1660 of the phenotypic data could be considered as being concordant with respect to their resistance call. No difference in the capacity to detect resistance associated with M184V, K103N and V106M mutations was noted between the two backbones. The following was concluded: (i A high level of concordance was shown between the two backbone phenotypic resistance profiles; (ii Assay variability is largely responsible for discordant results (i.e. for FC values close to BCO; (iii Confidence intervals should be given around the BCO's, when assessing resistance in HIV-1 subtype C; (iv No systematic resistance under- or overcalling of subtype C amplicons in the B-backbone was observed; (v Virus backbone subtype sequence variability outside the pol region does not contribute to phenotypic FC values. In conclusion the HXB2 virus backbone remains an acceptable vector for phenotyping HIV-1 subtype C pol amplicons.

  2. Determining protein function and interaction from genome analysis

    Science.gov (United States)

    Eisenberg, David; Marcotte, Edward M.; Thompson, Michael J.; Pellegrini, Matteo; Yeates, Todd O.

    2004-08-03

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

  3. In vivo protein synthesis determinations in human immune cells

    OpenAIRE

    Januszkiewicz, Anna

    2005-01-01

    Intact immune responses are essential for defeating severe infections in individual patients. Insufficient function of the immune system contributes to a poor prognosis in these patients, in particular the ICU patients. Nevertheless, the immune system function is not easily monitored and evaluated. The ongoing metabolic activity of immune competent cells is reflected by their in vivo protein synthesis rate. The aim of this thesis was to apply in vivo protein synthesis measur...

  4. Conformational changes in Bcl-2 pro-survival proteins determine their capacity to bind ligands.

    Science.gov (United States)

    Lee, Erinna F; Czabotar, Peter E; Yang, Hong; Sleebs, Brad E; Lessene, Guillaume; Colman, Peter M; Smith, Brian J; Fairlie, W Douglas

    2009-10-30

    Antagonists of anti-apoptotic Bcl-2 family members hold promise as cancer therapeutics. Apoptosis is triggered when a peptide containing a BH3 motif or a small molecule BH3 peptidomimetic, such as ABT 737, binds to the relevant Bcl-2 family members. ABT-737 is an antagonist of Bcl-2, Bcl-x(L), and Bcl-w but not of Mcl-1. Here we describe new structures of mutant BH3 peptides bound to Bcl-x(L) and Mcl-1. These structures suggested a rationale for the failure of ABT-737 to bind Mcl-1, but a designed variant of ABT-737 failed to acquire binding affinity for Mcl-1. Rather, it was selective for Bcl-x(L), a result attributable in part to significant backbone refolding and movements of helical segments in its ligand binding site. To date there are few reported crystal structures of organic ligands in complex with their pro-survival protein targets. Our structure of this new organic ligand provided insights into the structural transitions that occur within the BH3 binding groove, highlighting significant differences in the structural properties of members of the Bcl-2 pro-survival protein family. Such differences are likely to influence and be important in the quest for compounds capable of selectively antagonizing the different family members.

  5. Residue 259 in protein-tyrosine phosphatase PTP1B and PTPα determines the flexibility of glutamine 262

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Iversen, L.F.; Andersen, H.S.

    2004-01-01

    To study the flexibility of the substrate-binding site and in particular of Gln262, we have performed adiabatic conformational search and molecular dynamics simulations on the crystal structure of the catalytic domain of wild-type protein-tyrosine phosphatase (PTP) 1B, a mutant PTP1B(R47V),(D48N...... and second step of the phosphate hydrolysis. Analyses of the trajectories revealed that in the cysteine-phosphor complex of PTP1B, Gln262 oscillates freely between the bound phosphate group and Gly259 frequently forming, as observed in the crystal structure, a hydrogen bond with the backbone oxygen of Gly259...

  6. Determination of reversible protein equilibrium association coefficients using light scattering

    Science.gov (United States)

    Larkin, Michael

    2009-03-01

    The characterization in solution of reversible protein associations as well as associations between proteins and small molecules is essential in many areas of science. Understanding cellular function or developing and formulating pharmaceuticals or other biologically active materials often requires quantitation of such associations. Most pharmaceuticals have functionality due solely to association with molecules within the body, and the discovery and accurate characterization of these associations is a key element for pharmaceutical development. Unfortunately, most methods used to measure associations of proteins require either immobilizing the protein on a surface (e.g. surface plasmon resonance), which potentially alters the protein characteristics, or require considerable time and effort and large quantities of sample (e.g. analytical ultracentrifugation, isothermal titration calorimetry). Light scattering based measurements of reversible association coefficients require much less sample and may be performed much more rapidly than other free solution techniques. In this talk I describe how static and dynamic light scattering may each independently be used to measure equilibrium association coefficients between proteins in free solution, and may also be used to observe and quantitate the association of small molecules with them. I present background theory for both static and dynamic light scattering measurements of equilibrium associations, and examples of measurements made of both model systems and of systems with commercial relevance in the pharmaceutical industry.

  7. The Graphical Representation of the Digital Astronaut Physiology Backbone

    Science.gov (United States)

    Briers, Demarcus

    2010-01-01

    This report summarizes my internship project with the NASA Digital Astronaut Project to analyze the Digital Astronaut (DA) physiology backbone model. The Digital Astronaut Project (DAP) applies integrated physiology models to support space biomedical operations, and to assist NASA researchers in closing knowledge gaps related to human physiologic responses to space flight. The DA physiology backbone is a set of integrated physiological equations and functions that model the interacting systems of the human body. The current release of the model is HumMod (Human Model) version 1.5 and was developed over forty years at the University of Mississippi Medical Center (UMMC). The physiology equations and functions are scripted in an XML schema specifically designed for physiology modeling by Dr. Thomas G. Coleman at UMMC. Currently it is difficult to examine the physiology backbone without being knowledgeable of the XML schema. While investigating and documenting the tags and algorithms used in the XML schema, I proposed a standard methodology for a graphical representation. This standard methodology may be used to transcribe graphical representations from the DA physiology backbone. In turn, the graphical representations can allow examination of the physiological functions and equations without the need to be familiar with the computer programming languages or markup languages used by DA modeling software.

  8. Optimal sink placement in backbone assisted wireless sensor networks

    Directory of Open Access Journals (Sweden)

    I. Snigdh

    2016-07-01

    Full Text Available This article proposes a scheme for selecting the best site for sink placement in WSN applications employing backbone assisted communications. By placing the sink at a specific position, energy scavenging and delay constraints can effectively be controlled. In contrast to the conventional scheme for base station placement at the geographical centre or random placement at the end of the region of interest, the proposed scheme places the base station at either the graph theoretical centre or centroid of the backbone connecting nodes in the region of interest. This strategy shows a considerable reduction in the total number of hops that each packet needs to travel to reach the sink. The proposed scheme is applied on all the families of graphs prevalent in backbone assisted sensor networks to confirm the performance consistency and improvement in network parameters of the communication backbone measured in terms of delay, the carried load and the total energy consumption, eventually affected by the average number of hops for the message to reach the sink.

  9. Alkali metal salts of formazanate ligands : diverse coordination modes as a result of the nitrogen-rich [NNCNN] ligand backbone

    NARCIS (Netherlands)

    Travieso-Puente, Raquel; Chang, Mu-Chieh; Otten, Edwin

    2014-01-01

    Alkali metal salts of redox-active formazanate ligands were prepared, and their structures in the solid-state and in solution are determined. The nitrogen-rich [NNCNN] backbone of formazanates results in a varied coordination chemistry, with both the internal and terminal nitrogen atoms available fo

  10. Immunofluorescent determination of wheat protein in meat products

    Directory of Open Access Journals (Sweden)

    Michaela Petrášová

    2014-02-01

    Full Text Available In food industry nowadays, there are various plant-origin protein additives which are meant for production of meat products. Among the most frequent additives of this type there are different kinds of flour, starch, fiber, and plant-origin proteins. Their usage at present is limited by the existing legislation not to prevent consumer deception but also for reasons of possible influence on consumer health. Therefore, this problem is paid a lot of attention not only in the Czech Republic but also all over the world. The main risk is seen in the impossibility to choose a suitable foodstuff for an individual prone to allergic reactions. Potential allergens are also often plant-origin raw materials which are added into foodstuffs for their technological qualities and low price. Wheat is widely cultivated cereal as well as an important source of proteins. After ingestion or inhalation, wheat proteins may cause adverse reactions. These adverse effects include a wide range of disorders which are dependent on the method of contact with wheat protein. These adverse effects can then take the form of various clinical manifestations, such as celiac disease, T-cell mediated inflammatory bowel disease, dermatitis, skin rash, breathing difficulties, allergy to pollen or to wheat flour or food allergy to foodstuffs containing gluten. The only possible protection against adverse immune reactions for those with food allergies is strictly excluding the allergen from their diet. Although the number of studies dealing with the reduction or loss of allergenicity is increasing, yet these practices are not common. Most of the population suffering from food allergies is thus still dependent on strict exclusion of foodstuffs causing adverse allergic reactions from their diet. In order to avoid misleading consumers and also to protect allergic consumers, analytical methods applicable to all types of foodstuffs have been developed. Unfortunately, detection of allergens in

  11. Protein Energy Landscapes Determined by 5-Dimensional Crystallography

    CERN Document Server

    Schmidt, Marius; Henning, Robert; Ihee, Hyotcherl; Purwar, Namrta; Tenboer, Jason; Tripathi, Shailesh

    2013-01-01

    Free energy landscapes decisively determine the progress of enzymatically catalyzed reactions[1]. Time-resolved macromolecular crystallography unifies transient-state kinetics with structure determination [2-4] because both can be determined from the same set of X-ray data. We demonstrate here how barriers of activation can be determined solely from five-dimensional crystallography [5]. Directly linking molecular structures with barriers of activation between them allows for gaining insight into the structural nature of the barrier. We analyze comprehensive time series of crystal-lographic data at 14 different temperature settings and determine entropy and enthalpy contributions to the barriers of activation. 100 years after the discovery of X-ray scattering, we advance X-ray structure determination to a new frontier, the determination of energy landscapes.

  12. Structure determination of T-cell protein-tyrosine phosphatase

    DEFF Research Database (Denmark)

    Iversen, L.F.; Møller, K. B.; Pedersen, A.K.

    2002-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the high...

  13. Avirulence proteins of plant pathogens: determinants of victory and defeat

    NARCIS (Netherlands)

    Luderer, R.; Joosten, M.H.A.J.

    2001-01-01

    The simplest way to explain the biochemical basis of the gene-for-gene concept is by direct interaction between a pathogen-derived avirulence (Avr) gene product and a receptor protein, which is encoded by the matching resistance (R) gene of the host plant. The number of R genes for which the

  14. Circular Dichroism Method for Heat Capacity Determination of Proteins

    Science.gov (United States)

    Jones, Cecil L.; Bailey, Chris; Bheemarti, Kiran Kumar

    2006-01-01

    Circular dichroism spectroscopy was used to measure the thermal unfolding of bovine pancreatic ribonuclease A (RNase A) with various concentrations of guanidine hydrochloride (GuHCl). A red shift in transition midpoint temperatures, T[subscript m], occurred with increasing concentration of the strong protein denaturant. van Hoff enthalpy changes,…

  15. Protein energy landscapes determined by five-dimensional crystallography.

    Science.gov (United States)

    Schmidt, Marius; Srajer, Vukica; Henning, Robert; Ihee, Hyotcherl; Purwar, Namrta; Tenboer, Jason; Tripathi, Shailesh

    2013-12-01

    Free-energy landscapes decisively determine the progress of enzymatically catalyzed reactions [Cornish-Bowden (2012), Fundamentals of Enzyme Kinetics, 4th ed.]. Time-resolved macromolecular crystallography unifies transient-state kinetics with structure determination [Moffat (2001), Chem. Rev. 101, 1569-1581; Schmidt et al. (2005), Methods Mol. Biol. 305, 115-154; Schmidt (2008), Ultrashort Laser Pulses in Medicine and Biology] because both can be determined from the same set of X-ray data. Here, it is demonstrated how barriers of activation can be determined solely from five-dimensional crystallography, where in addition to space and time, temperature is a variable as well [Schmidt et al. (2010), Acta Cryst. A66, 198-206]. Directly linking molecular structures with barriers of activation between them allows insight into the structural nature of the barrier to be gained. Comprehensive time series of crystallographic data at 14 different temperature settings were analyzed and the entropy and enthalpy contributions to the barriers of activation were determined. One hundred years after the discovery of X-ray scattering, these results advance X-ray structure determination to a new frontier: the determination of energy landscapes.

  16. Determining the topology of stable protein-DNA complexes.

    Science.gov (United States)

    Darcy, Isabel K; Vazquez, Mariel

    2013-04-01

    Difference topology is an experimental technique that can be used to unveil the topological structure adopted by two or more DNA segments in a stable protein-DNA complex. Difference topology has also been used to detect intermediates in a reaction pathway and to investigate the role of DNA supercoiling. In the present article, we review difference topology as applied to the Mu transpososome. The tools discussed can be applied to any stable nucleoprotein complex.

  17. Hamiltonian replica-exchange simulations with adaptive biasing of peptide backbone and side chain dihedral angles.

    Science.gov (United States)

    Ostermeir, Katja; Zacharias, Martin

    2014-01-15

    A Hamiltonian Replica-Exchange Molecular Dynamics (REMD) simulation method has been developed that employs a two-dimensional backbone and one-dimensional side chain biasing potential specifically to promote conformational transitions in peptides. To exploit the replica framework optimally, the level of the biasing potential in each replica was appropriately adapted during the simulations. This resulted in both high exchange rates between neighboring replicas and improved occupancy/flow of all conformers in each replica. The performance of the approach was tested on several peptide and protein systems and compared with regular MD simulations and previous REMD studies. Improved sampling of relevant conformational states was observed for unrestrained protein and peptide folding simulations as well as for refinement of a loop structure with restricted mobility of loop flanking protein regions.

  18. [Spectrophotometric determination of protein content in THP-1 monocytes/macrophages - description of the method].

    Science.gov (United States)

    Wolska, Jolanta; Janda, Katarzyna; Gutowska, Izabela

    2015-01-01

    Proteins are the basic building block of tissue, and are part of enzymes and hormones regulating many important life processes. Changes in their concentration control the metabolic processes of the cell. Quantitative determination of the protein content is divided into indirect methods (e.g. Kjeldahl method) and direct methods (buret method, Lowry, immunoenzymatic, formol method, based on incorporation of dye in the range of ultraviolet spectrophotometry, and based on the phenomenon of selective absorption of radiation in the infrared range). One of the methods for the determination of protein content is the spectrophotometric method described by Bradford. The protein concentration assay procedure utilizes the phenomenon of formation of the dye (Coomassie Brillant Blue G-250)-protein and colour intensity is proportional to the protein content in the solution. The aim of this study was to verify the usefulness of this method for determining the protein content in THP-1 cells cultured with extracts of nettle fruit stalks (Urtica dioica L.). Aqueous and alcohol extracts at two concentrations were used. It has been shown that the spectrophotometric determination of protein content by the Bradford method is an effective and accurate method for determining the concentration of protein in THP-1 macrophages. The results indicate that this method can be recommended for the determination of the protein content in other cell cultures.

  19. Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

    Energy Technology Data Exchange (ETDEWEB)

    Eddy, Matthew T. [Massachusetts Institute of Technology, Department of Chemistry (United States); Belenky, Marina [Brandeis University, Department of Chemistry (United States); Sivertsen, Astrid C. [Massachusetts Institute of Technology, Francis Bitter Magnet Laboratory (United States); Griffin, Robert G. [Massachusetts Institute of Technology, Department of Chemistry (United States); Herzfeld, Judith, E-mail: herzfeld@brandeis.edu [Brandeis University, Department of Chemistry (United States)

    2013-10-15

    The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new 'redox' approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-{sup 13}C] acetate does not label {alpha} carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-{sup 13}C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra.

  20. Selectively Dispersed Isotope Labeling for Protein Structure Determination by Magic Angle Spinning NMR

    Science.gov (United States)

    Eddy, Matthew T.; Belenky, Marina; Sivertsen, Astrid; Griffin, Robert G.; Herzfeld, Judith

    2013-01-01

    The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-13C] acetate does not label α carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-13C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra. PMID:23990199

  1. X-ray Structure of Snow Flea Antifreeze Protein Determined by Racemic Crystallization of Synthetic Protein Enantiomers

    Energy Technology Data Exchange (ETDEWEB)

    Pentelute, Brad L.; Gates, Zachary P.; Tereshko, Valentina; Dashnau, Jennifer L.; Vanderkooi, Jane M.; Kossiakoff, Anthony A.; Kent, Stephen B.H. (UPENN); (UC)

    2008-08-20

    Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 {angstrom}. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.

  2. Evaluation of the refractometric method for the determination of total protein in avian plasma or serum.

    Science.gov (United States)

    Lumeij, J T; de Bruijne, J J

    1985-07-01

    Serum total protein concentrations in pigeon blood determined with the biuret method (TPB-se) were compared with total protein concentrations in plasma (TPR-pl) and serum (TPR-se) obtained by estimation from refractive index. The refractometric method consistently yielded higher values (Prefractometric method for determination of TP in pigeon blood is not recommended.

  3. Identification of systems containing nonlinear stiffnesses using backbone curves

    Science.gov (United States)

    Londoño, Julián M.; Cooper, Jonathan E.; Neild, Simon A.

    2017-02-01

    This paper presents a method for the dynamic identification of structures containing discrete nonlinear stiffnesses. The approach requires the structure to be excited at a single resonant frequency, enabling measurements to be made in regimes of large displacements where nonlinearities are more likely to be significant. Measured resonant decay data is used to estimate the system backbone curves. Linear natural frequencies and nonlinear parameters are identified using these backbone curves assuming a form for the nonlinear behaviour. Numerical and experimental examples, inspired by an aerospace industry test case study, are considered to illustrate how the method can be applied. Results from these models demonstrate that the method can successfully deliver nonlinear models able to predict the response of the test structure nonlinear dynamics.

  4. Determining and comparing protein function in Bacterial genome sequences

    DEFF Research Database (Denmark)

    Vesth, Tammi Camilla

    annotation of genes – the descriptions assigned to genes that describe the likely function of the encoded proteins. This process is limited by several factors, including the definition of a function which can be more or less specific as well as how many genes can actually be assigned a function based...... of this class have very little homology to other known genomes making functional annotation based on sequence similarity very difficult. Inspired in part by this analysis, an approach for comparative functional annotation was created based public sequenced genomes, CMGfunc. Functionally related groups...

  5. Backbone decomposition for continuous-state branching processes with immigration

    CERN Document Server

    Ren, A E Kyprianou Y-X

    2011-01-01

    In the spirit of Duqesne and Winkel (2007) and Berestycki et al. (2011) we show that supercritical continuous-state branching process with a general branching mechanism and general immigration mechanism is equal in law to a continuous-time Galton Watson process with immigration with Poissonian dressing. The result also characterises the limiting backbone decomposition which is predictable from the work on consistent growth of Galton-Watson trees with immigration in Cao and Winkel (2010).

  6. Spectrophotometric determination of total proteins in blood plasma: a comparative study among dye-binding methods

    OpenAIRE

    Dimas Augusto Morozin Zaia; Fábio Rangel Marques; Cássia Thaïs Bussamra Vieira Zaia

    2005-01-01

    A comparative study between the biuret method (standard method for total proteins) and spectrophotometric methods using dyes (Bradford, 3',3",5',5"-tetrabromophenolphthalein ethyl ester-TBPEE, and erythrosin-B) was carried out for the determination of total proteins in blood plasma from rats. Bradford method showed the highest sensitivity for proteins and biuret method showed the lowest. For all the methods, the absorbance for different proteins (BSA, casein, and egg albumin) was measured and...

  7. System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

    Science.gov (United States)

    Craig, George D.; Glass, Robert; Rupp, Bernhard

    1997-01-01

    A method for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10.sup.6 V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved.

  8. Extracting the multiscale backbone of complex weighted networks

    Science.gov (United States)

    Serrano, M. Ángeles; Boguñá, Marián; Vespignani, Alessandro

    2009-01-01

    A large number of complex systems find a natural abstraction in the form of weighted networks whose nodes represent the elements of the system and the weighted edges identify the presence of an interaction and its relative strength. In recent years, the study of an increasing number of large-scale networks has highlighted the statistical heterogeneity of their interaction pattern, with degree and weight distributions that vary over many orders of magnitude. These features, along with the large number of elements and links, make the extraction of the truly relevant connections forming the network's backbone a very challenging problem. More specifically, coarse-graining approaches and filtering techniques come into conflict with the multiscale nature of large-scale systems. Here, we define a filtering method that offers a practical procedure to extract the relevant connection backbone in complex multiscale networks, preserving the edges that represent statistically significant deviations with respect to a null model for the local assignment of weights to edges. An important aspect of the method is that it does not belittle small-scale interactions and operates at all scales defined by the weight distribution. We apply our method to real-world network instances and compare the obtained results with alternative backbone extraction techniques. PMID:19357301

  9. Determination of microbial protein in perennial ryegrass silage

    NARCIS (Netherlands)

    Driehuis, F.; Wikselaar, van P.G.

    2001-01-01

    The microbial matter fraction was determined in perennial ryegrass silages of different dry-matter (DM) contents, ensiled with or without Lactobacillus plantarum. 15N-Leucine and the bacterial cell wall constituent diaminopimelic acid (DAPA) were used as markers for microbial-N. Perennial ryegrass

  10. Determination of microbial protein in perennial ryegrass silage

    NARCIS (Netherlands)

    Driehuis, F.; Wikselaar, van P.G.

    2001-01-01

    The microbial matter fraction was determined in perennial ryegrass silages of different dry-matter (DM) contents, ensiled with or without Lactobacillus plantarum. 15N-Leucine and the bacterial cell wall constituent diaminopimelic acid (DAPA) were used as markers for microbial-N. Perennial ryegrass c

  11. Determination of the amount of protein and amino acids extracted from the microbial protein (SCP) of lignocellulosic wastes.

    Science.gov (United States)

    Ahmadi, A R; Ghoorchian, H; Hajihosaini, R; Khanifar, J

    2010-04-15

    With the increasing world population, the use of lignocellulosic wastes for production of microbial protein as animal feed becomes a necessity of our time. In order to verify the most productive protein, the amount of protein and amino acid extracted from Single Cell Protein (SCP) needs to be determined by an effective method. In this study Microbial protein was produced by treatment of wheat straw with Pleurotus florida; with heat at 100 degrees C and NaOH 2% as substrate by solid state fermentation. Concentration of protein was 62.8% per 100 g of dried microbial protein. Then the extracted protein hydrolyzed with HCl 6 Normal for 48 h under 110 degrees C temperature condition. Then the amino acids analyzed by using A-200 Amino Nova analyzer. The results of this study indicated that the ratio of essential amino acids to total amino acids was 65.6%. The concentration of essnyial amino acids were: Lysine = 9.5, histidine = 19.8, threonine = 0.6, valine = 6.6, methionine = 2.1, isoleucine = 7.3, leucine = 6.8, phenylalanine = 4.3 and arginine = 8.3 g/100 g of extracted protein that indicated the obtained microbial protein can be a good or suitable substitute in the food program of animal feed.

  12. An ancient protein-DNA interaction underlying metazoan sex determination.

    Science.gov (United States)

    Murphy, Mark W; Lee, John K; Rojo, Sandra; Gearhart, Micah D; Kurahashi, Kayo; Banerjee, Surajit; Loeuille, Guy-André; Bashamboo, Anu; McElreavey, Kenneth; Zarkower, David; Aihara, Hideki; Bardwell, Vivian J

    2015-06-01

    DMRT transcription factors are deeply conserved regulators of metazoan sexual development. They share the DM DNA-binding domain, a unique intertwined double zinc-binding module followed by a C-terminal recognition helix, which binds a pseudopalindromic target DNA. Here we show that DMRT proteins use a unique binding interaction, inserting two adjacent antiparallel recognition helices into a widened DNA major groove to make base-specific contacts. Versatility in how specific base contacts are made allows human DMRT1 to use multiple DNA binding modes (tetramer, trimer and dimer). Chromatin immunoprecipitation with exonuclease treatment (ChIP-exo) indicates that multiple DNA binding modes also are used in vivo. We show that mutations affecting residues crucial for DNA recognition are associated with an intersex phenotype in flies and with male-to-female sex reversal in humans. Our results illuminate an ancient molecular interaction underlying much of metazoan sexual development.

  13. Evaluation of a rapid protein analyzer for determination of protein in milk and cream.

    Science.gov (United States)

    Amamcharla, J K; Metzger, L E

    2010-08-01

    Accurate and rapid measurement of the protein content of milk is important from both a product quality and an economic standpoint. The Sprint rapid protein analyzer (CEM Corporation, Matthews, NC) is a commercial system based on a dye-binding technique and can be used for rapid measurement of protein in foods. The objective of the present study was to compare the Sprint method with the reference method (Kjeldahl method). Milk and cream samples were analyzed in duplicate for true protein and crude protein (CP) using the reference method as well as the rapid method. Method comparison statistics (regression analysis, graphical representation, standard deviation of residuals, repeatability, and so on) were used to evaluate the agreement between the 2 methods. Regression coefficients and the intercepts were not significantly different from 1 and zero for CP measurement in milk and cream, respectively. The average coefficient of variance between the duplicate CP measurements for the Sprint method was found to be 0.40, 0.49, and 0.76 for milk, light cream, and heavy cream, respectively. True protein measurement in milk and cream also followed a similar trend. Overall, there exists a sufficient level of agreement between the Sprint rapid protein analyzer and Kjeldahl method for true protein and CP measurement of milk and cream samples.

  14. Structure determination of uniformly {sup 13}C, {sup 15}N labeled protein using qualitative distance restraints from MAS solid-state {sup 13}C-NMR observed paramagnetic relaxation enhancement

    Energy Technology Data Exchange (ETDEWEB)

    Tamaki, Hajime [Hokkaido University, Graduate School of Life Science (Japan); Egawa, Ayako [Osaka University, Institute for Protein Research (Japan); Kido, Kouki [Hokkaido University, Graduate School of Life Science (Japan); Kameda, Tomoshi [National Institute of Advanced Industrial Science and Technology, Biotechnology Research Institute for Drug Discovery (Japan); Kamiya, Masakatsu; Kikukawa, Takashi; Aizawa, Tomoyasu [Hokkaido University, Faculty of Advanced Life Science (Japan); Fujiwara, Toshimichi [Osaka University, Institute for Protein Research (Japan); Demura, Makoto, E-mail: demura@sci.hokudai.ac.jp [Hokkaido University, Faculty of Advanced Life Science (Japan)

    2016-01-15

    Magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) is a powerful method for structure determination of insoluble biomolecules. However, structure determination by MAS solid-state NMR remains challenging because it is difficult to obtain a sufficient amount of distance restraints owing to spectral complexity. Collection of distance restraints from paramagnetic relaxation enhancement (PRE) is a promising approach to alleviate this barrier. However, the precision of distance restraints provided by PRE is limited in solid-state NMR because of incomplete averaged interactions and intermolecular PREs. In this report, the backbone structure of the B1 domain of streptococcal protein G (GB1) has been successfully determined by combining the CS-Rosetta protocol and qualitative PRE restraints. The derived structure has a Cα RMSD of 1.49 Å relative to the X-ray structure. It is noteworthy that our protocol can determine the correct structure from only three cysteine-EDTA-Mn{sup 2+} mutants because this number of PRE sites is insufficient when using a conventional structure calculation method based on restrained molecular dynamics and simulated annealing. This study shows that qualitative PRE restraints can be employed effectively for protein structure determination from a limited conformational sampling space using a protein fragment library.

  15. Exposing Hidden Alternative Backbone Conformations in X-ray Crystallography Using qFit.

    Science.gov (United States)

    Keedy, Daniel A; Fraser, James S; van den Bedem, Henry

    2015-10-01

    Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP) to evaluate an extremely large number of combinations of sidechain conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the "flap" regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Overall, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.

  16. Exposing Hidden Alternative Backbone Conformations in X-ray Crystallography Using qFit.

    Directory of Open Access Journals (Sweden)

    Daniel A Keedy

    2015-10-01

    Full Text Available Proteins must move between different conformations of their native ensemble to perform their functions. Crystal structures obtained from high-resolution X-ray diffraction data reflect this heterogeneity as a spatial and temporal conformational average. Although movement between natively populated alternative conformations can be critical for characterizing molecular mechanisms, it is challenging to identify these conformations within electron density maps. Alternative side chain conformations are generally well separated into distinct rotameric conformations, but alternative backbone conformations can overlap at several atomic positions. Our model building program qFit uses mixed integer quadratic programming (MIQP to evaluate an extremely large number of combinations of sidechain conformers and backbone fragments to locally explain the electron density. Here, we describe two major modeling enhancements to qFit: peptide flips and alternative glycine conformations. We find that peptide flips fall into four stereotypical clusters and are enriched in glycine residues at the n+1 position. The potential for insights uncovered by new peptide flips and glycine conformations is exemplified by HIV protease, where different inhibitors are associated with peptide flips in the "flap" regions adjacent to the inhibitor binding site. Our results paint a picture of peptide flips as conformational switches, often enabled by glycine flexibility, that result in dramatic local rearrangements. Our results furthermore demonstrate the power of large-scale computational analysis to provide new insights into conformational heterogeneity. Overall, improved modeling of backbone heterogeneity with high-resolution X-ray data will connect dynamics to the structure-function relationship and help drive new design strategies for inhibitors of biomedically important systems.

  17. Ultrafast colorimetric determination of predominant protein structure evolution with gold nanoplasmonic particles

    Science.gov (United States)

    Kim, Hye Young; Choi, Inhee

    2016-01-01

    The intracellular and extracellular accumulation of disordered proteins and aggregated proteins occurs in many protein conformational diseases, such as aging-related neurodegeneration and alcoholic liver diseases. However, the conventional methods to study protein structural changes are limited for the rapid detection and monitoring of protein aggregation because of long incubation times (i.e., usually several days), complicated sample pretreatment steps, and expensive instrumentation. Here, we describe an ultrafast colorimetric method for the real-time monitoring of protein structure evolution and the determination of predominant structures via nanoparticle-assisted protein aggregation. During the aggregation process, nanoparticles act as nucleation cores, which form networks depending on the structures of the protein aggregates, and accelerate the kinetics of the protein aggregation. Simultaneously, these nanoparticles exhibit colorimetric responses according to their embedded shapes (e.g., fibrillar and amorphous) on the protein aggregates. We observed distinct spectral shifts and concomitant colorimetric responses of concentration- and type-dependent protein aggregation with the naked eye within a few minutes (pH levels, high temperature, and chemicals. These findings suggest that the proposed method is an easy way to study the molecular biophysics of protein aggregation and to rapidly screen anti-aggregation drugs for protein conformational diseases.The intracellular and extracellular accumulation of disordered proteins and aggregated proteins occurs in many protein conformational diseases, such as aging-related neurodegeneration and alcoholic liver diseases. However, the conventional methods to study protein structural changes are limited for the rapid detection and monitoring of protein aggregation because of long incubation times (i.e., usually several days), complicated sample pretreatment steps, and expensive instrumentation. Here, we describe an

  18. Protein purification-free method of binding affinity determination by microscale thermophoresis.

    Science.gov (United States)

    Khavrutskii, Lyuba; Yeh, Joanna; Timofeeva, Olga; Tarasov, Sergey G; Pritt, Samuel; Stefanisko, Karen; Tarasova, Nadya

    2013-08-15

    Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

  19. An Improved Method of Predicting Extinction Coefficients for the Determination of Protein Concentration.

    Science.gov (United States)

    Hilario, Eric C; Stern, Alan; Wang, Charlie H; Vargas, Yenny W; Morgan, Charles J; Swartz, Trevor E; Patapoff, Thomas W

    2017-01-01

    Concentration determination is an important method of protein characterization required in the development of protein therapeutics. There are many known methods for determining the concentration of a protein solution, but the easiest to implement in a manufacturing setting is absorption spectroscopy in the ultraviolet region. For typical proteins composed of the standard amino acids, absorption at wavelengths near 280 nm is due to the three amino acid chromophores tryptophan, tyrosine, and phenylalanine in addition to a contribution from disulfide bonds. According to the Beer-Lambert law, absorbance is proportional to concentration and path length, with the proportionality constant being the extinction coefficient. Typically the extinction coefficient of proteins is experimentally determined by measuring a solution absorbance then experimentally determining the concentration, a measurement with some inherent variability depending on the method used. In this study, extinction coefficients were calculated based on the measured absorbance of model compounds of the four amino acid chromophores. These calculated values for an unfolded protein were then compared with an experimental concentration determination based on enzymatic digestion of proteins. The experimentally determined extinction coefficient for the native proteins was consistently found to be 1.05 times the calculated value for the unfolded proteins for a wide range of proteins with good accuracy and precision under well-controlled experimental conditions. The value of 1.05 times the calculated value was termed the predicted extinction coefficient. Statistical analysis shows that the differences between predicted and experimentally determined coefficients are scattered randomly, indicating no systematic bias between the values among the proteins measured. The predicted extinction coefficient was found to be accurate and not subject to the inherent variability of experimental methods. We propose the use of a

  20. Topographic patterns of vascular disease: HOX proteins as determining factors?

    Institute of Scientific and Technical Information of China (English)

    Richard; P; Visconti; Alexander; Awgulewitsch

    2015-01-01

    Steadily increasing evidence supports the idea that genetic diversities in the vascular bed are, in addition to hemodynamic influences, a major contributing factor in determining region-specific cardiovascular disease susceptibility. Members of the phylogenetically highly conserved Hox gene family of developmental regulators have to be viewed as prime candidates for determining these regional genetic differences in the vasculature. During embryonic patterning, the regionally distinct and precisely choreographed expression patterns of HOX transcription factors are essential for the correct specification of positional identities. Apparently, these topographic patterns are to some degree retained in certain adult tissues, including the circulatory system. While an understanding of the functional significance of these localized Hox activities in adult blood vessels is only beginning to emerge, an argument can be made for a role of Hox genes in the maintenance of vessel wall homeostasis and functional integrity on the one hand, and in regulating the development and progression of regionally restricted vascular pathologies, on the other. Initial functional studies in animal models, as well as data from clinical studies provide some level of support for this view. The data suggest that putative genetic regulatory networks of Hox-dependent cardiovascular disease processes include genes of diverse functional categories(extracellular matrix remodeling, transmembrane signaling, cell cycle control, inflammatory response, transcriptional control, etc.), as potential targets in both vascular smooth muscle and endothelial cells, as well as cell populations residing in the adventitia.

  1. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

    Science.gov (United States)

    Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

    2015-07-01

    Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool.

  2. Comparison of label-free quantification methods for the determination of protein complexes subunits stoichiometry

    Directory of Open Access Journals (Sweden)

    Bertrand Fabre

    2014-09-01

    Full Text Available Protein complexes are the main molecular machines that support all major cellular pathways and their in-depth characterization are essential to understand their functions. Determining the stoichiometry of the different subunits of a protein complex still remains challenging. Recently, many label-free quantitative proteomic approaches have been developed to study the composition of protein complexes. It is therefore of great interest to evaluate these different methods in a stoichiometry oriented objective. Here we compare the ability of four absolute quantitative label-free methods currently used in proteomic studies to determine the stoichiometry of a well-characterized protein complex, the 26S proteasome.

  3. A simple method for the determination of reduction potentials in heme proteins.

    Science.gov (United States)

    Efimov, Igor; Parkin, Gary; Millett, Elizabeth S; Glenday, Jennifer; Chan, Cheuk K; Weedon, Holly; Randhawa, Harpreet; Basran, Jaswir; Raven, Emma L

    2014-03-03

    We describe a simple method for the determination of heme protein reduction potentials. We use the method to determine the reduction potentials for the PAS-A domains of the regulatory heme proteins human NPAS2 (Em=-115 mV ± 2 mV, pH 7.0) and human CLOCK (Em=-111 mV ± 2 mV, pH 7.0). We suggest that the method can be easily and routinely applied to the determination of reduction potentials across the family of heme proteins.

  4. RNA-Redesign: a web server for fixed-backbone 3D design of RNA.

    Science.gov (United States)

    Yesselman, Joseph D; Das, Rhiju

    2015-07-01

    RNA is rising in importance as a design medium for interrogating fundamental biology and for developing therapeutic and bioengineering applications. While there are several online servers for design of RNA secondary structure, there are no tools available for the rational design of 3D RNA structure. Here we present RNA-Redesign (http://rnaredesign.stanford.edu), an online 3D design tool for RNA. This resource utilizes fixed-backbone design to optimize the sequence identity and nucleobase conformations of an RNA to match a desired backbone, analogous to fundamental tools that underlie rational protein engineering. The resulting sequences suggest thermostabilizing mutations that can be experimentally verified. Further, sequence preferences that differ between natural and computationally designed sequences can suggest whether natural sequences possess functional constraints besides folding stability, such as cofactor binding or conformational switching. Finally, for biochemical studies, the designed sequences can suggest experimental tests of 3D models, including concomitant mutation of base triples. In addition to the designs generated, detailed graphical analysis is presented through an integrated and user-friendly environment.

  5. On the role of thermal backbone fluctuations in myoglobin ligand gate dynamics

    CERN Document Server

    Krokhotin, Andrey; Peng, Xubiao

    2012-01-01

    We construct an energy function that describes the crystallographic structure of spermwhale myoglobin backbone. As a model in our construction, we use the Protein Data Bank entry 1ABS that has been measured at liquid helium temperature. Consequently the thermal B-factor fluctuations are very small, which is an advantage in our construction. The energy function that we utilize resembles that of the discrete non-linear Schrodinger equation. Likewise, ours supports solitons as local minimum energy configurations. We describe the 1ABS backbone in terms of solitons with a precision that deviates from 1ABS by an average root-mean-square distance, which is less than the experimentally observed Debye-Waller B-factor fluctuation distance. We then subject the multisoliton solution to extensive numerical heating and cooling experiments, over a very wide range of temperatures. We concentrate in particular to temperatures above 300K and below the theta-point unfolding temperature, which is around 348K. We confirm that the...

  6. Structural determinants for protein adsorption/non-adsorption to silica surface.

    Directory of Open Access Journals (Sweden)

    Christelle Mathé

    Full Text Available The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nanotechnology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many π-π interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption.

  7. ROMP- and RAFT-Based Guanidinium-Containing Polymers as Scaffolds for Protein Mimic Synthesis.

    Science.gov (United States)

    Sarapas, Joel M; Backlund, Coralie M; deRonde, Brittany M; Minter, Lisa M; Tew, Gregory N

    2017-05-17

    Cell-penetrating peptides are an important class of molecules with promising applications in bioactive cargo delivery. A diverse series of guanidinium-containing polymeric cell-penetrating peptide mimics (CPPMs) with varying backbone chemistries was synthesized and assessed for delivery of both GFP and fluorescently tagged siRNA. Specifically, we examined CPPMs based on norbornene, methacrylate, and styrene backbones to determine how backbone structure impacted internalization of these two cargoes. Either charge content or degree of polymerization was held constant at 20, with diguanidinium norbornene molecules being polymerized to both 10 and 20 repeat units. Generally, homopolymer CPPMs delivered low amounts of siRNA into Jurkat T cells, with no apparent backbone dependence; however, by adding a short hydrophobic methyl methacrylate block to the guanidinium-rich methacrylate polymer, siRNA delivery to nearly the entire cell population was achieved. Protein internalization yielded similar results for most of the CPPMs, though the block polymer was unable to deliver proteins. In contrast, the styrene-based CPPM yielded the highest internalization for GFP (≈40 % of cells affected), showing that indeed backbone chemistry impacts protein delivery, specifically through the incorporation of an aromatic group. These results demonstrate that an understanding of how polymer structure affects cargo-dependent internalization is critical to designing new, more effective CPPMs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Small envelope protein E of SARS:cloning,expression, purification, CD determination, and bioinformatics analysis

    Institute of Scientific and Technical Information of China (English)

    SHENXu; XUEJian-Hua; YUChang-Ying; LUOHai-Bin; QINLei; YUXiao-Jing; CHENJing; CHENLi-Li; XIONGBin; YUELi-Duo; CAIJian-Hua; SHENJian-Hua; LUOXiao-Min; CHENKai-Xian; SHITie-Liu; LIYi-Xue; HUGeng-Xi; JIANGHua-Liang

    2003-01-01

    AIM:To obtain the pure sample of SARS small envelope E protein (SARS E protein), study its properties and analyze its possible functions. METHODS: The plasmid of SARS E protein was constructed by the polymerase chain reaction (PCR), and the protein was expressed in the E coli strain. The secondary structure feature of the protein was determined by circular dichroism (CD) technique. The possible functions of this protein were annotated by bioinformatics methods, and its possible three-dimensional model was constructed by molecular modeling. RESULTS: The pure sample of SARS E protein was obtained. The secondary structure feature derived from CD determination is similar to that from the secondary structure prediction. Bioinformatics analysis indicated that the key residues of SARS E protein were much conserved compared to the E proteins of other coronaviruses. In particular, the primary amino acid sequence of SARS E protien is much more similar to that of murine hepatitis virus(MHV) and other mammal coronaviruses. The transmembrane (TM) segment of the SARS E protein is relatively more conserved in the whole protein than other regions. CONCLUSION: The success of expressing the SARS E protein is a good starting point for investigating the structure and functions of this protein and SARS coronavirus itself as well. The SARS E protein may fold in water solution in a similar way as it in membrane-water mixed environment. It is possible that β-sheet I of the SARS E protein interacts with the membrane surface via hydrogen bonding, this β-sheet may uncoil to a random structure in water solution.

  9. Resistance of Feynman diagrams and the percolation backbone dimension.

    Science.gov (United States)

    Janssen, H K; Stenull, O; Oerding, K

    1999-06-01

    We present an alternative view of Feynman diagrams for the field theory of random resistor networks, in which the diagrams are interpreted as being resistor networks themselves. This simplifies the field theory considerably as we demonstrate by calculating the fractal dimension D(B) of the percolation backbone to three loop order. Using renormalization group methods we obtain D(B)=2+epsilon/21-172epsilon(2)/9261+2epsilon(3)[-74 639+22 680zeta(3)]/4 084 101, where epsilon=6-d with d being the spatial dimension and zeta(3)=1.202 057... .

  10. Application of Multicast-based Video Conference on CERNET Backbone

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Multicast-based video conference is a representative application in advanced network. In multi-point video conference using multicast can get better efficiency facilitated by inner-group broadcast mechanism. In the application, the multicast-based network resources assignment, management and security should be considered together. This paper presents a framework model of multicast-based video conferencing application with three layers. And a practical multicast-based video conferencing is implemented in CERNET(China Education and Research Network) backbone. The practice is valuable for the development of multicast-based video conferencing application in China.

  11. Design of an IPTV Multicast System for Internet Backbone Networks

    OpenAIRE

    T. H. Szymanski; Gilbert, D

    2010-01-01

    The design of an IPTV multicast system for the Internet backbone network is presented and explored through extensive simulations. In the proposed system, a resource reservation algorithm such as RSVP, IntServ, or DiffServ is used to reserve resources (i.e., bandwidth and buffer space) in each router in an IP multicast tree. Each router uses an Input-Queued, Output-Queued, or Crosspoint-Queued switch architecture with unity speedup. A recently proposed Recursive Fair Stochastic Matrix Decompos...

  12. Unique optimal solution instance and computational complexity of backbone in the graph bi-partitioning problem

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    As an important tool for heuristic design of NP-hard problems, backbone analysis has become a hot spot in theoretical computer science in recent years. Due to the difficulty in the research on computational complexity of the backbone, many researchers analyzed the backbone by statistic ways. Aiming to increase the backbone size which is usually very small by the existing methods, the unique optimal solution instance construction (UOSIC) is proposed for the graph bi-partitioning problem (GBP). Also, we prove by using the UOSIC that it is NP-hard to obtain the backbone, i.e. no algorithm exists to obtain the backbone of a GBP in polynomial time under the assumption that P ( NP. Our work expands the research area of computational complexity of the backbone. And the UOSIC provides a new way for heuristic design of NP-hard problems.

  13. Protein fold determination from sparse distance restraints: The restrained generic protein direct Monte Carlo method

    Energy Technology Data Exchange (ETDEWEB)

    Debe, D.A.; Carlson, M.J.; Chan, S.I; Goddard, W.A. III [California Inst. of Tech., Pasadena, CA (United States); Sadanobu, Jiro [Teijin Limited, Iwakuni, Yamaguchi (Japan). Polymer and Materials Research Labs.

    1999-04-15

    The authors present the generate-and-select hierarchy for tertiary protein structure prediction. The foundation of this hierarchy is the Restrained Generic Protein (RGP) Direct Monte Carlo method. The RGP method is a highly efficient off-lattice residue buildup procedure that can quickly generate the complete set of topologies that satisfy a very small number of interresidue distance restraints. For three restraints uniformly distributed in a 72-residue protein, the authors demonstrate that the size of this set is {approximately}10{sup 4}. The RGP method can generate this set of structures in less than 1 h using a Silicon Graphics R10000 single processor workstation. Following structure generation, a simple criterion that measures the burial of hydrophobic and hydrophilic residues can reliably select a reduced set of {approximately}10{sup 2} structures that contains the native topology. A minimization of the structures in the reduced set typically ranks the native topology in the five lowest energy folds. Thus, using this hierarchical approach, the authors suggest that de novo prediction of moderate resolution globular protein structure can be achieved in just a few hours on a single processor workstation.

  14. Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds

    DEFF Research Database (Denmark)

    Pattison, D I; Davies, Michael Jonathan

    2001-01-01

    , absolute second-order rate constants for the reactions of HOCl with protein side chains, model compounds, and backbone amide (peptide) bonds have been determined at physiological pH values. The reactivity of HOCl with potential reactive sites in proteins is summarized by the series: Met (3.8 x 10(7) M(-1......) x s(-1)) > backbone amides (10-10(-3) M(-1) x s(-1)) > Gln(0.03 M(-1) x s(-1)) approximately Asn (0.03 M(-1) x s(-1)). The rate constants for reaction of HOCl with backbone amides (peptide bonds) vary by 4 orders of magnitude with uncharged peptide bonds reacting more readily with HOCl than those...

  15. Distribution of protein I in mammalian brain as determined by a detergent-based radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Goelz, S.E.; Nestler, E.J.; Chehrazi, B.; Greengard, P.

    1981-04-01

    A radioimmunoassay has been developed for measuring protein I, a basic, neuron-specific protein associated with nerve terminals. The procedure utilizes the detergents NaDodSO/sub 4/ and Nonidet P-40 to prevent nonspecific adsorption of this highly charged protein to various surfaces. By use of this procedure, it has been possible to show that protein I comprises approximately 0.4% of the total protein in cerebral cortex of several mammalian species. In addition, the amount of protein I was determined in about 40 regions of cat brain. The results suggest that measurement of protein I may provide a quantitative method for estimating the density of nerve terminals in various regions of the mammalian nervous system.

  16. Bioluminescent indicator for determining protein-protein interactions using intramolecular complementation of split click beetle luciferase.

    Science.gov (United States)

    Kim, Sung Bae; Otani, Yosuke; Umezawa, Yoshio; Tao, Hiroaki

    2007-07-01

    Click beetle luciferase (CBLuc) is insensitive to pH, temperature, and heavy metals, and emits a stable, highly tissue-transparent red light with luciferin in physiological circumstances. Thus, the luminescence signal is optimal for a bioanalytical index reporting the magnitude of a signal transduction of interest. Here, we validated a single-molecule-format complementation system of split CBLuc to study signal-controlled protein-protein (peptide) interactions. First, we generated 10 pairs of N- and C-terminal fragments of CBLuc to examine respectively whether a significant recovery of the activity occurs through the intramolecular complementation. The ligand binding domain of androgen receptor (AR LBD) was connected to a functional peptide sequence through a flexible linker. The fusion protein was then sandwiched between the dissected N- and C-terminal fragments of CBLuc. Androgen induces the association between AR LBD and a functional peptide and the subsequent complementation of N- and C-terminal fragments of split CBLuc inside the single-molecule-format probe, which restores the activities of CBLuc. The examination about the dissection sites of CBLuc revealed that the dissection positions next to the amino acids D412 and I439 admit a stable recovery of CBLuc activity through an intramolecular complementation. The ligand sensitivity and kinetics of the single molecular probe with split CBLuc were discussed in various cell lines and in different protein-peptide binding models. The probe is applicable to developing biotherapeutic agents on the AR signaling and for screening adverse chemicals that possibly influence the signal transduction of proteins in living cells or animals.

  17. A Rapid Method for Determining the Concentration of Recombinant Protein Secreted from Pichia pastoris

    Science.gov (United States)

    Sun, L. W.; Zhao, Y.; Niu, L. P.; Jiang, R.; Song, Y.; Feng, H.; feng, K.; Qi, C.

    2011-02-01

    Pichia secretive expression system is one of powerful eukaryotic expression systems in genetic engineering, which is especially suitable for industrial utilization. Because of the low concentration of the target protein in initial experiment, the methods and conditions for expression of the target protein should be optimized according to the protein yield repetitively. It is necessary to set up a rapid, simple and convenient analysis method for protein expression levels instead of the generally used method such as ultrafiltration, purification, dialysis, lyophilization and so on. In this paper, acetone precipitation method was chosen to concentrate the recombinant protein firstly after comparing with four different protein precipitation methods systematically, and then the protein was analyzed by SDS-Polyacrylamide Gel Electrophoresis. The recombinant protein was determined with the feature of protein band by the Automated Image Capture and 1-D Analysis Software directly. With this method, the optimized expression conditions of basic fibroblast growth factor secreted from pichia were obtained, which is as the same as using traditional methods. Hence, a convenient tool to determine the optimized conditions for the expression of recombinant proteins in Pichia was established.

  18. [Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

    Science.gov (United States)

    Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

    2013-02-01

    To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

  19. Peptide backbone orientation and dynamics in spider dragline silk and two-photon excitation in nuclear magnetic and quadrupole resonance

    Energy Technology Data Exchange (ETDEWEB)

    Eles, P.T

    2005-07-01

    In the first part of the dissertation, spider dragline silk is studied by solid state NMR techniques. The dependence of NMR frequency on molecular orientation is exploited using the DECODER experiment to determine the orientation of the protein backbone within the silk fibre. Practical experimental considerations require that the silk fibres be wound about a cylindrical axis perpendicular to the external magnetic field, complicating the reconstruction of the underlying orientation distribution and necessitating the development of numerical techniques for this purpose. A two-component model of silk incorporating static b-sheets and polyglycine II helices adequately fits the NMR data and suggests that the b-sheets are well aligned along the silk axis (20 FWHM) while the helices are poorly aligned (68 FWHM). The effects of fibre strain, draw rate and hydration on orientation are measured. Measurements of the time-scale for peptide backbone motion indicate that when wet, a strain-dependent fraction of the poorly aligned component becomes mobile. This suggests a mechanism for the supercontraction of silk involving latent entropic springs that undergo a local strain-dependent phase transition, driving supercontraction. In the second part of this dissertation a novel method is developed for exciting NMR and nuclear quadrupole resonance (NQR) by rf irradiation at multiple frequencies that sum to (or differ by) the resonance frequency. This is fundamentally different than traditional NMR experiments where irradiation is applied on-resonance. With excitation outside the detection bandwidth, two-photon excitation allows for detection of free induction signals during excitation, completely eliminating receiver dead-time. A theoretical approach to describing two-photon excitation is developed based on average Hamiltonian theory. An intuition for two-photon excitation is gained by analogy to the coherent absorption of multiple photons requiring conservation of total energy and

  20. Method for the rapid determination of protein in meats using the CEM Sprint protein analyzer: collaborative study.

    Science.gov (United States)

    Moser, Cindy; Herman, Kathy

    2011-01-01

    A collaborative study was conducted to determine the protein content of raw and processed meat products by a protein-tagging and colorimetric technique. Meat products were prepared following AOAC Official Method 983.18 and analyzed using CEM Corporation's Sprint Rapid Protein Analyzer. Sprint provides protein results by combining an accurately weighed test portion with a known amount of dye-binding agent. The dye-binding agent binds with the lysine, histidine, and arginine, as well as the n-terminus of the proteins commonly found in raw meat and processed meat products. Results are displayed and reported by the Sprint as a percentage (g/100 g) of protein. Ten blind duplicate study samples were sent to 10 collaborating laboratories in the United States. The within-laboratory (repeatability) relative standard deviation (RSD(r)) ranged from 0.91 to 3.04%, and between-laboratories (reproducibility) relative standard deviation (RSDR) ranged from 1.50 to 3.41% for protein. The method is recommended for Official First Action.

  1. Process-based network decomposition reveals backbone motif structure.

    Science.gov (United States)

    Wang, Guanyu; Du, Chenghang; Chen, Hao; Simha, Rahul; Rong, Yongwu; Xiao, Yi; Zeng, Chen

    2010-06-08

    A central challenge in systems biology today is to understand the network of interactions among biomolecules and, especially, the organizing principles underlying such networks. Recent analysis of known networks has identified small motifs that occur ubiquitously, suggesting that larger networks might be constructed in the manner of electronic circuits by assembling groups of these smaller modules. Using a unique process-based approach to analyzing such networks, we show for two cell-cycle networks that each of these networks contains a giant backbone motif spanning all the network nodes that provides the main functional response. The backbone is in fact the smallest network capable of providing the desired functionality. Furthermore, the remaining edges in the network form smaller motifs whose role is to confer stability properties rather than provide function. The process-based approach used in the above analysis has additional benefits: It is scalable, analytic (resulting in a single analyzable expression that describes the behavior), and computationally efficient (all possible minimal networks for a biological process can be identified and enumerated).

  2. Constructing Battery-Aware Virtual Backbones in Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Chi Ma

    2007-05-01

    Full Text Available A critical issue in battery-powered sensor networks is to construct energy efficient virtual backbones for network routing. Recent study in battery technology reveals that batteries tend to discharge more power than needed and reimburse the over-discharged power if they are recovered. In this paper we first provide a mathematical battery model suitable for implementation in sensor networks. We then introduce the concept of battery-aware connected dominating set (BACDS and show that in general the minimum BACDS (MBACDS can achieve longer lifetime than the previous backbone structures. Then we show that finding a MBACDS is NP-hard and give a distributed approximation algorithm to construct the BACDS. The resulting BACDS constructed by our algorithm is at most (8+Δopt size, where Δ is the maximum node degree and opt is the size of an optimal BACDS. Simulation results show that the BACDS can save a significant amount of energy and achieve up to 30% longer network lifetime than previous schemes.

  3. Constructing Battery-Aware Virtual Backbones in Wireless Sensor Networks

    Directory of Open Access Journals (Sweden)

    Yang Yuanyuan

    2007-01-01

    Full Text Available A critical issue in battery-powered sensor networks is to construct energy efficient virtual backbones for network routing. Recent study in battery technology reveals that batteries tend to discharge more power than needed and reimburse the over-discharged power if they are recovered. In this paper we first provide a mathematical battery model suitable for implementation in sensor networks. We then introduce the concept of battery-aware connected dominating set (BACDS and show that in general the minimum BACDS (MBACDS can achieve longer lifetime than the previous backbone structures. Then we show that finding a MBACDS is NP-hard and give a distributed approximation algorithm to construct the BACDS. The resulting BACDS constructed by our algorithm is at most opt size, where is the maximum node degree and opt is the size of an optimal BACDS. Simulation results show that the BACDS can save a significant amount of energy and achieve up to longer network lifetime than previous schemes.

  4. Determination of phosphorus in small amounts of protein samples by ICP-MS.

    Science.gov (United States)

    Becker, J Sabine; Boulyga, Sergei F; Pickhardt, Carola; Becker, J; Buddrus, Stefan; Przybylski, Michael

    2003-02-01

    Inductively coupled plasma mass spectrometry (ICP-MS) is used for phosphorus determination in protein samples. A small amount of solid protein sample (down to 1 micro g) or digest (1-10 micro L) protein solution was denatured in nitric acid and hydrogen peroxide by closed-microvessel microwave digestion. Phosphorus determination was performed with an optimized analytical method using a double-focusing sector field inductively coupled plasma mass spectrometer (ICP-SFMS) and quadrupole-based ICP-MS (ICP-QMS). For quality control of phosphorus determination a certified reference material (CRM), single cell proteins (BCR 273) with a high phosphorus content of 26.8+/-0.4 mg g(-1), was analyzed. For studies on phosphorus determination in proteins while reducing the sample amount as low as possible the homogeneity of CRM BCR 273 was investigated. Relative standard deviation and measurement accuracy in ICP-QMS was within 2%, 3.5%, 11% and 12% when using CRM BCR 273 sample weights of 40 mg, 5 mg, 1 mg and 0.3 mg, respectively. The lowest possible sample weight for an accurate phosphorus analysis in protein samples by ICP-MS is discussed. The analytical method developed was applied for the analysis of homogeneous protein samples in very low amounts [1-100 micro g of solid protein sample, e.g. beta-casein or down to 1 micro L of protein or digest in solution (e.g., tau protein)]. A further reduction of the diluted protein solution volume was achieved by the application of flow injection in ICP-SFMS, which is discussed with reference to real protein digests after protein separation using 2D gel electrophoresis.The detection limits for phosphorus in biological samples were determined by ICP-SFMS down to the ng g(-1) level. The present work discusses the figure of merit for the determination of phosphorus in a small amount of protein sample with ICP-SFMS in comparison to ICP-QMS.

  5. Backbone dynamics of a biologically active human FGF-1 monomer, complexed to a hexasaccharide heparin-analogue, by {sup 15}N NMR relaxation methods

    Energy Technology Data Exchange (ETDEWEB)

    Canales-Mayordomo, Angeles; Fayos, Rosa [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain); Angulo, Jesus; Ojeda, Rafael [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Martin-Pastor, Manuel [Unidad de RM y Unidad de RMN de Biomoleculas Asociada al CSIC, Laboratorio de Estructura e Estructura de Biomoleculas Jose Carracido (Spain); Nieto, Pedro M.; Martin-Lomas, Manuel [Instituto de Investigaciones Quimicas, CSIC, Grupo de Carbohidratos (Spain); Lozano, Rosa; Gimenez-Gallego, Guillermo; Jimenez-Barbero, Jesus [Centro de Investigaciones Biologicas, CSIC, Departamento de Estructura y Funcion de Proteinas (Spain)], E-mail: jjbarbero@cib.csic.es

    2006-08-15

    The binding site and backbone dynamics of a bioactive complex formed by the acidic fibroblast growth factor (FGF-1) and a specifically designed heparin hexasaccharide has been investigated by HSQC and relaxation NMR methods. The comparison of the relaxation data for the free and bound states has allowed showing that the complex is monomeric, and still induces mutagenesis, and that the protein backbone presents reduced motion in different timescale in its bound state, except in certain points that are involved in the interaction with the fibroblast growth factor receptor (FGFR)

  6. A fast method for the determination of fractional contributions to solvation in proteins

    Science.gov (United States)

    Talavera, David; Morreale, Antonio; Meyer, Tim; Hospital, Adam; Ferrer-Costa, Carles; Gelpi, Josep Lluis; de la Cruz, Xavier; Soliva, Robert; Luque, F. Javier; Orozco, Modesto

    2006-01-01

    A fast method for the calculation of residue contributions to protein solvation is presented. The approach uses the exposed polar and apolar surface of protein residues and has been parametrized from the fractional contributions to solvation determined from linear response theory coupled to molecular dynamics simulations. Application of the method to a large subset of proteins taken from the Protein Data Bank allowed us to compute the expected fractional solvation of residues. This information is used to discuss when a residue or a group of residues presents an uncommon solvation profile. PMID:17001031

  7. 1H, 15N and 13C backbone resonance assignments of the archetypal serpin α1-antitrypsin.

    Science.gov (United States)

    Nyon, Mun Peak; Kirkpatrick, John; Cabrita, Lisa D; Christodoulou, John; Gooptu, Bibek

    2012-10-01

    Alpha(1)-antitrypsin is a 45-kDa (394-residue) serine protease inhibitor synthesized by hepatocytes, which is released into the circulatory system and protects the lung from the actions of neutrophil elastase via a conformational transition within a dynamic inhibitory mechanism. Relatively common point mutations subvert this transition, causing polymerisation of α(1)-antitrypsin and deficiency of the circulating protein, predisposing carriers to severe lung and liver disease. We have assigned the backbone resonances of α(1)-antitrypsin using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for a detailed solution state characterization of the structural properties of this highly dynamic protein via NMR methods.

  8. Determining Membrane Protein-Lipid Binding Thermodynamics Using Native Mass Spectrometry.

    Science.gov (United States)

    Cong, Xiao; Liu, Yang; Liu, Wen; Liang, Xiaowen; Russell, David H; Laganowsky, Arthur

    2016-04-06

    Membrane proteins are embedded in the biological membrane where the chemically diverse lipid environment can modulate their structure and function. However, the thermodynamics governing the molecular recognition and interaction of lipids with membrane proteins is poorly understood. Here, we report a method using native mass spectrometry (MS), to determine thermodynamics of individual ligand binding events to proteins. Unlike conventional methods, native MS can resolve individual ligand binding events and, coupled with an apparatus to control the temperature, determine binding thermodynamic parameters, such as for protein-lipid interactions. We validated our approach using three soluble protein-ligand systems (maltose binding protein, lysozyme, and nitrogen regulatory protein) and obtained similar results to those using isothermal titration calorimetry and surface plasmon resonance. We also determined for the first time the thermodynamics of individual lipid binding to the ammonia channel (AmtB), an integral membrane protein from Escherichia coli. Remarkably, we observed distinct thermodynamic signatures for the binding of different lipids and entropy-enthalpy compensation for binding lipids of variable chain length. Additionally, using a mutant form of AmtB that abolishes a specific phosphatidylglycerol (PG) binding site, we observed distinct changes in the thermodynamic signatures for binding PG, implying these signatures can identify key residues involved in specific lipid binding and potentially differentiate between specific lipid binding sites.

  9. Recognition determinants for proteins and antibiotics within 23S rRNA

    DEFF Research Database (Denmark)

    Douthwaite, Stephen Roger; Voldborg, Bjørn Gunnar Rude; Hansen, Lykke Haastrup

    1995-01-01

    Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination of molecu......Ribosomal RNAs fold into phylogenetically conserved secondary and tertiary structures that determine their function in protein synthesis. We have investigated Escherichia coli 23S rRNA to identify structural elements that interact with antibiotic and protein ligands. Using a combination......-proteins L10.(L12)4 and L11 and is inhibited by interaction with the antibiotic thiostrepton. The peptidyltransferase center within domain V is inhibited by macrolide, lincosamide, and streptogramin B antibiotics, which interact with the rRNA around nucleotide A2058. Drug resistance is conferred by mutations...

  10. Blind testing of routine, fully automated determination of protein structures from NMR data.

    Science.gov (United States)

    Rosato, Antonio; Aramini, James M; Arrowsmith, Cheryl; Bagaria, Anurag; Baker, David; Cavalli, Andrea; Doreleijers, Jurgen F; Eletsky, Alexander; Giachetti, Andrea; Guerry, Paul; Gutmanas, Aleksandras; Güntert, Peter; He, Yunfen; Herrmann, Torsten; Huang, Yuanpeng J; Jaravine, Victor; Jonker, Hendrik R A; Kennedy, Michael A; Lange, Oliver F; Liu, Gaohua; Malliavin, Thérèse E; Mani, Rajeswari; Mao, Binchen; Montelione, Gaetano T; Nilges, Michael; Rossi, Paolo; van der Schot, Gijs; Schwalbe, Harald; Szyperski, Thomas A; Vendruscolo, Michele; Vernon, Robert; Vranken, Wim F; Vries, Sjoerd de; Vuister, Geerten W; Wu, Bin; Yang, Yunhuang; Bonvin, Alexandre M J J

    2012-02-08

    The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by using a specific computer program. To assess whether it is indeed possible to generate in a fully automated manner NMR structures adequate for deposition in the Protein Data Bank, we gathered 10 experimental data sets with unassigned nuclear Overhauser effect spectroscopy (NOESY) peak lists for various proteins of unknown structure, computed structures for each of them using different, fully automatic programs, and compared the results to each other and to the manually solved reference structures that were not available at the time the data were provided. This constitutes a stringent "blind" assessment similar to the CASP and CAPRI initiatives. This study demonstrates the feasibility of routine, fully automated protein structure determination by NMR.

  11. Determining the Sub-Cellular Localization of Proteins within Caenorhabditis elegans Body Wall Muscle

    Science.gov (United States)

    Viveiros, Ryan; Warner, Adam; Plastino, Lorena; Lorch, Adam; Granger, Laure; Segalat, Laurent; Moerman, Donald G.

    2011-01-01

    Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive “localizome” for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function. PMID:21611156

  12. Solitons and Collapse in the lambda-repressor protein

    CERN Document Server

    Krokhotin, Andrey; Niemi, Antti J

    2012-01-01

    The enterobacteria lambda phage is a paradigm temperate bacteriophage. Its lysogenic and lytic life cycles echo competition between the DNA binding $\\lambda$-repressor (CI) and CRO proteins. Here we scrutinize the structure, stability and folding pathways of the $\\lambda$-repressor protein, that controls the transition from the lysogenic to the lytic state. We first investigate the super-secondary helix-loop-helix composition of its backbone. We use a discrete Frenet framing to resolve the backbone spectrum in terms of bond and torsion angles. Instead of four, there appears to be seven individual loops. We model the putative loops using an explicit soliton Ansatz. It is based on the standard soliton profile of the continuum nonlinear Schr\\"odinger equation. The accuracy of the Ansatz far exceeds the B-factor fluctuation distance accuracy of the experimentally determined protein configuration. We then investigate the folding pathways and dynamics of the $\\lambda$-repressor protein. We introduce a coarse-graine...

  13. The Nanomechanical Properties of Lactococcus lactis Pili Are Conditioned by the Polymerized Backbone Pilin.

    Directory of Open Access Journals (Sweden)

    Mickaël Castelain

    Full Text Available Pili produced by Lactococcus lactis subsp. lactis are putative linear structures consisting of repetitive subunits of the major pilin PilB that forms the backbone, pilin PilA situated at the distal end of the pilus, and an anchoring pilin PilC that tethers the pilus to the peptidoglycan. We determined the nanomechanical properties of pili using optical-tweezers force spectroscopy. Single pili were exposed to optical forces that yielded force-versus-extension spectra fitted using the Worm-Like Chain model. Native pili subjected to a force of 0-200 pN exhibit an inextensible, but highly flexible ultrastructure, reflected by their short persistence length. We tested a panel of derived strains to understand the functional role of the different pilins. First, we found that both the major pilin PilB and sortase C organize the backbone into a full-length organelle and dictate the nanomechanical properties of the pili. Second, we found that both PilA tip pilin and PilC anchoring pilin were not essential for the nanomechanical properties of pili. However, PilC maintains the pilus on the bacterial surface and may play a crucial role in the adhesion- and biofilm-forming properties of L. lactis.

  14. Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings.

    Science.gov (United States)

    Alexander, Crispin G; Wanner, Randy; Johnson, Christopher M; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

    2014-12-01

    Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein-ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide-PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes.

  15. Sequential backbone assignment based on dipolar amide-to-amide correlation experiments.

    Science.gov (United States)

    Xiang, ShengQi; Grohe, Kristof; Rovó, Petra; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan; Linser, Rasmus

    2015-07-01

    Proton detection in solid-state NMR has seen a tremendous increase in popularity in the last years. New experimental techniques allow to exploit protons as an additional source of information on structure, dynamics, and protein interactions with their surroundings. In addition, sensitivity is mostly improved and ambiguity in assignment experiments reduced. We show here that, in the solid state, sequential amide-to-amide correlations turn out to be an excellent, complementary way to exploit amide shifts for unambiguous backbone assignment. For a general assessment, we compare amide-to-amide experiments with the more common (13)C-shift-based methods. Exploiting efficient CP magnetization transfers rather than less efficient INEPT periods, our results suggest that the approach is very feasible for solid-state NMR.

  16. Sequential backbone assignment based on dipolar amide-to-amide correlation experiments

    Energy Technology Data Exchange (ETDEWEB)

    Xiang, ShengQi; Grohe, Kristof; Rovó, Petra; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan; Linser, Rasmus, E-mail: rali@nmr.mpibpc.mpg.de [Max Planck Institute for Biophysical Chemistry, Department for NMR-Based Structural Biology (Germany)

    2015-07-15

    Proton detection in solid-state NMR has seen a tremendous increase in popularity in the last years. New experimental techniques allow to exploit protons as an additional source of information on structure, dynamics, and protein interactions with their surroundings. In addition, sensitivity is mostly improved and ambiguity in assignment experiments reduced. We show here that, in the solid state, sequential amide-to-amide correlations turn out to be an excellent, complementary way to exploit amide shifts for unambiguous backbone assignment. For a general assessment, we compare amide-to-amide experiments with the more common {sup 13}C-shift-based methods. Exploiting efficient CP magnetization transfers rather than less efficient INEPT periods, our results suggest that the approach is very feasible for solid-state NMR.

  17. Side chain and backbone contributions of Phe508 to CFTR folding

    Energy Technology Data Exchange (ETDEWEB)

    Thibodeau, Patrick H.; Brautigam, Chad A.; Machius, Mischa; Thomas, Philip J. (U. of Texas-SMED)

    2010-12-07

    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein, cause cystic fibrosis (CF). The most common CF-causing mutant, deletion of Phe508, fails to properly fold. To elucidate the role Phe508 plays in the folding of CFTR, missense mutations at this position were generated. Only one missense mutation had a pronounced effect on the stability and folding of the isolated domain in vitro. In contrast, many substitutions, including those of charged and bulky residues, disrupted folding of full-length CFTR in cells. Structures of two mutant nucleotide-binding domains (NBDs) reveal only local alterations of the surface near position 508. These results suggest that the peptide backbone plays a role in the proper folding of the domain, whereas the side chain plays a role in defining a surface of NBD1 that potentially interacts with other domains during the maturation of intact CFTR.

  18. Review of methods for determination of total protein and peptide concentration in biological samples.

    Science.gov (United States)

    Sapan, Christine V; Lundblad, Roger L

    2015-04-01

    Clinical proteomics can be defined as the use of proteomic technologies to identify and measure biomarkers in fluids and tissues. The current work is intended to review various methods used for the determination of the total concentration of protein or peptide in fluids and tissues and the application of such methods to clinical proteomics. Specifically, this article considers the approaches to the measurement of total protein concentration, not the measurement of the concentration of a specific protein or group of proteins in a larger mixture of proteins. The necessity of understanding various concepts such as fit-for-use, quality-by-design, and other regulatory elements is discussed, as is the significance of using suitable standards for the protein quality of various samples.

  19. Blind testing of routine, fully automated determination of protein structures from NMR data.

    NARCIS (Netherlands)

    Rosato, A.; Aramini, J.M.; Arrowsmith, C.; Bagaria, A.; Baker, D.; Cavalli, A.; Doreleijers, J.; Eletsky, A.; Giachetti, A.; Guerry, P.; Gutmanas, A.; Guntert, P.; He, Y.; Herrmann, T.; Huang, Y.J.; Jaravine, V.; Jonker, H.R.; Kennedy, M.A.; Lange, O.F.; Liu, G.; Malliavin, T.E.; Mani, R.; Mao, B.; Montelione, G.T.; Nilges, M.; Rossi, P.; Schot, G. van der; Schwalbe, H.; Szyperski, T.A.; Vendruscolo, M.; Vernon, R.; Vranken, W.F.; Vries, S.D. de; Vuister, G.W.; Wu, B.; Yang, Y.; Bonvin, A.M.

    2012-01-01

    The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by us

  20. Dispersion of Nanoparticles in Different Media Importantly Determines the Composition of Their Protein Corona

    Science.gov (United States)

    Strojan, Klemen; Leonardi, Adrijana; Bregar, Vladimir B.; Križaj, Igor; Svete, Jurij; Pavlin, Mojca

    2017-01-01

    Protein corona of nanoparticles (NPs), which forms when these particles come in to contact with protein-containing fluids, is considered as an overlooked factor in nanomedicine. Through numerous studies it has been becoming increasingly evident that it importantly dictates the interaction of NPs with their surroundings. Several factors that determine the compositions of NPs protein corona have been identified in recent years, but one has remained largely ignored—the composition of media used for dispersion of NPs. Here, we determined the effect of dispersion media on the composition of protein corona of polyacrylic acid-coated cobalt ferrite NPs (PAA NPs) and silica NPs. Our results confirmed some of the basic premises such as NPs type-dependent specificity of the protein corona. But more importantly, we demonstrated the effect of the dispersion media on the protein corona composition. The differences between constituents of the media used for dispersion of NPs, such as divalent ions and macromolecules were responsible for the differences in protein corona composition formed in the presence of fetal bovine serum (FBS). Our results suggest that the protein corona composition is a complex function of the constituents present in the media used for dispersion of NPs. Regardless of the dispersion media and FBS concentration, majority of proteins from either PAA NPs or silica NPs coronas were involved in the process of transport and hemostasis. Interestingly, corona of silica NPs contained three complement system related proteins: complement factor H, complement C3 and complement C4 while PAA NPs bound only one immune system related protein, α-2-glycoprotein. Importantly, relative abundance of complement C3 protein in corona of silica NPs was increased when NPs were dispersed in NaCl, which further implies the relevance of dispersion media used to prepare NPs. PMID:28052135

  1. Importance of native-state topology for determining the folding rate of two-state proteins.

    Science.gov (United States)

    Gromiha, M Michael

    2003-01-01

    Understanding the relationship between amino acid sequences and folding rate of proteins is a challenging task similar to protein folding problem. In this work, we have analyzed the relative importance of protein sequence and structure for predicting the protein folding rates in terms of amino acid properties and contact distances, respectively. We found that the parameters derived with protein sequence (physical-chemical, energetic, and conformational properties of amino acid residues) show very weak correlation (|r| proteins, indicating that the sequence information alone is not sufficient to understand the folding rates of two-state proteins. However, the maximum positive correlation obtained for the properties, number of medium-range contacts, and alpha-helical tendency reveals the importance of local interactions to initiate protein folding. On the other hand, a remarkable correlation (r varies from -0.74 to -0.88) has been obtained between structural parameters (contact order, long-range order, and total contact distance) and protein folding rates. Further, we found that the secondary structure content and solvent accessibility play a marginal role in determining the folding rates of two-state proteins. Multiple regression analysis carried out with the combination of three properties, beta-strand tendency, enthalpy change, and total contact distance improved the correlation to 0.92 with protein folding rates. The relative importance of existing methods along with multiple-regression model proposed in this work will be discussed. Our results demonstrate that the native-state topology is the major determinant for the folding rates of two-state proteins.

  2. Dispersion of Nanoparticles in Different Media Importantly Determines the Composition of Their Protein Corona.

    Science.gov (United States)

    Strojan, Klemen; Leonardi, Adrijana; Bregar, Vladimir B; Križaj, Igor; Svete, Jurij; Pavlin, Mojca

    2017-01-01

    Protein corona of nanoparticles (NPs), which forms when these particles come in to contact with protein-containing fluids, is considered as an overlooked factor in nanomedicine. Through numerous studies it has been becoming increasingly evident that it importantly dictates the interaction of NPs with their surroundings. Several factors that determine the compositions of NPs protein corona have been identified in recent years, but one has remained largely ignored-the composition of media used for dispersion of NPs. Here, we determined the effect of dispersion media on the composition of protein corona of polyacrylic acid-coated cobalt ferrite NPs (PAA NPs) and silica NPs. Our results confirmed some of the basic premises such as NPs type-dependent specificity of the protein corona. But more importantly, we demonstrated the effect of the dispersion media on the protein corona composition. The differences between constituents of the media used for dispersion of NPs, such as divalent ions and macromolecules were responsible for the differences in protein corona composition formed in the presence of fetal bovine serum (FBS). Our results suggest that the protein corona composition is a complex function of the constituents present in the media used for dispersion of NPs. Regardless of the dispersion media and FBS concentration, majority of proteins from either PAA NPs or silica NPs coronas were involved in the process of transport and hemostasis. Interestingly, corona of silica NPs contained three complement system related proteins: complement factor H, complement C3 and complement C4 while PAA NPs bound only one immune system related protein, α-2-glycoprotein. Importantly, relative abundance of complement C3 protein in corona of silica NPs was increased when NPs were dispersed in NaCl, which further implies the relevance of dispersion media used to prepare NPs.

  3. Predicting protein dynamics from structural ensembles

    CERN Document Server

    Copperman, J

    2015-01-01

    The biological properties of proteins are uniquely determined by their structure and dynamics. A protein in solution populates a structural ensemble of metastable configurations around the global fold. From overall rotation to local fluctuations, the dynamics of proteins can cover several orders of magnitude in time scales. We propose a simulation-free coarse-grained approach which utilizes knowledge of the important metastable folded states of the protein to predict the protein dynamics. This approach is based upon the Langevin Equation for Protein Dynamics (LE4PD), a Langevin formalism in the coordinates of the protein backbone. The linear modes of this Langevin formalism organize the fluctuations of the protein, so that more extended dynamical cooperativity relates to increasing energy barriers to mode diffusion. The accuracy of the LE4PD is verified by analyzing the predicted dynamics across a set of seven different proteins for which both relaxation data and NMR solution structures are available. Using e...

  4. Determination of protein rotational correlation time from NMR relaxation data at various solvent viscosities

    Energy Technology Data Exchange (ETDEWEB)

    Korchuganov, Denis S.; Gagnidze, Ivan E.; Tkach, Elena N.; Schulga, Alexey A.; Kirpichnikov, Mikhail P.; Arseniev, Alexander S. [Russian Academy of Sciences, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry (Russian Federation)], E-mail: aars@nmr.ru

    2004-12-15

    An accurate determination of the overall rotation of a protein plays a crucial role in the investigation of its internal motions by NMR. In the present work, an innovative approach to the determination of the protein rotational correlation time {tau}{sub R} from the heteronuclear relaxation data is proposed. The approach is based on a joint fit of relaxation data acquired at several viscosities of a protein solution. The method has been tested on computer simulated relaxation data as compared to the traditional {tau}{sub R} determination method from T{sub 1}/T{sub 2} ratio. The approach has been applied to ribonuclease barnase from Bacillus amyloliquefaciens dissolved in an aqueous solution and deuterated glycerol as a viscous component. The resulting rotational correlation time of 5.56 {+-} 0.01 ns and other rotational diffusion tensor parameters are in good agreement with those determined from T{sub 1}/T{sub 2} ratio.

  5. Heat stability of proteins in desiccation tolerant cattail pollen (Typha latifolia): A Fourier transform infrared spectroscopic study.

    NARCIS (Netherlands)

    Wolkers, W.F.; Hoekstra, F.A.

    1997-01-01

    Secondary structure and aggregation behavior of proteins, as determined in situ in Typha latifolia pollen, were studied by means of Fourier transform infrared microspectroscopy. The amide-I band, arising from the peptide backbone, was recorded over a temperature range from -50 to 120°C at different

  6. Plasma Membrane Protein Ubiquitylation and Degradation as Determinants of Positional Growth in Plants

    Institute of Scientific and Technical Information of China (English)

    Barbara Korbei; Christian Luschnig

    2013-01-01

    Being sessile organisms, plants evolved an unparalleled plasticity in their post-embryonic development, allowing them to adapt and fine-tune their vital parameters to an ever-changing environment. Cross-talk between plants and their environment requires tight regulation of information exchange at the plasma membrane (PM). Plasma membrane proteins mediate such communication, by sensing variations in nutrient availability, external cues as well as by controlled solute transport across the membrane border. Localiza-tion and steady-state levels are essential for PM protein function and ongoing research identified cis- and trans-acting determinants, involved in control of plant PM protein localization and turnover. In this overview, we summarize recent progress in our understanding of plant PM protein sorting and degradation via ubiquitylation, a post-translational and reversible modification of proteins. We highlight characterized components of the machinery involved in sorting of ubiquitylated PM proteins and discuss consequences of protein ubiquitylation on fate of selected PM proteins. Specifically, we focus on the role of ubiquitylation and PM protein degradation in the regulation of polar auxin transport (PAT). We combine this regulatory circuit with further aspects of PM protein sorting control, to address the interplay of events that might control PAT and polarized growth in higher plants.

  7. Marburg virus VP35 can both fully coat the backbone and cap the ends of dsRNA for interferon antagonism.

    Directory of Open Access Journals (Sweden)

    Shridhar Bale

    2012-09-01

    Full Text Available Filoviruses, including Marburg virus (MARV and Ebola virus (EBOV, cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (dsRNA-binding domain (RBD of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.

  8. Studies on BN rats model to determine the potential allergenicity of proteins from genetically modified foods

    Institute of Scientific and Technical Information of China (English)

    Xu-Dong Jia; Ning Li; Yong-Ning Wu; Xiao-Guang Yang

    2005-01-01

    AIM: To develop a Brown Norway (BN) rat model to determine the potential allergenicity of novel proteins in genetically modified food.METHODS: The allergenicity of different proteins were compared, including ovalbumin (OVA), a potent respiratory and food allergen, bovine serum albumin (BSA), a protein that is considered to have a lesser allergenic potential,and potato acid phosphatase (PAP), a non-allergenic protein when administered to BN rats via different routes of exposure (intraperitoneally or by gavage). IgG and IgE antibody responses were determined by ELISA and PCA,respectively. An immunoassay kit was used to determine the plasma histamine level. In addition, possible systemic effect of allergens was investigated by monitoring blood pressure.RESULTS: OVA provoked very vigorous protein-specific IgG and IgE responses, low grade protein-specific IgG and IgE responses were elicited by BSA, while by neither route did PAP elicit anything. In either routes of exposure,plasma histamine level in BN rats sensitized with OVA was higher than that of BSA or PAP. In addition, an oral challenge with BSA and PAP did not induce any effect on blood pressure, while a temporary drop in systolic blood pressure in few animals of each routes of exposure was found by an oral challenge with OVA.CONCLUSION: BN rat model might be a useful and predictive animal model to study the potential allergenicity of novel food proteins.

  9. Thermodynamic contribution of backbone conformational entropy in the binding between SH3 domain and proline-rich motif.

    Science.gov (United States)

    Zeng, Danyun; Shen, Qingliang; Cho, Jae-Hyun

    2017-02-26

    Biological functions of intrinsically disordered proteins (IDPs), and proteins containing intrinsically disordered regions (IDRs) are often mediated by short linear motifs, like proline-rich motifs (PRMs). Upon binding to their target proteins, IDPs undergo a disorder-to-order transition which is accompanied by a large conformational entropy penalty. Hence, the molecular mechanisms underlying control of conformational entropy are critical for understanding the binding affinity and selectivity of IDPs-mediated protein-protein interactions (PPIs). Here, we investigated the backbone conformational entropy change accompanied by binding of the N-terminal SH3 domain (nSH3) of CrkII and PRM derived from guanine nucleotide exchange factor 1 (C3G). In particular, we focused on the estimation of conformational entropy change of disordered PRM upon binding to the nSH3 domain. Quantitative characterization of conformational dynamics of disordered peptides like PRMs is limited. Hence, we combined various methods, including NMR model-free analysis, δ2D, DynaMine, and structure-based calculation of entropy loss. This study demonstrates that the contribution of backbone conformational entropy change is significant in the PPIs mediated by IDPs/IDRs.

  10. Present and future of membrane protein structure determination by electron crystallography.

    Science.gov (United States)

    Ubarretxena-Belandia, Iban; Stokes, David L

    2010-01-01

    Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins.

  11. [Determination of protein and gossypol content in cotton kernel powder with near infrared reflectance spectroscopy].

    Science.gov (United States)

    Qin, Li; Shen, Xiao-Jia; Chen, Jin-Hong; Zhu, Shui-Jin

    2010-03-01

    Near-infrared reflectance spectroscopy (NIRS) was used as a rapid and nondestructive method to determine the protein content and gossypol content in cotton kernel powder samples, using 49 upland cotton (Gossypium hirsutum L.) germplasms and 188 recombinant inbred lines (RILs). The cottonseed samples harvested from the upland cotton germplasms and RILs grown in different cotton growing regions in different years were analyzed chemically for protein and gossypol contents, as well as scanned in the reflectance mode of a scanning monochromator. Using ISI software for scanning and data analysis, protein and gossypol calibration equations were obtained with a standard normal variate + detrending scatter correction and a 2, 4, 4, 1 math treatment and modified partial least square (MPLS) as the regression method. The protein content calibration results revealed that the multiple correlation coefficients (RSQ) and statistic 1--variance ratio (1-VR) for the determination of protein content in cottonseed kernels were 0.933 and 0.929, respectively, and its standard error of calibration (SEC) and standard error of cross validation (SECV) were 0.623 and 0.638, respectively. As the calibration equations were judged by the calibration RSQ (or 1-VR) and SEC (or SECV), the results indicated that NIRS is comparable to chemical methods in both accuracy and prediction and is reliable in the determination of protein content in cottonseed kernels. However, the RSQ, SEC, 1-VR and SECV for gossypol content determination of NIRS were 0.836, 0.811, 0.074 and 0.079, respectively. Although it was weaker than that of protein content, the NIRS method is still good enough for the determination and prediction of the gossypol content in cottonseed kernels. Therefore, NIRS models were successfully developed for protein content and gossypol content analysis of cotton kernel powder sample in the present study and they could be introduced into the cotton germplasm evaluation and breeding program for

  12. Acrylamide concentration determines the direction and magnitude of helical membrane protein gel shifts

    Science.gov (United States)

    Rath, Arianna; Cunningham, Fiona; Deber, Charles M.

    2013-01-01

    SDS/PAGE is universally used in biochemistry, cell biology, and immunology to resolve minute protein amounts readily from tissue and cell extracts. Although molecular weights of water-soluble proteins are reliably determined from their SDS/PAGE mobility, most helical membrane proteins, which comprise 20–30% of the human genome and the majority of drug targets, migrate to positions that have for decades been unpredictably slower or faster than their actual formula weight, often confounding their identification. Using de novo designed transmembrane-mimetic polypeptides that match the composition of helical membrane-spanning sequences, we quantitate anomalous SDS/PAGE fractionation of helical membrane proteins by comparing the relative mobilities of these polypeptides with typical water-soluble reference proteins on Laemmli gels. We find that both the net charge and effective molecular size of the migrating particles of transmembrane-mimetic species exceed those of the corresponding reference proteins and that gel acrylamide concentration dictates the impact of these two factors on the direction and magnitude of anomalous migration. Algorithms we derived from these data compensate for this differential effect of acrylamide concentration on the SDS/PAGE mobility of a variety of natural membrane proteins. Our results provide a unique means to predict anomalous migration of membrane proteins, thereby facilitating straightforward determination of their molecular weights via SDS/PAGE. PMID:24019476

  13. Immunochemical analytical methods for the determination of peanut proteins in foods.

    Science.gov (United States)

    Whitaker, Thomas B; Williams, Kristina M; Trucksess, Mary W; Slate, Andrew B

    2005-01-01

    Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as "Bio-Kit" for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 microg whole peanut per g food; the spiked levels were 0.0, 5, 10, and 20 microg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred

  14. Phase Transitions and Backbones of the Asymmetric Traveling Salesman Problem

    CERN Document Server

    Zhang, W

    2011-01-01

    In recent years, there has been much interest in phase transitions of combinatorial problems. Phase transitions have been successfully used to analyze combinatorial optimization problems, characterize their typical-case features and locate the hardest problem instances. In this paper, we study phase transitions of the asymmetric Traveling Salesman Problem (ATSP), an NP-hard combinatorial optimization problem that has many real-world applications. Using random instances of up to 1,500 cities in which intercity distances are uniformly distributed, we empirically show that many properties of the problem, including the optimal tour cost and backbone size, experience sharp transitions as the precision of intercity distances increases across a critical value. Our experimental results on the costs of the ATSP tours and assignment problem agree with the theoretical result that the asymptotic cost of assignment problem is pi ^2 /6 the number of cities goes to infinity. In addition, we show that the average computation...

  15. Transforming plastic surfaces with electrophilic backbones from hydrophobic to hydrophilic.

    Science.gov (United States)

    Kim, Samuel; Bowen, Raffick A R; Zare, Richard N

    2015-01-28

    We demonstrate a simple nonaqueous reaction scheme for transforming the surface of plastics from hydrophobic to hydrophilic. The chemical modification is achieved by base-catalyzed trans-esterification with polyols. It is permanent, does not release contaminants, and causes no optical or mechanical distortion of the plastic. We present contact angle measurements to show successful modification of several types of plastics including poly(ethylene terephthalate) (PET) and polycarbonate (PC). Its applicability to blood analysis is explored using chemically modified PET blood collection tubes and found to be quite satisfactory. We expect this approach will reduce the cost of manufacturing plastic devices with optimized wettability and can be generalized to other types of plastic materials having an electrophilic linkage as its backbone.

  16. Design of an IPTV Multicast System for Internet Backbone Networks

    Directory of Open Access Journals (Sweden)

    T. H. Szymanski

    2010-01-01

    Full Text Available The design of an IPTV multicast system for the Internet backbone network is presented and explored through extensive simulations. In the proposed system, a resource reservation algorithm such as RSVP, IntServ, or DiffServ is used to reserve resources (i.e., bandwidth and buffer space in each router in an IP multicast tree. Each router uses an Input-Queued, Output-Queued, or Crosspoint-Queued switch architecture with unity speedup. A recently proposed Recursive Fair Stochastic Matrix Decomposition algorithm used to compute near-perfect transmission schedules for each IP router. The IPTV traffic is shaped at the sources using Application-Specific Token Bucker Traffic Shapers, to limit the burstiness of incoming network traffic. The IPTV traffic is shaped at the destinations using Application-Specific Playback Queues, to remove residual network jitter and reconstruct the original bursty IPTV video streams at each destination. All IPTV traffic flows are regenerated at the destinations with essentially zero delay jitter and essentially-perfect QoS. The destination nodes deliver the IPTV streams to the ultimate end users using the same IPTV multicast system over a regional Metropolitan Area Network. It is shown that all IPTV traffic is delivered with essentially-perfect end-to-end QoS, with deterministic bounds on the maximum delay and jitter on each video frame. Detailed simulations of an IPTV distribution system, multicasting several hundred high-definition IPTV video streams over several essentially saturated IP backbone networks are presented.

  17. NIRS and MIRS technique for the determination of protein and fat content in milk powder

    Science.gov (United States)

    Wu, Di; Feng, Shuijuan; He, Chao; He, Yong

    2008-03-01

    It is very important to detect the protein and fat content in milk powder fast and non-destructively. Near-infrared (NIR) and mid-infrared(MIR) spectroscopy techniques have been compared and evaluated for the determination of the protein and fat content in milk powder with the use of Least-squares support vector machines (LS-SVM). LS-SVM models have been developed by using both NIR and MIR spectra. Both methods have shown good correlations between infrared transmission values and two nutrition contents. MIRS provided better prediction performance over NIRS. It is concluded that infrared spectroscopy technique can quantify of the protein and fat content in milk powder fast and nondestructively. The process is simple and easy to operate than chemistry methods. The results can be beneficial for designing a simple and non-destructive instrument with MIRS or NIRS spectral sensor for the determination of the protein fat content in milk powder.

  18. New insights into structural determinants of prion protein folding and stability.

    Science.gov (United States)

    Benetti, Federico; Legname, Giuseppe

    2015-01-01

    Prions are the etiological agent of fatal neurodegenerative diseases called prion diseases or transmissible spongiform encephalopathies. These maladies can be sporadic, genetic or infectious disorders. Prions are due to post-translational modifications of the cellular prion protein leading to the formation of a β-sheet enriched conformer with altered biochemical properties. The molecular events causing prion formation in sporadic prion diseases are still elusive. Recently, we published a research elucidating the contribution of major structural determinants and environmental factors in prion protein folding and stability. Our study highlighted the crucial role of octarepeats in stabilizing prion protein; the presence of a highly enthalpically stable intermediate state in prion-susceptible species; and the role of disulfide bridge in preserving native fold thus avoiding the misfolding to a β-sheet enriched isoform. Taking advantage from these findings, in this work we present new insights into structural determinants of prion protein folding and stability.

  19. Determination of adsorbed protein concentration in aluminum hydroxide suspensions by near-infrared transmittance Spectroscopy

    DEFF Research Database (Denmark)

    Lai, Xuxin; Zheng, Yiwu; Jacobsen, Susanne

    2008-01-01

    Analysis of aluminum hydroxide based vaccines is difficult after antigen adsorption. Adsorbed protein is often assessed by measuring residual unadsorbed protein for quality control. A new method for the direct determination of adsorbed protein concentration in suspension using near-infrared (NIR......) transmittance spectroscopy is proposed here. A simple adsorption system using albumin from bovine serum (BSA) and aluminum hydroxide as a model system is employed. The results show that the NIR absorbance at 700-1300 nm is correlated to the adsorbed BSA concentration, measured by the ultraviolet (UV) method...... reduce the batch influence, and the NIR predicted results show good agreement with the reference values. The NIR method might be the only direct method for the determination of adsorbed protein concentration in suspension so far. It is a nondestructive method, and it has great advantage for use...

  20. Protein profile of exhaled breath condensate determined by high resolution mass spectrometry.

    Science.gov (United States)

    Muccilli, Vera; Saletti, Rosaria; Cunsolo, Vincenzo; Ho, Jenny; Gili, Elisa; Conte, Enrico; Sichili, Stefania; Vancheri, Carlo; Foti, Salvatore

    2015-02-01

    A method based on liquid chromatography/high resolution tandem mass spectrometry coupled with electrophoretic separation, for determination and relative quantification of the protein composition of exhaled breath condensate (EBC), was developed. Application of the procedure to a sample of EBC, pooled from nine healthy subjects, resulted in the identification of 167 unique gene products, 113 of which not previously reported in EBC samples. The abundance of the protein identified was estimated by means of the exponentially modified protein abundance index protocol (emPAI). Cytokeratins were by far the most abundant proteins in EBC samples. Many of the identified proteins were associated with multiple cellular location with cytoplasm constituting the largest group. Cytosol, nucleus, membrane, cytoskeleton and extracellular were other abundantly represented locations. No amylase was detected, suggesting the absence of saliva protein contamination. The profile obtained represents the most comprehensive protein characterization of EBC so far reported and demonstrates that this approach provides a powerful tool for investigating the protein profile of EBC samples. Compared with analogous investigations, this study also shows that the protein profile of EBC is strongly affected by the sampling method adopted.

  1. Novel DNPH-based method for determination of protein carbonylation in muscle and meat.

    Science.gov (United States)

    Soglia, Francesca; Petracci, Massimiliano; Ertbjerg, Per

    2016-04-15

    Protein oxidation is considered an ongoing deteriorative process during storage of fresh and processed meat. Carbonyl compounds have traditionally been detected spectrophotometrically after derivatization with 2,4-dinitrophenylhydrazine (DNPH) to form protein-bound hydrazones with absorbance at 370 nm. Here we describe a novel DNPH-based method to quantify protein carbonylation in muscle and meat. The additional steps of the novel method aimed at increasing the protein solubility and inducing protein unfolding before labeling with DNPH. Compared to the traditional method, the new procedure reflected an increased protein carbonylation level measuring overall two to fourfold more carbonyls in muscles from different species as well as in soluble, salt-soluble and insoluble protein fractions. The study suggested that protein unfolding is a more important phenomenon than solubilization for increased DNPH labeling. The novel method resulted in three to fourfold larger carbonyl content determined in chicken, pork and beef (2.8, 3.6 and 3.1 nmol/mg of protein, respectively). Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. A convenient method for rapid determination of proteins in rices and rice products

    OpenAIRE

    光永, 俊郎; 安藤, ひとみ

    1990-01-01

    [Author abstract]In the biuret determination of protein in rices and rice products, starch and lipids extractable with an alkaline solution such as biuret reagent were found to interfere with the biuret method. Comparative evaluation of their effects revealed that starch, a major component of rice, has the most significant effect on the biuret method. In the presence of starch, the correlation between Kjeldahl protein and the optical density of biuret was poor. It was found that several perha...

  3. Determination of protein-ligand interactions using accelerator mass spectrometry: modified crosslinking assay.

    Science.gov (United States)

    Hah, Sang Soo

    2009-05-01

    A highly sensitive detection method for the determination of protein-ligand interactions has been developed. Radiocarbon-labeled 17beta-estradiol was incubated with estrogen receptor-alpha; as a selective binding partner, and covalently attached using crosslinking agents, to form covalently linked protein-ligand complexes. After separation using a denaturing gel, the (14)C content in the sliced gels was identified by accelerator mass spectrometry. The obtained data demonstrated specific binding of the small molecule to its binding partner. In theory, this method can be applied to most protein-ligand interaction studies.

  4. Determination of functional collective motions in a protein at atomic resolution using coherent neutron scattering

    Science.gov (United States)

    Hong, Liang; Jain, Nitin; Cheng, Xiaolin; Bernal, Ana; Tyagi, Madhusudan; Smith, Jeremy C.

    2016-01-01

    Protein function often depends on global, collective internal motions. However, the simultaneous quantitative experimental determination of the forms, amplitudes, and time scales of these motions has remained elusive. We demonstrate that a complete description of these large-scale dynamic modes can be obtained using coherent neutron-scattering experiments on perdeuterated samples. With this approach, a microscopic relationship between the structure, dynamics, and function in a protein, cytochrome P450cam, is established. The approach developed here should be of general applicability to protein systems. PMID:27757419

  5. A robust method of determination of high concentrations of peptides and proteins

    DEFF Research Database (Denmark)

    Levashov, Pavel A; Sutherland, Duncan S; Besenbacher, Flemming

    2009-01-01

    In this paper, we pioneer application of a unique method of protein determination by coloring peptide bonds for analysis of a variety of biomolecules with different grades of purity (e.g., oligopeptides, membrane, and glycol proteins). We demonstrated that the calibration curve for all studied...... molecules is universal and linear within 0.1 to 1.2mg protein content range. The assay thus can be used to analyze peptides without preliminary dilutions and calibration in up to 1g/ml solutions of peptides, which is crucial for many biotechnological processes, such as development of coatings, scaffolds...

  6. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    Science.gov (United States)

    Xie, Jianming; Wang, Lei; Wu, Ning; Schultz, Peter G.

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  7. A hierarchical virtual backbone construction protocol for mobile ad hoc networks

    Directory of Open Access Journals (Sweden)

    Bharti Sharma

    2016-07-01

    Full Text Available We propose a hierarchical backbone construction protocol for mobile ad hoc networks. Our protocol is based on the idea of using an efficient extrema finding method to create clusters comprising the nodes that are within certain prespecified wireless hop distance. Afterward, we apply our ‘diameter’ algorithm among clusters to identify the dominating nodes that are, finally, connected via multi-hop virtual links to construct the backbone. We present the analytic as well as simulation study of our algorithm and also a method for the dynamic maintenance of constructed backbone. In the end, we illustrate the use of the virtual backbone with the help of an interesting application.

  8. Thin Films Formed from Conjugated Polymers with Ionic, Water-Soluble Backbones.

    Science.gov (United States)

    Voortman, Thomas P; Chiechi, Ryan C

    2015-12-30

    This paper compares the morphologies of films of conjugated polymers in which the backbone (main chain) and pendant groups are varied between ionic/hydrophilic and aliphatic/hydrophobic. We observe that conjugated polymers in which the pendant groups and backbone are matched, either ionic-ionic or hydrophobic-hydrophobic, form smooth, structured, homogeneous films from water (ionic) or tetrahydrofuran (hydrophobic). Mismatched conjugated polymers, by contrast, form inhomogeneous films with rough topologies. The polymers with ionic backbone chains are conjugated polyions (conjugated polymers with closed-shell charges in the backbone), which are semiconducting materials with tunable bad-gaps, not unlike uncharged conjugated polymers.

  9. Protein-nanoparticle interactions evaluation by immunomethods: Surfactants can disturb quantitative determinations.

    Science.gov (United States)

    Fornaguera, Cristina; Calderó, Gabriela; Solans, Conxita; Vauthier, Christine

    2015-08-01

    The adsorption of proteins on nanoparticle surface is one of the first events that occur when nanoparticles enter in the blood stream, which influences nanoparticles lifetime and further biodistribution. Albumin, which is the most abundant protein in serum and which has been deeply characterized, is an interesting model protein to investigate nanoparticle-protein interactions. Therefore, the interaction of nanoparticles with serum albumin has been widely studied. Immunomethods were suggested for the investigation of adsorption isotherms because of their ease to quantify the non-adsorbed bovine serum albumin without the need of applying separation methods that could modify the balance between the adsorbed and non-adsorbed proteins. The present work revealed that this method should be applied with caution. Artifacts in the determination of free protein can be generated by the presence of surfactants such as polysorbate 80, widely used in the pharmaceutical and biomedical field, that are needed to preserve the stability of nanoparticle dispersions. It was shown that the presence of traces of polysorbate 80 in the dispersion leads to an overestimation of the amount of bovine serum albumin remaining free in the dispersion medium when determined by both radial immunodiffusion and rocket immunoelectrophoresis. However, traces of poloxamer 188 did not result in clear perturbed migrations. These methods are not appropriate to perform adsorption isotherms of proteins on nanoparticle dispersions containing traces of remaining free surfactant. They should only be applied on dispersions that are free of surfactant that is not associated with nanoparticles.

  10. Novel determination of protein, fat, and lactose of milk by liquid scintillation counter

    Energy Technology Data Exchange (ETDEWEB)

    Noble, R.C.; Shand, J.H.; West, I.G.

    1981-01-01

    A method for routine determination of protein, fat, and lactose contents of milk is based on the ability of a scintillation counter to measure coloration or opalescence through attenuation of photons emitted from sealed miniature carbon-14 and hydrogen-3 radioactive standards. A series of simplified and accurate analytical procedures enable full advantage to be taken of the automatic facilities on the modern liquid scintillation counter. The methods provide several advantages over existing procedures. Accuracy of quantification was high as assessed by comparing the results with those derived by recommended Kjeldahl, Gerber, and colorimetric procedures for protein, fat, and lactose determinations, respectively.

  11. Kjeldahl nitrogen analysis as a reference method for protein determination in dairy products.

    Science.gov (United States)

    Lynch, J M; Barbano, D M

    1999-01-01

    Measurement of total nitrogen by Kjeldahl analysis is the historical reference method for determination of the protein content of dairy products and is used for both calibration and validation of alternative methods for protein determination. Accurate evaluation of alternative methods is not possible if there is large uncertainty regarding the reference values. When Kjeldahl analysis is used to establish reference values, the performance of the Kjeldahl testing must be verified and within established expectations. Advice is given for Kjeldahl system optimization, evaluation of test results, and trouble-shooting. Techniques for successful Kjeldahl nitrogen analysis of dairy products other than milk are discussed.

  12. The backbone of the post-synaptic density originated in a unicellular ancestor of choanoflagellates and metazoans

    Directory of Open Access Journals (Sweden)

    Manuel Michaël

    2010-02-01

    Full Text Available Abstract Background Comparative genomics of the early diverging metazoan lineages and of their unicellular sister-groups opens new window to reconstructing the genetic changes which preceded or accompanied the evolution of multicellular body plans. A recent analysis found that the genome of the nerve-less sponges encodes the homologues of most vertebrate post-synaptic proteins. In vertebrate excitatory synapses, these proteins assemble to form the post-synaptic density, a complex molecular platform linking membrane receptors, components of their signalling pathways, and the cytoskeleton. Newly available genomes from Monosiga brevicollis (a member of Choanoflagellata, the closest unicellular relatives of animals and Trichoplax adhaerens (a member of Placozoa: besides sponges, the only nerve-less metazoans offer an opportunity to refine our understanding of post-synaptic protein evolution. Results Searches for orthologous proteins and reconstruction of gene gains/losses based on the taxon phylogeny indicate that post-synaptic proteins originated in two main steps. The backbone scaffold proteins (Shank, Homer, DLG and some of their partners were acquired in a unicellular ancestor of choanoflagellates and metazoans. A substantial additional set appeared in an exclusive ancestor of the Metazoa. The placozoan genome contains most post-synaptic genes but lacks some of them. Notably, the master-scaffold protein Shank might have been lost secondarily in the placozoan lineage. Conclusions The time of origination of most post-synaptic proteins was not concomitant with the acquisition of synapses or neural-like cells. The backbone of the scaffold emerged in a unicellular context and was probably not involved in cell-cell communication. Based on the reconstructed protein composition and potential interactions, its ancestral function could have been to link calcium signalling and cytoskeleton regulation. The complex later became integrated into the evolving

  13. Leucine content of dietary proteins is a determinant of postprandial skeletal muscle protein synthesis in adult rats

    Directory of Open Access Journals (Sweden)

    Norton Layne E

    2012-07-01

    Full Text Available Abstract Background Leucine (Leu regulates muscle protein synthesis (MPS producing dose-dependent plasma Leu and MPS responses from free amino acid solutions. This study examined the role of Leu content from dietary proteins in regulation of MPS after complete meals. Methods Experiment 1 examined 4 protein sources (wheat, soy, egg, and whey with different Leu concentrations (6.8, 8.0, 8.8, and 10.9% (w/w, respectively on the potential to increase plasma Leu, activate translation factors, and stimulate MPS. Male rats (~250 g were trained for 14 day to eat 3 meals/day consisting of 16/54/30% of energy from protein, carbohydrates and fats. Rats were killed on d14 either before or 90 min after consuming a 4 g breakfast meal. Experiment 2 compared feeding wheat, whey, and wheat + Leu to determine if supplementing the Leu content of the wheat meal would yield similar anabolic responses as whey. Results In Experiment 1, only whey and egg groups increased post-prandial plasma Leu and stimulated MPS above food-deprived controls. Likewise, greater phosphorylation of p70 S6 kinase 1 (S6K1 and 4E binding protein-1 (4E-BP1 occurred in whey and egg groups versus wheat and soy groups. Experiment 2 demonstrated that supplementing wheat with Leu to equalize the Leu content of the meal also equalized the rates of MPS. Conclusion These findings demonstrate that Leu content is a critical factor for evaluating the quantity and quality of proteins necessary at a meal for stimulation of MPS.

  14. Determining novel functions of Arabidopsis 14-3-3 proteins in central metabolic processes

    Directory of Open Access Journals (Sweden)

    Diaz Celine

    2011-11-01

    Full Text Available Abstract Background 14-3-3 proteins are considered master regulators of many signal transduction cascades in eukaryotes. In plants, 14-3-3 proteins have major roles as regulators of nitrogen and carbon metabolism, conclusions based on the studies of a few specific 14-3-3 targets. Results In this study, extensive novel roles of 14-3-3 proteins in plant metabolism were determined through combining the parallel analyses of metabolites and enzyme activities in 14-3-3 overexpression and knockout plants with studies of protein-protein interactions. Decreases in the levels of sugars and nitrogen-containing-compounds and in the activities of known 14-3-3-interacting-enzymes were observed in 14-3-3 overexpression plants. Plants overexpressing 14-3-3 proteins also contained decreased levels of malate and citrate, which are intermediate compounds of the tricarboxylic acid (TCA cycle. These modifications were related to the reduced activities of isocitrate dehydrogenase and malate dehydrogenase, which are key enzymes of TCA cycle. In addition, we demonstrated that 14-3-3 proteins interacted with one isocitrate dehydrogenase and two malate dehydrogenases. There were also changes in the levels of aromatic compounds and the activities of shikimate dehydrogenase, which participates in the biosynthesis of aromatic compounds. Conclusion Taken together, our findings indicate that 14-3-3 proteins play roles as crucial tuners of multiple primary metabolic processes including TCA cycle and the shikimate pathway.

  15. Topology of Endoplasmic Reticulum-Associated Cellular and Viral Proteins Determined with Split-GFP.

    Science.gov (United States)

    Hyun, Seong-In; Maruri-Avidal, Liliana; Moss, Bernard

    2015-07-01

    The split green fluorescent protein (GFP) system was adapted for investigation of the topology of ER-associated proteins. A 215-amino acid fragment of GFP (S1-10) was expressed in the cytoplasm as a free protein or fused to the N-terminus of calnexin and in the ER as an intraluminal protein or fused to the C-terminus of calnexin. A 16-amino acid fragment of GFP (S11) was fused to the N- or C-terminus of the target protein. Fluorescence occurred when both GFP fragments were in the same intracellular compartment. After validation with the cellular proteins PDI and tapasin, we investigated two vaccinia virus proteins (L2 and A30.5) of unknown topology that localize to the ER and are required for assembly of the viral membrane. Our results indicated that the N- and C-termini of L2 faced the cytoplasmic and luminal sides of the ER, respectively. In contrast both the N- and C-termini of A30.5 faced the cytoplasm. The system offers advantages for quickly determining the topology of intracellular proteins: the S11 tag is similar in length to commonly used epitope tags; multiple options are available for detecting fluorescence in live or fixed cells; transfection protocols are adaptable to numerous expression systems and can enable high throughput applications.

  16. The ratio of intracellular CRY proteins determines the clock period length

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yang [College of Biological Sciences, China Agricultural University, Beijing 100083 (China); National Institute of Biological Sciences, Beijing 102206 (China); Xiong, Wei [National Institute of Biological Sciences, Beijing 102206 (China); School of Life Sciences, Peking University, Beijing 100871 (China); Zhang, Eric Erquan, E-mail: zhangerquan@nibs.ac.cn [National Institute of Biological Sciences, Beijing 102206 (China)

    2016-04-08

    Although a deficiency in CRY1 or CRY2 correlates with a shorter or longer circadian period, the regulation of CRY proteins in the circadian period has not been well studied. In this study, we found that both CRY1 and CRY2 were able to rescue oscillation in CRY null cells and that they displayed different periods. Furthermore, we demonstrated that protein nuclear import rates, not protein stability, regulate the period-length at the cellular level. Co-transfection of CRY1 and CRY2 in various ratios in the same cells gives rise to the predicted period length in a dose-dependent manner. Given the distinct characteristics of the C-terminal tails of the CRY1 and CRY2 proteins, our study addresses a long-standing hypothesis that the ratio of these two CRY molecules affects the clock period. - Highlights: • Rhythmic CRY2, like CRY1, in the correct CRY1 phase is sufficient to rescue clock oscillation in CRY null cells. • The short-period mammalian CRY2 protein is more stable than the CRY1 protein. • The N-terminal polypeptide of CRY2 contributes to its stability and Per2 repression, but it does not affect the period. • The C-terminal tails of CRYs regulate their protein stability and nuclear import, but the import rate governs the period. • The ratio, rather than the absolute amounts of CRY1 and CRY2 proteins, determines the period in mammalian cells.

  17. Aqueous self-assembly of hydrophobic macromolecules with adjustable rigidity of the backbone.

    Science.gov (United States)

    Guan, Zhou; Liu, Dapeng; Lin, Jiaping; Wang, Xiaosong

    2017-08-02

    P(FpC3P) (Fp: CpFe(CO)2; C3P: propyl diphenyl phosphine) has a helical backbone, resulting from piano stool metal coordination geometry, which is rigid with intramolecular aromatic interaction of the phenyl groups. The macromolecule is hydrophobic, but the polarized CO groups can interact with water for aqueous self-assembly. The stiffness of P(FpC3P), which is adjustable by temperature, is an important factor influencing the morphologies of kinetically trapped assemblies. P(FpC3P)7 self-assembles in DMSO/water (10/90 by volume) into lamellae at 25 °C, vesicles at 40 °C and irregular aggregates at higher temperatures (60 and 70 °C). The colloidal stability decreases in the order of lamellae, vesicles and irregular aggregates. Dissipative particle dynamics (DPD) simulation reveals the same temperature-dependent self-assembled morphologies with an interior of hydrophobic aromatic groups covered with the metal coordination units. The rigid backbone at 25 °C accounts for the formation of the layered morphology, while the reduced rigidity of the same P(FpC3P)7 at 40 °C curves up the lamellae into vesicles. At a higher temperature (60 or 70 °C), P(FpC3P)7 behaves as a random coil without obvious amphiphilic segregation, resulting in irregular aggregates. The stiffness is, therefore, a crucial factor for the aqueous assembly of macromolecules without obvious amphiphilic segregation, which is reminiscent of the solution behavior observed for many hydrophobic biological macromolecules such as proteins.

  18. Comparison of the backbone dynamics of a natural and a consensus designed 3-TPR domain.

    Science.gov (United States)

    Jarymowycz, Virginia A; Cortajarena, Aitziber L; Regan, Lynne; Stone, Martin J

    2008-07-01

    The tetratricopeptide repeat (TPR) is a 34-amino acid helix-turn-helix motif that occurs in tandem arrays in numerous proteins. Here we compare the backbone dynamics of a natural 3-repeat TPR domain, from the protein UBP, with the behavior of a designed protein CTPR3, which consists of three identical consensus TPR units. Although the three tandem TPR repeats in both CTPR3 and UBP behave as a single unit, with no evidence of independent repeat motions, the data indicate that certain positions in UBP are significantly more flexible than are the corresponding positions in CTPR3. Most of the dynamical changes occur at or adjacent to positions that are involved in intra-repeat packing interactions. These observations lead us to suggest that the three-TPR domain of UBP does not incorporate optimized packing, compared to that seen in the idealized CTPR. The natural TPR domain is not only less stable overall than CTPR3, but also presents increased local flexibility at the positions where the sequences differs from the conserved consensus.

  19. Protein Secondary Structure Determination by Constrained Single-Particle Cryo-Electron Tomography

    OpenAIRE

    Bartesaghi, Alberto; Lecumberry, Federico; Sapiro, Guillermo; Subramaniam, Sriram

    2012-01-01

    Cryo-electron microscopy (cryo-EM) is a powerful technique for 3D structure determination of protein complexes by averaging information from individual molecular images. The resolutions that can be achieved with single-particle cryo-EM are frequently limited by inaccuracies in assigning molecular orientations based solely on 2D projection images. Tomographic data collection schemes, however, provide powerful constraints that can be used to more accurately determine molecular orientations nece...

  20. Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

    Science.gov (United States)

    Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2013-09-01

    In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Structure and orientation of interfacial proteins determined by sum frequency generation vibrational spectroscopy: method and application.

    Science.gov (United States)

    Ye, Shuji; Wei, Feng; Li, Hongchun; Tian, Kangzhen; Luo, Yi

    2013-01-01

    In situ and real-time characterization of molecular structures and orientation of proteins at interfaces is essential to understand the nature of interfacial protein interaction. Such work will undoubtedly provide important clues to control biointerface in a desired manner. Sum frequency generation vibrational spectroscopy (SFG-VS) has been demonstrated to be a powerful technique to study the interfacial structures and interactions at the molecular level. This paper first systematically introduced the methods for the calculation of the Raman polarizability tensor, infrared transition dipole moment, and SFG molecular hyperpolarizability tensor elements of proteins/peptides with the secondary structures of α-helix, 310-helix, antiparallel β-sheet, and parallel β-sheet, as well as the methodology to determine the orientation of interfacial protein secondary structures using SFG amide I spectra. After that, recent progresses on the determination of protein structure and orientation at different interfaces by SFG-VS were then reviewed, which provides a molecular-level understanding of the structures and interactions of interfacial proteins, specially understanding the nature of driving force behind such interactions. Although this review has focused on analysis of amide I spectra, it will be expected to offer a basic idea for the spectral analysis of amide III SFG signals and other complicated molecular systems such as RNA and DNA. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Active-state models of ternary GPCR complexes: determinants of selective receptor-G-protein coupling.

    Directory of Open Access Journals (Sweden)

    Ralf C Kling

    Full Text Available Based on the recently described crystal structure of the β2 adrenergic receptor--Gs-protein complex, we report the first molecular-dynamics simulations of ternary GPCR complexes designed to identify the selectivity determinants for receptor-G-protein binding. Long-term molecular dynamics simulations of agonist-bound β2AR-Gαs and D2R-Gαi complexes embedded in a hydrated bilayer environment and computational alanine-scanning mutagenesis identified distinct residues of the N-terminal region of intracellular loop 3 to be crucial for coupling selectivity. Within the G-protein, specific amino acids of the α5-helix, the C-terminus of the Gα-subunit and the regions around αN-β1 and α4-β6 were found to determine receptor recognition. Knowledge of these determinants of receptor-G-protein binding selectivity is essential for designing drugs that target specific receptor/G-protein combinations.

  3. Design and synthesis of peptide YY analogues with c-terminal backbone amide-to-ester modifications

    DEFF Research Database (Denmark)

    Albertsen, Louise; Andersen, J.J.; Paulsson, J.F.;

    2013-01-01

    Peptide YY (PYY) is a gut hormone that activates the G protein-coupled neuropeptide Y (NPY) receptors, and because of its appetite reducing actions, it is evaluated as an antiobesity drug candidate. The C-terminal tail of PYY is crucial for activation of the NPY receptors. Here, we describe...... the design and preparation of a series of PYY(3-36) depsipeptide analogues, in which backbone amide-to-ester modifications were systematically introduced in the C-terminal. Functional NPY receptor assays and circular dichroism revealed that the ψ(CONH) bonds at positions 30-31 and 33-34 are particularly...

  4. Determining protein structures by combining semireliable data with atomistic physical models by Bayesian inference.

    Science.gov (United States)

    MacCallum, Justin L; Perez, Alberto; Dill, Ken A

    2015-06-02

    More than 100,000 protein structures are now known at atomic detail. However, far more are not yet known, particularly among large or complex proteins. Often, experimental information is only semireliable because it is uncertain, limited, or confusing in important ways. Some experiments give sparse information, some give ambiguous or nonspecific information, and others give uncertain information-where some is right, some is wrong, but we don't know which. We describe a method called Modeling Employing Limited Data (MELD) that can harness such problematic information in a physics-based, Bayesian framework for improved structure determination. We apply MELD to eight proteins of known structure for which such problematic structural data are available, including a sparse NMR dataset, two ambiguous EPR datasets, and four uncertain datasets taken from sequence evolution data. MELD gives excellent structures, indicating its promise for experimental biomolecule structure determination where only semireliable data are available.

  5. EMSCOPE - Electromagnetic Component of EarthScope Backbone and Transportable Array Experiments 2006-2008

    Science.gov (United States)

    Egbert, G.; Evans, R.; Ingate, S.; Livelybrooks, D.; Mickus, K.; Park, S.; Schultz, A.; Unsworth, M.; Wannamaker, P.

    2007-12-01

    USArray (http://www.iris.edu/USArray) in conjunction with EMSOC (Electromagnetic Studies of the Continents) (http://emsoc.ucr.edu/emsoc) is installing magnetotelluric (MT) stations as part of Earthscope. The MT component of Earthscope consists of permanent (Backbone) and transportable long period stations to record naturally occurring, time varying electric and magnetic fields to produce a regional lithospheric/asthensospheric electrical conductivity map of the United States. The recent arrival of 28 long period MT instruments allows for the final installation of the Backbone stations throughout the US and yearly transportable array studies. The Backbone MT survey consists of 7 stations spaced throughout the continental US with preliminary installation at Soap Creek, Oregon; Parkfield, California; Braden, Missouri and Socorro, New Mexico.Siting and permitting are underway or completed at stations in eastern Montana, northern Wisconsin and Virginia. These stations will be recording for at least five years to determine electrical conductivities at depths that extend into the mantle transition zone. The first transportable array experiment was performed in the summer and fall of 2006 in central and eastern Oregon (Oregon Pilot Project) using equipment loaned from EMSOC. Thirty-one long period MT stations were recorded with 14 to 21 day occupations. Preliminary 3D inverse models indicate several lithospheric electrical conductivity anomalies including a linear zone marked by low-high conductivity transition along the Klamath-Blue Mountain Lineament associated with a linear trend of gravity minima. High electrical conductivity values occur in the upper crust under the accreted terrains in the Blue Mountains region. The second transportable array experiment was performed in the summer and fall of 2007 and completes coverage of the Oregon, Washington, and western Idaho, targeting the Cascadia subduction zone, Precambrian boundaries, and sub-basalt lithologies. The 2008

  6. Study of nuclear reaction method for the determination of protein content in wheat seeds

    Institute of Scientific and Technical Information of China (English)

    LUO Xiao-Bing; ZHANG Ruan-Yu; CHEN Shi-Guo; LI Tai-Hua; AN Zhu; CAO Yang-Shu; HE Fu-Qing; PENG Xiu-Feng; PAN Shi-Biao

    2005-01-01

    A 13.4 MeV deuteron beam from the 1.2 meter cyclotron of Sichuan University was used to determine the protein content in wheat seeds on the basis of (d,p) reactions. The influence of the variation of the water content in seeds has been investigated.

  7. Minor component effects on the determination of fat, protein, and lactose in milk by FTIR spectroscopy

    Science.gov (United States)

    Hop, E.; Lutz, E. T.; Luinge, Hendrik J.; de Jong, E. A.; van Hemert, H. A.

    1994-01-01

    The determination of fat, protein and lactose in milk by infrared spectrometry may be affected by the presence of minor components absorbing in interfering spectral regions. The size of these effects is reported. Also, analyses with conventional filter infrared spectrometers are compared with newly developed FT-IR based methods in combination with multivariate calibration techniques.

  8. A novel strategy for NMR resonance assignment and protein structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Lemak, Alexander [University of Toronto, Ontario Cancer Institute and Campbell Family Cancer Research Institute, Department of Medical Biophysics (Canada); Gutmanas, Aleksandras [European Bioinformatics Institute, Protein Data Bank Europe (United Kingdom); Chitayat, Seth; Karra, Murthy [University of Toronto, Ontario Cancer Institute and Campbell Family Cancer Research Institute, Department of Medical Biophysics (Canada); Fares, Christophe [Max-Planck-Institut f. Kohlenforschung (Germany); Sunnerhagen, Maria [Linkoeping University, Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology (Sweden); Arrowsmith, Cheryl H., E-mail: carrow@uhnres.utoronto.ca [University of Toronto, Ontario Cancer Institute and Campbell Family Cancer Research Institute, Department of Medical Biophysics (Canada)

    2011-01-15

    The quality of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy is contingent on the number and quality of experimentally-derived resonance assignments, distance and angular restraints. Two key features of protein NMR data have posed challenges for the routine and automated structure determination of small to medium sized proteins; (1) spectral resolution - especially of crowded nuclear Overhauser effect spectroscopy (NOESY) spectra, and (2) the reliance on a continuous network of weak scalar couplings as part of most common assignment protocols. In order to facilitate NMR structure determination, we developed a semi-automated strategy that utilizes non-uniform sampling (NUS) and multidimensional decomposition (MDD) for optimal data collection and processing of selected, high resolution multidimensional NMR experiments, combined it with an ABACUS protocol for sequential and side chain resonance assignments, and streamlined this procedure to execute structure and refinement calculations in CYANA and CNS, respectively. Two graphical user interfaces (GUIs) were developed to facilitate efficient analysis and compilation of the data and to guide automated structure determination. This integrated method was implemented and refined on over 30 high quality structures of proteins ranging from 5.5 to 16.5 kDa in size.

  9. Abdominal obesity and smoking are important determinants of C-reactive protein in renal transplant recipients

    NARCIS (Netherlands)

    van Ree, RM; de Vries, APJ; Oterdoom, LH; The, TH; Gansevoort, RT; van der Heide, JJH; van Son, WJ; Ploeg, RJ; de Jong, PE; Gans, ROB; Bakker, SJL

    2005-01-01

    Background. C-reactive protein (CRP) is a predictor of coronary heart disease, total mortality and chronic allograft nephropathy in renal transplant recipients. The determinants of CRP have been investigated in the general population, but not in renal transplant recipients. CRP might reflect metabol

  10. Rumen escape protein in concentrate ingredients determined with the nylon bag and enzymatic techniques

    NARCIS (Netherlands)

    Cone, J.W.; Kamman, A.A.; Gelder, A.H. van; Hindle, V.A.

    2002-01-01

    Rumen escape protein (REP) was determined for 26 concentrate feed ingredients using the nylon bag technique and in vitro using a proteolytic enzyme preparation of Streptomyces griseus. In vitro, the samples were incubated for 0, 1, 6 and 24 h. The highest correlation was observed between percentage

  11. Interference from alpha-amino acid and protein on determination of formaldehyde in food

    Science.gov (United States)

    Lu, Xiumin; Zhang, Xiaofeng; Fu, Yujie; Xiang, Jinxin

    2005-12-01

    The disturbance of alpha-amino acids and proteins on the analysis of formaldehyde content in food was investigated by electrochemical assay. Results show that the pH decreases gradually from 9.91 to 4.36 with increasing aspartic acid concentration. The recovery rate changes from 8% to 100% after different amounts of formaldehyde were added into protein solutions. For edible bamboo shoots, the recovery rate of formaldehyde is 80% to 100%. For shrimp kernel, however, the recovery rate of formaldehyde is 8% to 60%. These results indicate that the consumed quantity of formaldehyde is correlative with the protein concentration in foods. Therefore, the determinate formaldehyde content in food is actually not the totally applied amount, but just the residue after its reaction with the alpha-amino acids or free amino groups on the protein surface.

  12. A practical method for extending the biuret assay to protein determination of corn-based products.

    Science.gov (United States)

    Liu, Zelong; Pan, Junhui

    2017-06-01

    A modified biuret method suitable for protein determination of corn-based products was developed by introducing a combination of an alkaline reagent with sodium dodecyl sulfate (reagent A) and heat treatments. The method was tested on seven corn-based samples. The results showed mostly good agreement (P>0.05) as compared to the Kjeldahl values. The proposed method was found to enhance the accuracy of prediction on zein content using bovine serum albumin as standard. Reagent A and sample treatment were proved to effectively improve protein solubilization for the thermally-dried corn-based products, e.g. corn gluten meal. The absorbance was stable for at least 1-h. Moreover, the whole measurement of protein content only needs 15-20min more than the traditional biuret assay, and can be performed in batches. The findings suggest that the proposed method could be a timesaving alternative for routine protein analyses in corn processing factories.

  13. Determination of the structure of P II protein from H. seropedicae

    Energy Technology Data Exchange (ETDEWEB)

    Benelli, M.; Souza, E.M.; Delboni, F.; Pedrosa, F.O. [Parana Univ., Curitiba, PR (Brazil). Dept. de Bioquimica; Buck, M. [Imperial Coll. of Science and Technology and Medicine, London (United Kingdom). Dept. of Biology; Moore, M.; Harper, A. [New York Univ., NY (United States). Dept. of Chemistry

    1997-12-31

    Full text. The P II protein, the product of the glnB gene is involved in a signal transducing cascade that controls nitrogen fixation in diazotrophs. The PII protein of H. seropedicae was over-produced in Escherichia coli RB9065 (glnB, glnD mutant), as PII-His from the plasmid pEMB101.7 and native PII from the plasmid pEMB101.8. Both plasmids yielded soluble proteins which were purified, the PII protein was crystallized and its structure solved at 3.0 A resolution by molecular replacement, using the E. coli PII structure as a model. The protein is a trimer and the monomer has a double bab motif. Comparison of the PII proteins of both organisms showed that the main structural difference was in the C-terminal. The T loop that contains the Tyr 51 residue was not visible in the H. seropedicae PII crystal structure. To determine the T loop structure and improve the resolution, the PII protein from H. seropedicae is being co-crystallized in the presence of small ligands, ATP and 2-ketoglutarate. (author)

  14. Dietary Proteins as Determinants of Metabolic and Physiologic Functions of the Gastrointestinal Tract

    Directory of Open Access Journals (Sweden)

    G. Harvey Anderson

    2011-05-01

    Full Text Available Dietary proteins elicit a wide range of nutritional and biological functions. Beyond their nutritional role as the source of amino acids for protein synthesis, they are instrumental in the regulation of food intake, glucose and lipid metabolism, blood pressure, bone metabolism and immune function. The interaction of dietary proteins and their products of digestion with the regulatory functions of the gastrointestinal (GI tract plays a dominant role in determining the physiological properties of proteins. The site of interaction is widespread, from the oral cavity to the colon. The characteristics of proteins that influence their interaction with the GI tract in a source-dependent manner include their physico-chemical properties, their amino acid composition and sequence, their bioactive peptides, their digestion kinetics and also the non-protein bioactive components conjugated with them. Within the GI tract, these products affect several regulatory functions by interacting with receptors releasing hormones, affecting stomach emptying and GI transport and absorption, transmitting neural signals to the brain, and modifying the microflora. This review discusses the interaction of dietary proteins during digestion and absorption with the physiological and metabolic functions of the GI tract, and illustrates the importance of this interaction in the regulation of amino acid, glucose, lipid metabolism, and food intake.

  15. In-situ protein determination to monitor contamination in a centrifugal partition chromatograph.

    Science.gov (United States)

    Bouiche, Feriel; Faure, Karine

    2017-05-15

    Centrifugal partition chromatography (CPC) works with biphasic liquid systems including aqueous two-phase systems. Metallic rotors are able to retain an aqueous stationary phase able to purify proteins. But the adhesion of proteins to solid surface may pose a cross-contamination risk during downstream processes. So it is of utmost importance to ensure the cleanliness of the equipment and detect possible protein contamination in a timely manner. Thereby, a direct method that allows the determination of the effective presence of proteins and the extent of contamination in the metallic CPC rotors was developed. This in-situ method is derived from the Amino Density Estimation by Colorimetric Assay (ADECA) which is based on the affinity of a dye, Coomassie Brillant Blue (CBB), with protonated N(+) groups of the proteins. In this paper, the ADECA method was developed dynamically, on a 25 mL stainless-steel rotor with various extents of protein contaminations using bovine serum albumin (BSA) as a fouling model. The eluted CBB dye was quantified and found to respond linearly to BSA contamination up to 70 mg injected. Limits of detection and quantification were recorded as 0.9 mg and 3.1 mg, respectively. While the non-specific interactions between the dye and the rotor cannot currently be neglected, this method allows for in situ determination of proteins contamination and should contribute to the development of CPC as a separation tool in protein purification processes. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. The dominant role of side-chain backbone interactions in structural realization of amino acid code. ChiRotor: a side-chain prediction algorithm based on side-chain backbone interactions.

    Science.gov (United States)

    Spassov, Velin Z; Yan, Lisa; Flook, Paul K

    2007-03-01

    The basic differences between the 20 natural amino acid residues are due to differences in their side-chain structures. This characteristic design of protein building blocks implies that side-chain-side-chain interactions play an important, even dominant role in 3D-structural realization of amino acid codes. Here we present the results of a comparative analysis of the contributions of side-chain-side-chain (s-s) and side-chain-backbone (s-b) interactions to the stabilization of folded protein structures within the framework of the CHARMm molecular data model. Contrary to intuition, our results suggest that side-chain-backbone interactions play the major role in side-chain packing, in stabilizing the folded structures, and in differentiating the folded structures from the unfolded or misfolded structures, while the interactions between side chains have a secondary effect. An additional analysis of electrostatic energies suggests that combinatorial dominance of the interactions between opposite charges makes the electrostatic interactions act as an unspecific folding force that stabilizes not only native structure, but also compact random conformations. This observation is in agreement with experimental findings that, in the denatured state, the charge-charge interactions stabilize more compact conformations. Taking advantage of the dominant role of side-chain-backbone interactions in side-chain packing to reduce the combinatorial problem, we developed a new algorithm, ChiRotor, for rapid prediction of side-chain conformations. We present the results of a validation study of the method based on a set of high resolution X-ray structures.

  17. Thin Films Formed from Conjugated Polymers with Ionic, Water-Soluble Backbones

    NARCIS (Netherlands)

    Voortman, Thomas P; Chiechi, Ryan C

    2015-01-01

    This paper compares the morphologies of films of conjugated polymers in which the backbone (main chain) and pendant groups are varied between ionic/hydrophilic and aliphatic/hydrophobic. We observe that conjugated polymers in which the pendant groups and backbone are matched, either ionic-ionic or h

  18. Backbone of complex networks of corporations: The flow of control

    Science.gov (United States)

    Glattfelder, J. B.; Battiston, S.

    2009-09-01

    We present a methodology to extract the backbone of complex networks based on the weight and direction of links, as well as on nontopological properties of nodes. We show how the methodology can be applied in general to networks in which mass or energy is flowing along the links. In particular, the procedure enables us to address important questions in economics, namely, how control and wealth are structured and concentrated across national markets. We report on the first cross-country investigation of ownership networks, focusing on the stock markets of 48 countries around the world. On the one hand, our analysis confirms results expected on the basis of the literature on corporate control, namely, that in Anglo-Saxon countries control tends to be dispersed among numerous shareholders. On the other hand, it also reveals that in the same countries, control is found to be highly concentrated at the global level, namely, lying in the hands of very few important shareholders. Interestingly, the exact opposite is observed for European countries. These results have previously not been reported as they are not observable without the kind of network analysis developed here.

  19. Data acquisition backbone core DABC release v1.0

    Energy Technology Data Exchange (ETDEWEB)

    Adamczewski-Musch, Joern; Essel, Hans G.; Kurz, Nikolaus; Linev, S. [GSI Helmholtzzentrum fuer Schwerionenforschung, Darmstadt (Germany)

    2010-07-01

    The new experiments at FAIR require new concepts of data acquisition systems for the distribution of self-triggered, time stamped data streams over high performance networks for event building. The Data Acquisition Backbone Core (DABC) is a general purpose software framework developed for the implementation of such data acquisition systems. A DABC application consists of functional components like data input, combiner, scheduler, event builder, filter, analysis and storage which can be configured at runtime. Application specific code including the support of all kinds of data channels (front-end systems) is implemented by C++ program plug-ins. DABC is also well suited as environment for various detector and readout components test beds. A set of DABC plug-ins has been developed for the FAIR experiment CBM (Compressed Baryonic Matter) at GSI. This DABC application is used as DAQ system for test beamtimes. Front-end boards equipped with n-XYTER ASICs and ADCs are connected to read-out controller boards (ROC). From there the data is sent over Ethernet (UDP), or over optics and PCIe interface cards into Linux PCs. DABC does the controlling, event building, archiving and data serving. The first release of DABC was published in 2009 and is available under GPL license.

  20. Backbone of complex networks of corporations: the flow of control.

    Science.gov (United States)

    Glattfelder, J B; Battiston, S

    2009-09-01

    We present a methodology to extract the backbone of complex networks based on the weight and direction of links, as well as on nontopological properties of nodes. We show how the methodology can be applied in general to networks in which mass or energy is flowing along the links. In particular, the procedure enables us to address important questions in economics, namely, how control and wealth are structured and concentrated across national markets. We report on the first cross-country investigation of ownership networks, focusing on the stock markets of 48 countries around the world. On the one hand, our analysis confirms results expected on the basis of the literature on corporate control, namely, that in Anglo-Saxon countries control tends to be dispersed among numerous shareholders. On the other hand, it also reveals that in the same countries, control is found to be highly concentrated at the global level, namely, lying in the hands of very few important shareholders. Interestingly, the exact opposite is observed for European countries. These results have previously not been reported as they are not observable without the kind of network analysis developed here.

  1. Refined solution structure and backbone dynamics of 15N-labeled C12A-p8MTCP studied by NMR relaxation

    Energy Technology Data Exchange (ETDEWEB)

    Barthe, Philippe; Chiche, Laurent; Declerck, Nathalie; Delsuc, Marc-Andre [Universite de Montpellier I, Faculte de Pharmacie, Centre de Biochimie Structurale, CNRS-UMR 9955, INSERM-U414 (France); Lefevre, Jean-Francois [Universite Louis Pasteur, CNRS UPR-9003, ESBS (France); Malliavin, Therese [Universite de Montpellier I, Faculte de Pharmacie, Centre de Biochimie Structurale, CNRS-UMR 9955, INSERM-U414 (France); Mispelter, Joel [Centre Universitaire Bat 112, INSERM-U350, Institut Curie, Biologie (France); Stern, Marc-Henri [Hopital Saint-Louis, Unite INSERM-U462 (France); Lhoste, Jean-Marc; Roumestand, Christian [Universite de Montpellier I, Faculte de Pharmacie, Centre de Biochimie Structurale, CNRS-UMR 9955, INSERM-U414 (France)

    1999-12-15

    MTCP1 (for Mature-T-Cell Proliferation) was the first gene unequivocally identified in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8{sup MTCP} protein encoded by the MTCP1 oncogene has been previously determined by homonuclear proton two-dimensional NMR methods at 600 MHz: it consists of an original scaffold comprising three {alpha}-helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disulfide bridges, forming an {alpha}-hairpin which resembles an antiparallel coiled-coil. The third helix is orientated roughly parallel to the plane defined by the {alpha}-antiparallel motif and appears less well defined. In order to gain more insight into the details of this new scaffold, we uniformly labeled with nitrogen-15 a mutant of this protein (C12A-p8{sup MTCP1}) in which the unbound cysteine at position 12 has been replaced by an alanine residue, thus allowing reproducibly high yields of recombinant protein. The refined structure benefits from 211 additional NOEs, extracted from {sup 15}N-edited 3D experiments, and from a nearly complete set of {phi} angular restraints allowing the estimation of the helical content of the structured part of the protein. Moreover, measurements of {sup 15} N spin relaxation times and heteronuclear {sup 15} N{sup 1}HNOEs provided additional insights into the dynamics of the protein backbone. The analysis of the linear correlation between J(0) and J({omega}) was used to interpret relaxation parameters. It appears that the apparent relative disorder seen in helix III is not simply due to a lack of experimental constraints, but associated with substantial contributions of sub-nanosecond motions in this segment.

  2. Identification of hydroxyl protons, determination of their exchange dynamics, and characterization of hydrogen bonding in a microcrystallin protein.

    Science.gov (United States)

    Agarwal, Vipin; Linser, Rasmus; Fink, Uwe; Faelber, Katja; Reif, Bernd

    2010-03-10

    Heteronuclear correlation experiments employing perdeuterated proteins enable the observation of all hydroxyl protons in a microcrystalline protein by MAS solid-state NMR. Dipolar-based sequences allow magnetization transfers that are >50 times faster compared to scalar-coupling-based sequences, which significantly facilitates their assignment. Hydroxyl exchange rates were measured using EXSY-type experiments. We find a biexponential decay behavior for those hydroxyl groups that are involved in side chain-side chain C-O-H...O horizontal lineC hydrogen bonds. The quantification of the distances between the hydroxyl proton and the carbon atoms in the hydrogen-bonding donor as well as acceptor group is achieved via a REDOR experiment. In combination with X-ray data and isotropic proton chemical shifts, availability of (1)H,(13)C distance information can aid in the quantitative description of the geometry of these hydrogen bonds. Similarly, correlations between backbone amide proton and carbonyl atoms are observed, which will be useful in the analysis of the registry of beta-strand arrangement in amyloid fibrils.

  3. Improving the chances of successful protein structure determination with a random forest classifier

    Energy Technology Data Exchange (ETDEWEB)

    Jahandideh, Samad [Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92307 (United States); Joint Center for Structural Genomics, (United States); Jaroszewski, Lukasz; Godzik, Adam, E-mail: adam@burnham.org [Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92307 (United States); Joint Center for Structural Genomics, (United States); University of California, San Diego, La Jolla, California (United States)

    2014-03-01

    Using an extended set of protein features calculated separately for protein surface and interior, a new version of XtalPred based on a random forest classifier achieves a significant improvement in predicting the success of structure determination from the primary amino-acid sequence. Obtaining diffraction quality crystals remains one of the major bottlenecks in structural biology. The ability to predict the chances of crystallization from the amino-acid sequence of the protein can, at least partly, address this problem by allowing a crystallographer to select homologs that are more likely to succeed and/or to modify the sequence of the target to avoid features that are detrimental to successful crystallization. In 2007, the now widely used XtalPred algorithm [Slabinski et al. (2007 ▶), Protein Sci.16, 2472–2482] was developed. XtalPred classifies proteins into five ‘crystallization classes’ based on a simple statistical analysis of the physicochemical features of a protein. Here, towards the same goal, advanced machine-learning methods are applied and, in addition, the predictive potential of additional protein features such as predicted surface ruggedness, hydrophobicity, side-chain entropy of surface residues and amino-acid composition of the predicted protein surface are tested. The new XtalPred-RF (random forest) achieves significant improvement of the prediction of crystallization success over the original XtalPred. To illustrate this, XtalPred-RF was tested by revisiting target selection from 271 Pfam families targeted by the Joint Center for Structural Genomics (JCSG) in PSI-2, and it was estimated that the number of targets entered into the protein-production and crystallization pipeline could have been reduced by 30% without lowering the number of families for which the first structures were solved. The prediction improvement depends on the subset of targets used as a testing set and reaches 100% (i.e. twofold) for the top class of predicted

  4. RNABC: forward kinematics to reduce all-atom steric clashes in RNA backbone.

    Science.gov (United States)

    Wang, Xueyi; Kapral, Gary; Murray, Laura; Richardson, David; Richardson, Jane; Snoeyink, Jack

    2008-01-01

    Although accurate details in RNA structure are of great importance for understanding RNA function, the backbone conformation is difficult to determine, and most existing RNA structures show serious steric clashes (>or= 0.4 A overlap) when hydrogen atoms are taken into account. We have developed a program called RNABC (RNA Backbone Correction) that performs local perturbations to search for alternative conformations that avoid those steric clashes or other local geometry problems. Its input is an all-atom coordinate file for an RNA crystal structure (usually from the MolProbity web service), with problem areas specified. RNABC rebuilds a suite (the unit from sugar to sugar) by anchoring the phosphorus and base positions, which are clearest in crystallographic electron density, and reconstructing the other atoms using forward kinematics. Geometric parameters are constrained within user-specified tolerance of canonical or original values, and torsion angles are constrained to ranges defined through empirical database analyses. Several optimizations reduce the time required to search the many possible conformations. The output results are clustered and presented to the user, who can choose whether to accept one of the alternative conformations. Two test evaluations show the effectiveness of RNABC, first on the S-motifs from 42 RNA structures, and second on the worst problem suites (clusters of bad clashes, or serious sugar pucker outliers) in 25 unrelated RNA structures. Among the 101 S-motifs, 88 had diagnosed problems, and RNABC produced clash-free conformations with acceptable geometry for 71 of those (about 80%). For the 154 worst problem suites, RNABC proposed alternative conformations for 72. All but 8 of those were judged acceptable after examining electron density (where available) and local conformation. Thus, even for these worst cases, nearly half the time RNABC suggested corrections suitable to initiate further crystallographic refinement. The program is

  5. Structural determinants of protein partitioning into ordered membrane domains and lipid rafts.

    Science.gov (United States)

    Lorent, Joseph Helmuth; Levental, Ilya

    2015-11-01

    Increasing evidence supports the existence of lateral nanoscopic lipid domains in plasma membranes, known as lipid rafts. These domains preferentially recruit membrane proteins and lipids to facilitate their interactions and thereby regulate transmembrane signaling and cellular homeostasis. The functionality of raft domains is intrinsically dependent on their selectivity for specific membrane components; however, while the physicochemical determinants of raft association for lipids are known, very few systematic studies have focused on the structural aspects that guide raft partitioning of proteins. In this review, we describe biophysical and thermodynamic aspects of raft-mimetic liquid ordered phases, focusing on those most relevant for protein partitioning. Further, we detail the variety of experimental models used to study protein-raft interactions. Finally, we review the existing literature on mechanisms for raft targeting, including lipid post-translational modifications, lipid binding, and transmembrane domain features. We conclude that while protein palmitoylation is a clear raft-targeting signal, few other general structural determinants for raft partitioning have been revealed, suggesting that many discoveries lie ahead in this burgeoning field.

  6. A multiplex serum protein assay for determining the probability of colorectal cancer.

    Science.gov (United States)

    Brock, Randall; Xiong, Bob; Li, Lily; Vanbogelen, Ruth A; Christman, Lori

    2012-01-01

    Our purpose is to develop a serum assay to determine an individual's probability of having colorectal cancer (CRC). We have discovered a protein panel yielding encouraging, clinically significant results. We evaluated 431 serum samples from donors screened for CRC by colonoscopy. We compared the concentration of seven proteins in individuals with CRC versus individuals found to be CRC free. The assay monitored a single peptide from each of seven proteins. Comparing CRC to normal samples in univariate two-sample t-tests, 6 of the 7 proteins yielded a p-value less than 0.01. Logistic regression was used to construct a model for determination of CRC probability. The model was fit on a randomly chosen training set of 321 samples. Using 6 of the 7 proteins (ORM1, GSN, C9, HABP2, SAA2, and C3) and a cut point of 0.4, an independent test set of 110 samples yielded a sensitivity of 93.75%, a specificity of 82.89% and a prevalence-adjusted negative predictive value (NPV) of 99.9775% for the assay. The results demonstrate that the assay has promise as a sensitive, non-invasive diagnostic test to provide individuals with an understanding of their own probability of having CRC.

  7. Computational tools for experimental determination and theoretical prediction of protein structure

    Energy Technology Data Exchange (ETDEWEB)

    O`Donoghue, S.; Rost, B.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. The authors intend to review the state of the art in the experimental determination of protein 3D structure (focus on nuclear magnetic resonance), and in the theoretical prediction of protein function and of protein structure in 1D, 2D and 3D from sequence. All the atomic resolution structures determined so far have been derived from either X-ray crystallography (the majority so far) or Nuclear Magnetic Resonance (NMR) Spectroscopy (becoming increasingly more important). The authors briefly describe the physical methods behind both of these techniques; the major computational methods involved will be covered in some detail. They highlight parallels and differences between the methods, and also the current limitations. Special emphasis will be given to techniques which have application to ab initio structure prediction. Large scale sequencing techniques increase the gap between the number of known proteins sequences and that of known protein structures. They describe the scope and principles of methods that contribute successfully to closing that gap. Emphasis will be given on the specification of adequate testing procedures to validate such methods.

  8. Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester substitution.

    Science.gov (United States)

    Desai, Bijoy J; Goto, Yuki; Cembran, Alessandro; Fedorov, Alexander A; Almo, Steven C; Gao, Jiali; Suga, Hiroaki; Gerlt, John A

    2014-10-21

    Hydrogen bonds between backbone amide groups of enzymes and their substrates are often observed, but their importance in substrate binding and/or catalysis is not easy to investigate experimentally. We describe the generation and kinetic characterization of a backbone amide to ester substitution in the orotidine 5'-monophosphate (OMP) decarboxylase from Methanobacter thermoautotrophicum (MtOMPDC) to determine the importance of a backbone amide-substrate hydrogen bond. The MtOMPDC-catalyzed reaction is characterized by a rate enhancement (∼10(17)) that is among the largest for enzyme-catalyzed reactions. The reaction proceeds through a vinyl anion intermediate that may be stabilized by hydrogen bonding interaction between the backbone amide of a conserved active site serine residue (Ser-127) and oxygen (O4) of the pyrimidine moiety and/or electrostatic interactions with the conserved general acidic lysine (Lys-72). In vitro translation in conjunction with amber suppression using an orthogonal amber tRNA charged with L-glycerate ((HO)S) was used to generate the ester backbone substitution (S127(HO)S). With 5-fluoro OMP (FOMP) as substrate, the amide to ester substitution increased the value of Km by ∼1.5-fold and decreased the value of kcat by ∼50-fold. We conclude that (i) the hydrogen bond between the backbone amide of Ser-127 and O4 of the pyrimidine moiety contributes a modest factor (∼10(2)) to the 10(17) rate enhancement and (ii) the stabilization of the anionic intermediate is accomplished by electrostatic interactions, including its proximity of Lys-72. These conclusions are in good agreement with predictions obtained from hybrid quantum mechanical/molecular mechanical calculations.

  9. Protein Adsorption Patterns and Analysis on IV Nanoemulsions—The Key Factor Determining the Organ Distribution

    Directory of Open Access Journals (Sweden)

    Mirko Jansch

    2012-12-01

    Full Text Available Intravenous nanoemulsions have been on the market for parenteral nutrition since the 1950s; meanwhile, they have also been used successfully for IV drug delivery. To be well tolerable, the emulsions should avoid uptake by the MPS cells of the body; for drug delivery, they should be target-specific. The organ distribution is determined by the proteins adsorbing them after injection from the blood (protein adsorption pattern, typically analyzed by two-dimensional polyacrylamide gel electrophoresis, 2-D PAGE. The article reviews the 2-D PAGE method, the analytical problems to be faced and the knowledge available on how the composition of emulsions affects the protein adsorption patterns, e.g., the composition of the oil phase, stabilizer layer and drug incorporation into the interface or oil core. Data were re-evaluated and compared, and the implications for the in vivo distribution are discussed. Major results are that the interfacial composition of the stabilizer layer is the main determining factor and that this composition can be modulated by simple processes. Drug incorporation affects the pattern depending on the localization of the drug (oil core versus interface. The data situation regarding in vivo effects is very limited; mainly, it has to be referred to in the in vivo data of polymeric nanoparticles. As a conclusion, determination of the protein adsorption patterns can accelerate IV nanoemulsion formulation development regarding optimized organ distribution and related pharmacokinetics.

  10. Intrinsic Disorder in Male Sex Determination: Disorderedness of Proteins from the Sry Transcriptional Network.

    Science.gov (United States)

    Merone, Jean; Nwogu, Onyekahi; Redington, Jennifer M; Uversky, Vladimir N

    2016-10-28

    Sex differentiation is a complex process where sexually indifferent embryo progressively acquires male or female characteristics via tightly controlled, perfectly timed, and sophisticatedly intertwined chain of events. This process is controlled and regulated by a set of specific proteins, with one of the first steps in sex differentiation being the activation of the Y-chromosomal Sry gene (sex-determining region Y) in males that acts as a switch from undifferentiated gonad somatic cells to testis development. There are several key players in this process, which constitute the Sry transcriptional network, and collective action of which governs testis determination. Although it is accepted now that many proteins engaged in signal transduction as well as regulation and control of various biological processes are intrinsically disordered (i.e., do not have unique structure and remain unstructured, or incompletely structured, under physiological conditions), the roles and profusion of intrinsic disorder in proteins involved in the male sex determination have not been accessed as of yet. The goal of this study is to cover this gap by analyzing some key players of the Sry transcriptional network. To this end, we employed a broad set of computational tools for intrinsic disorder analysis and conducted intensive literature search in order to gain information on the structural peculiarities of the Sry network-related proteins, their intrinsic disorder predispositions, and the roles of intrinsic disorder in their functions.

  11. Interplay between hydrophobicity and the positive-inside rule in determining membrane-protein topology.

    Science.gov (United States)

    Elazar, Assaf; Weinstein, Jonathan Jacob; Prilusky, Jaime; Fleishman, Sarel Jacob

    2016-09-13

    The energetics of membrane-protein interactions determine protein topology and structure: hydrophobicity drives the insertion of helical segments into the membrane, and positive charges orient the protein with respect to the membrane plane according to the positive-inside rule. Until recently, however, quantifying these contributions met with difficulty, precluding systematic analysis of the energetic basis for membrane-protein topology. We recently developed the dsTβL method, which uses deep sequencing and in vitro selection of segments inserted into the bacterial plasma membrane to infer insertion-energy profiles for each amino acid residue across the membrane, and quantified the insertion contribution from hydrophobicity and the positive-inside rule. Here, we present a topology-prediction algorithm called TopGraph, which is based on a sequence search for minimum dsTβL insertion energy. Whereas the average insertion energy assigned by previous experimental scales was positive (unfavorable), the average assigned by TopGraph in a nonredundant set is -6.9 kcal/mol. By quantifying contributions from both hydrophobicity and the positive-inside rule we further find that in about half of large membrane proteins polar segments are inserted into the membrane to position more positive charges in the cytoplasm, suggesting an interplay between these two energy contributions. Because membrane-embedded polar residues are crucial for substrate binding and conformational change, the results implicate the positive-inside rule in determining the architectures of membrane-protein functional sites. This insight may aid structure prediction, engineering, and design of membrane proteins. TopGraph is available online (topgraph.weizmann.ac.il).

  12. DETERMINATION OF PROTEIN CATABOLIC RATE IN PATIENTS ON CHRONIC INTERMITTENT HEMODIALYSIS - UREA OUTPUT MEASUREMENTS COMPARED WITH DIETARY-PROTEIN INTAKE AND WITH CALCULATION OF UREA GENERATION RATE

    NARCIS (Netherlands)

    STEGEMAN, CA; HUISMAN, RM; DEROUW, B; JOOSTEMA, A; DEJONG, PE

    1995-01-01

    We assessed the agreement between different methods of determining protein catabolic rate (PCR) in hemodialysis patients and the possible influence of postdialysis urea rebound and the length of the interdialytic interval on the PCR determination. Protein catabolic rate derived from measured total u

  13. Enhancing backbone sampling in Monte Carlo simulations using internal coordinates normal mode analysis.

    Science.gov (United States)

    Gil, Victor A; Lecina, Daniel; Grebner, Christoph; Guallar, Victor

    2016-10-15

    Normal mode methods are becoming a popular alternative to sample the conformational landscape of proteins. In this study, we describe the implementation of an internal coordinate normal mode analysis method and its application in exploring protein flexibility by using the Monte Carlo method PELE. This new method alternates two different stages, a perturbation of the backbone through the application of torsional normal modes, and a resampling of the side chains. We have evaluated the new approach using two test systems, ubiquitin and c-Src kinase, and the differences to the original ANM method are assessed by comparing both results to reference molecular dynamics simulations. The results suggest that the sampled phase space in the internal coordinate approach is closer to the molecular dynamics phase space than the one coming from a Cartesian coordinate anisotropic network model. In addition, the new method shows a great speedup (∼5-7×), making it a good candidate for future normal mode implementations in Monte Carlo methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Determination of fat and total protein content in milk using conventional digital imaging

    DEFF Research Database (Denmark)

    Kucheryavskiy, Sergey; Melenteva, Anastasiia; Bogomolov, Andrey

    2014-01-01

    The applicability of conventional digital imaging to quantitative determination of fat and total protein in cow’s milk, based on the phenomenon of light scatter, has been proved. A new algorithm for extracting features from digital images of milk samples has been developed. The algorithm takes...... with cross-validated R2=0.890 for fat content and R2=0.720 for protein content). The results have been compared with previously published Vis/SW-NIR spectroscopic study of similar samples....

  15. Group B Streptococcus surface proteins as major determinants for meningeal tropism.

    Science.gov (United States)

    Tazi, Asmaa; Bellais, Samuel; Tardieux, Isabelle; Dramsi, Shaynoor; Trieu-Cuot, Patrick; Poyart, Claire

    2012-02-01

    Streptococcus agalactiae (group B Streptococcus, GBS), a normal constituent of the intestinal microbiota is the major cause of human neonatal infections and a worldwide spread 'hypervirulent' clone, GBS ST-17, is strongly associated with neonatal meningitis. Adhesion to epithelial and endothelial cells constitutes a key step of the infectious process. Therefore GBS surface-anchored proteins are obvious potential adhesion mediators of barrier crossing and determinant of hypervirulence. This review addresses the most recent molecular insights gained from studies on GBS surface proteins proven to be involved in the crossing of the brain-blood barrier and emphasizes on the specificity of a hypervirulent clone that displays meningeal tropism.

  16. Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

    Science.gov (United States)

    Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

    2016-04-01

    A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

  17. Determinants of antigenicity and specificity in immune response for protein sequences

    Directory of Open Access Journals (Sweden)

    Li Cheng

    2011-06-01

    . Conclusions Together our results suggest that antigenicity is a local property of the protein sequences and that protein sequence properties of composition, secondary structure, solvent accessibility and evolutionary conservation are the determinants of antigenicity and specificity in immune response. Moreover, specificity in immune response could also be accurately predicted for large protein regions without the knowledge of the protein tertiary structure or the presence of discontinuous epitopes. The dataset prepared in this work and the classifier models are available for download at https://sites.google.com/site/oracleclassifiers/.

  18. Structure determination of an integral membrane protein at room temperature from crystals in situ

    Energy Technology Data Exchange (ETDEWEB)

    Axford, Danny [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Foadi, James [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Hu, Nien-Jen; Choudhury, Hassanul Ghani [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Iwata, So [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Kyoto University, Kyoto 606-8501 (Japan); Beis, Konstantinos [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom); Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Alguel, Yilmaz, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE (United Kingdom); Imperial College London, London SW7 2AZ (United Kingdom); Rutherford Appleton Laboratory, Oxfordshire OX11 0FA (United Kingdom)

    2015-05-14

    The X-ray structure determination of an integral membrane protein using synchrotron diffraction data measured in situ at room temperature is demonstrated. The structure determination of an integral membrane protein using synchrotron X-ray diffraction data collected at room temperature directly in vapour-diffusion crystallization plates (in situ) is demonstrated. Exposing the crystals in situ eliminates manual sample handling and, since it is performed at room temperature, removes the complication of cryoprotection and potential structural anomalies induced by sample cryocooling. Essential to the method is the ability to limit radiation damage by recording a small amount of data per sample from many samples and subsequently assembling the resulting data sets using specialized software. The validity of this procedure is established by the structure determination of Haemophilus influenza TehA at 2.3 Å resolution. The method presented offers an effective protocol for the fast and efficient determination of membrane-protein structures at room temperature using third-generation synchrotron beamlines.

  19. 3D structure determination of protein using TEM single particle analysis.

    Science.gov (United States)

    Sato, Chikara; Mio, Kazuhiro; Kawata, Masaaki; Ogura, Toshihiko

    2014-11-01

    Proteins play important roles in cell functions such as enzymes, cell trafficking, neurotransmission, muscle contraction and hormone secretion. However, some proteins are very difficult to be crystallized and their structures are undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Among them, electron microscopy based single particle reconstruction (SPA) technique is a computer-aided structure determination method. This method reconstructs the 3D structure from projection images of dispersed protein. A large number of two-dimensional particle images are picked up from EM films, aligned and classified to generate 2D averages, and used to reconstruct the 3D structure by assigning the Euler angle of each 2D average. Due to the necessity of elaborate collaboration between the classical biology and the innovative information technology including parallel computing, scientists needed to break unseen barriers to get a start of this analysis. However, recent progresses in electron microscopes, mathematical algorithms, and computational abilities greatly reduced the height of barriers and expanded targets that are considered to be primarily addressable using single particle analysis. Membrane proteins are one of these targets to which the single particle analysis is successfully applied for the understanding of their 3D structures. For this purpose, we have developed various SPA methods [1-5] and applied them to different proteins [6-8].Here, we introduce reconstructed proteins, and discuss the availability of this technique. The intramembrane-cleaving proteases (I-CLiPs) that sever the transmembrane domains of their substrates have been identified in a range of organisms and play a variety of roles in biological conditions. I-CLiPs have been classified into three groups: serine-, aspartyl- and metalloprotease

  20. Role of pathogenicity determinant protein C (PdpC in determining the virulence of the Francisella tularensis subspecies tularensis SCHU.

    Directory of Open Access Journals (Sweden)

    Akihiko Uda

    Full Text Available Francisella tularensis subspecies tularensis, the etiological agent of tularemia, is highly pathogenic to humans and animals. However, the SCHU strain of F. tularensis SCHU P0 maintained by passaging in artificial media has been found to be attenuated. To better understand the molecular mechanisms behind the pathogenicity of F. tularensis SCHU, we attempted to isolate virulent bacteria by serial passages in mice. SCHU P5 obtained after 5th passages in mice remained avirulent, while SCHU P9 obtained after 9th passages was completely virulent in mice. Moreover, SCHU P9 grew more efficiently in J774.1 murine macrophages compared with that in the less pathogenic SCHU P0 and P5. Comparison of the nucleotide sequences of the whole genomes of SCHU P0, P5, and P9 revealed only 1 nucleotide difference among P0, P5 and P9 in 1 of the 2 copies of pathogenicity determinant protein C (pdpC gene. An adenine residue deletion was observed in the pdpC1 gene of SCHU P0, P5, and P9 and in the pdpC2 gene of SCHU P0, and P5, while P9 was characterized by the wild type pdpC2 gene. Thus, SCHU P0 and P5 expressed only truncated forms of PdpC protein, while SCHU P9 expressed both wild type and truncated versions. To validate the pathogenicity of PdpC, both copies of the pdpC gene in SCHU P9 have been inactivated by Targetron mutagenesis. SCHU P9 mutants with inactivated pdpC gene showed low intracellular growth in J774.1 cells and did not induce severe disease in experimentally infected mice, while virulence of the mutants was restored by complementation with expression of the intact PdpC. These results demonstrate that PdpC is crucial in determining the virulence of F. tularensis SCHU.

  1. The Population Genomics of Sunflowers and Genomic Determinants of Protein Evolution Revealed by RNAseq

    Directory of Open Access Journals (Sweden)

    Loren H. Rieseberg

    2012-10-01

    determinants of rates of protein evolution and the impact of selection on patterns of polymorphism and divergence.

  2. Use of electrophoretic techniques in determining the composition of seed storage proteins in alfalfa.

    Science.gov (United States)

    Krochko, J E; Bewley, J D

    1988-11-01

    Holoprotein molecular weights and polypeptide composition can be determined for complex mixtures of oligomeric proteins using two-dimensional electrophoretic techniques. The variety of two-dimensional analyses presented here is a reflection of the general usefulness of each method for the identification and characterization of the different classes of seed storage proteins in alfalfa. These techniques can be applied to studies of storage proteins in other seeds as well as non-seed storage proteins. The major seed storage proteins in alfalfa are medicagin (a legumin-like globulin), alfin (a vicilin-like globulin) and a family of lower molecular weight albumins (LMW1-3). These comprise 30%, 10%, and 20%, respectively, of the total extractable protein from cotyledons of mature seeds. Alfin is a heterogeneous oligomeric protein (Mr approximately 150,000) composed of polypeptides ranging in size from Mr 14,000 to 50,000 (alpha 1-alpha 6; 50,000, 38,000, 32,000, 20,000, 16,000 and 14,000, respectively). Medicagin is also a high molecular weight oligomeric protein, but requires high concentrations of salt for solubilisation. It is comprised of a family of individually distinct subunits, each composed of an acidic polypeptide (A1-A9; Mr 49,000 to 39,000) linked via disulphide bond(s) to a basic polypeptide (B1, B2, B3; Mr 24,000, 23,000 and 20,000, respectively). This pairing is highly specific and two families are recognizable on the basis of the B polypeptide (B3 or B1/B2). Subunits (Mr approximately 50,000-65,000) are assembled as trimers (8S) or larger oligomers (12S-15S) in mature seeds. The lower molecular weight albumins (LMW1-3) are acidic (pI less than 6), and consist of sets of disulphide-bonded polypeptides (Mr 15,000 and 11,000).

  3. The Population Genomics of Sunflowers and Genomic Determinants of Protein Evolution Revealed by RNAseq.

    Science.gov (United States)

    Renaut, Sébastien; Grassa, Christopher J; Moyers, Brook T; Kane, Nolan C; Rieseberg, Loren H

    2012-10-25

    Few studies have investigated the causes of evolutionary rate variation among plant nuclear genes, especially in recently diverged species still capable of hybridizing in the wild. The recent advent of Next Generation Sequencing (NGS) permits investigation of genome wide rates of protein evolution and the role of selection in generating and maintaining divergence. Here, we use individual whole-transcriptome sequencing (RNAseq) to refine our understanding of the population genomics of wild species of sunflowers (Helianthus spp.) and the factors that affect rates of protein evolution. We aligned 35 GB of transcriptome sequencing data and identified 433,257 polymorphic sites (SNPs) in a reference transcriptome comprising 16,312 genes. Using SNP markers, we identified strong population clustering largely corresponding to the three species analyzed here (Helianthus annuus, H. petiolaris, H. debilis), with one distinct early generation hybrid. Then, we calculated the proportions of adaptive substitution fixed by selection (alpha) and identified gene ontology categories with elevated values of alpha. The "response to biotic stimulus" category had the highest mean alpha across the three interspecific comparisons, implying that natural selection imposed by other organisms plays an important role in driving protein evolution in wild sunflowers. Finally, we examined the relationship between protein evolution (dN/dS ratio) and several genomic factors predicted to co-vary with protein evolution (gene expression level, divergence and specificity, genetic divergence [FST], and nucleotide diversity pi). We find that variation in rates of protein divergence was correlated with gene expression level and specificity, consistent with results from a broad range of taxa and timescales. This would in turn imply that these factors govern protein evolution both at a microevolutionary and macroevolutionary timescale. Our results contribute to a general understanding of the determinants of

  4. Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids

    DEFF Research Database (Denmark)

    Holm, Lars; O'Rourke, Bruce; Ebenstein, David

    2013-01-01

    A method to determine the rate of protein breakdown in individual proteins was developed and tested in rats and confirmed in humans, using administration of deuterium oxide and incorporation of the deuterium into alanine that was subsequently incorporated into body proteins. Measurement of the fr....... The applicability of this protein-specific FBR approach is suitable for human in vivo experimentation. The labeling period of deuterium oxide administration is dependent on the turnover rate of the protein of interest.......A method to determine the rate of protein breakdown in individual proteins was developed and tested in rats and confirmed in humans, using administration of deuterium oxide and incorporation of the deuterium into alanine that was subsequently incorporated into body proteins. Measurement...... of the fractional breakdown rate of proteins was determined from the rate of disappearance of deuterated alanine from the proteins. The rate of disappearance of deuterated alanine from the proteins was calculated using an exponential decay, giving the fractional breakdown rate (FBR) of the proteins...

  5. Protein structure validation and refinement using amide proton chemical shifts derived from quantum mechanics

    DEFF Research Database (Denmark)

    Christensen, Anders Steen; Linnet, Troels Emtekær; Borg, Mikael;

    2013-01-01

    We present the ProCS method for the rapid and accurate prediction of protein backbone amide proton chemical shifts - sensitive probes of the geometry of key hydrogen bonds that determine protein structure. ProCS is parameterized against quantum mechanical (QM) calculations and reproduces high level...... QM results obtained for a small protein with an RMSD of 0.25 ppm (r = 0.94). ProCS is interfaced with the PHAISTOS protein simulation program and is used to infer statistical protein ensembles that reflect experimentally measured amide proton chemical shift values. Such chemical shift...

  6. Determination of the protein content in brine from salted herring using near-infrared spectroscopy

    DEFF Research Database (Denmark)

    Svensson, V.T.; Nielsen, Henrik Hauch; Bro, Rasmus

    2004-01-01

    Near-infrared reflectance (NIR) spectroscopy in the spectral range of 1000-2500 nm, was measured directly on brine from barrel salted herring, to investigate the potential of NIR as a fast method to determine the protein content. A principal component analysis performed on the NIR spectra shows two...... clearly indicate that NIR spectroscopy has a potential as a fast and noninvasive method for assessing the protein content in brine from barrel salted herring, which again may be used as an indicator for the ripening quality of barrel salted herring....... groups, separating the first 100 days of storage from the storage time exceeding 100 days. A partial least-squares regression model between selected regions of the NIR spectra and the protein content yields a correlation coefficient of 0.93 and a prediction error (RMSECV) of 0.25 g/100 g. The results...

  7. Determining effects of non-synonymous SNPs on protein-protein interactions using supervised and semi-supervised learning.

    Directory of Open Access Journals (Sweden)

    Nan Zhao

    2014-05-01

    Full Text Available Single nucleotide polymorphisms (SNPs are among the most common types of genetic variation in complex genetic disorders. A growing number of studies link the functional role of SNPs with the networks and pathways mediated by the disease-associated genes. For example, many non-synonymous missense SNPs (nsSNPs have been found near or inside the protein-protein interaction (PPI interfaces. Determining whether such nsSNP will disrupt or preserve a PPI is a challenging task to address, both experimentally and computationally. Here, we present this task as three related classification problems, and develop a new computational method, called the SNP-IN tool (non-synonymous SNP INteraction effect predictor. Our method predicts the effects of nsSNPs on PPIs, given the interaction's structure. It leverages supervised and semi-supervised feature-based classifiers, including our new Random Forest self-learning protocol. The classifiers are trained based on a dataset of comprehensive mutagenesis studies for 151 PPI complexes, with experimentally determined binding affinities of the mutant and wild-type interactions. Three classification problems were considered: (1 a 2-class problem (strengthening/weakening PPI mutations, (2 another 2-class problem (mutations that disrupt/preserve a PPI, and (3 a 3-class classification (detrimental/neutral/beneficial mutation effects. In total, 11 different supervised and semi-supervised classifiers were trained and assessed resulting in a promising performance, with the weighted f-measure ranging from 0.87 for Problem 1 to 0.70 for the most challenging Problem 3. By integrating prediction results of the 2-class classifiers into the 3-class classifier, we further improved its performance for Problem 3. To demonstrate the utility of SNP-IN tool, it was applied to study the nsSNP-induced rewiring of two disease-centered networks. The accurate and balanced performance of SNP-IN tool makes it readily available to study the

  8. Determination of Signaling Pathways in Proteins through Network Theory: Importance of the Topology.

    Science.gov (United States)

    Ribeiro, Andre A S T; Ortiz, Vanessa

    2014-04-08

    Network theory methods are being increasingly applied to proteins to investigate complex biological phenomena. Residues that are important for signaling processes can be identified by their condition as critical nodes in a protein structure network. This analysis involves modeling the protein as a graph in which each residue is represented as a node and edges are drawn between nodes that are deemed connected. In this paper, we show that the results obtained from this type of network analysis (i.e., signaling pathways, key residues for signal transmission, etc.) are profoundly affected by the topology of the network, with normally used determination of network edges by geometrical cutoff schemes giving rise to substantial statistical errors. We propose a method of determining protein structure networks by calculating inter-residue interaction energies and show that it gives an accurate and reliable description of the signal-propagation properties of a known allosteric enzyme. We also show that including covalent interactions in the network topology is essential for accurate results to be obtained.

  9. Determination of walnut protein in processed foods by enzyme-linked immunosorbent assay: interlaboratory study.

    Science.gov (United States)

    Sakai, Shinobu; Adachi, Reiko; Akiyama, Hiroshi; Teshima, Reiko; Doi, Hirotoshi; Shibata, Haruki

    2010-01-01

    Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; "walnut kit") has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 microg walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.8-9.9% RSDR) and a high level of recovery (81-119%) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0%. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.

  10. Fecal collection methods for the determination of protein digestibility in bullfrogs

    Directory of Open Access Journals (Sweden)

    Marta Verardino De Stéfani

    2015-08-01

    Full Text Available Adequate methods for the determination of protein digestibility in bullfrogs are important for the understanding of nutrient utilization. Therefore, this study evaluated two methods of feces collection: intestinal dissection and fecal decantation, using cylindric-conical tanks. Frogs were fed with a commercial diet (45% crude protein which was ground and supplemented with 0.5% chromium oxide III. The frogs were fasted 48h before force-feeding (5% of the animal's live weight. For the decantation method, the animals were sacrificed 36 h after force-feeding and feces were collected directly from the large intestine. For the sedimentation method, feces were collected when they appeared in the tubes attached to the front end of the cylindric tanks. No significant difference (P>0.05 in the apparent digestibility coefficients of crude protein for dietary was observed between the methods tested (74.0% and 76.4% for the dissection and decantation methods, respectively. In conclusion, both methods can be used for the determination of protein digestibility of bullfrog feeds

  11. Conformation-specific spectroscopy of capped glutamine-containing peptides: role of a single glutamine residue on peptide backbone preferences.

    Science.gov (United States)

    Walsh, Patrick S; Dean, Jacob C; McBurney, Carl; Kang, Hyuk; Gellman, Samuel H; Zwier, Timothy S

    2016-04-28

    The conformational preferences of a series of short, aromatic-capped, glutamine-containing peptides have been studied under jet-cooled conditions in the gas phase. This work seeks a bottom-up understanding of the role played by glutamine residues in directing peptide structures that lead to neurodegenerative diseases. Resonant ion-dip infrared (RIDIR) spectroscopy is used to record single-conformation infrared spectra in the NH stretch, amide I and amide II regions. Comparison of the experimental spectra with the predictions of calculations carried out at the DFT M05-2X/6-31+G(d) level of theory lead to firm assignments for the H-bonding architectures of a total of eight conformers of four molecules, including three in Z-Gln-OH, one in Z-Gln-NHMe, three in Ac-Gln-NHBn, and one in Ac-Ala-Gln-NHBn. The Gln side chain engages actively in forming H-bonds with nearest-neighbor amide groups, forming C8 H-bonds to the C-terminal side, C9 H-bonds to the N-terminal side, and an amide-stacked geometry, all with an extended (C5) peptide backbone about the Gln residue. The Gln side chain also stabilizes an inverse γ-turn in the peptide backbone by forming a pair of H-bonds that bridge the γ-turn and stabilize it. Finally, the entire conformer population of Ac-Ala-Gln-NHBn is funneled into a single structure that incorporates the peptide backbone in a type I β-turn, stabilized by the Gln side chain forming a C7 H-bond to the central amide group in the β-turn not otherwise involved in a hydrogen bond. This β-turn backbone structure is nearly identical to that observed in a series of X-(AQ)-Y β-turns in the protein data bank, demonstrating that the gas-phase structure is robust to perturbations imposed by the crystalline protein environment.

  12. MCBT: Multi-Hop Cluster Based Stable Backbone Trees for Data Collection and Dissemination in WSNs.

    Science.gov (United States)

    Shin, Inyoung; Kim, Moonseong; Mutka, Matt W; Choo, Hyunseung; Lee, Tae-Jin

    2009-01-01

    We propose a stable backbone tree construction algorithm using multi-hop clusters for wireless sensor networks (WSNs). The hierarchical cluster structure has advantages in data fusion and aggregation. Energy consumption can be decreased by managing nodes with cluster heads. Backbone nodes, which are responsible for performing and managing multi-hop communication, can reduce the communication overhead such as control traffic and minimize the number of active nodes. Previous backbone construction algorithms, such as Hierarchical Cluster-based Data Dissemination (HCDD) and Multicluster, Mobile, Multimedia radio network (MMM), consume energy quickly. They are designed without regard to appropriate factors such as residual energy and degree (the number of connections or edges to other nodes) of a node for WSNs. Thus, the network is quickly disconnected or has to reconstruct a backbone. We propose a distributed algorithm to create a stable backbone by selecting the nodes with higher energy or degree as the cluster heads. This increases the overall network lifetime. Moreover, the proposed method balances energy consumption by distributing the traffic load among nodes around the cluster head. In the simulation, the proposed scheme outperforms previous clustering schemes in terms of the average and the standard deviation of residual energy or degree of backbone nodes, the average residual energy of backbone nodes after disseminating the sensed data, and the network lifetime.

  13. SPEER-SERVER: a web server for prediction of protein specificity determining sites

    Science.gov (United States)

    Chakraborty, Abhijit; Mandloi, Sapan; Lanczycki, Christopher J.; Panchenko, Anna R.; Chakrabarti, Saikat

    2012-01-01

    Sites that show specific conservation patterns within subsets of proteins in a protein family are likely to be involved in the development of functional specificity. These sites, generally termed specificity determining sites (SDS), might play a crucial role in binding to a specific substrate or proteins. Identification of SDS through experimental techniques is a slow, difficult and tedious job. Hence, it is very important to develop efficient computational methods that can more expediently identify SDS. Herein, we present Specificity prediction using amino acids’ Properties, Entropy and Evolution Rate (SPEER)-SERVER, a web server that predicts SDS by analyzing quantitative measures of the conservation patterns of protein sites based on their physico-chemical properties and the heterogeneity of evolutionary changes between and within the protein subfamilies. This web server provides an improved representation of results, adds useful input and output options and integrates a wide range of analysis and data visualization tools when compared with the original standalone version of the SPEER algorithm. Extensive benchmarking finds that SPEER-SERVER exhibits sensitivity and precision performance that, on average, meets or exceeds that of other currently available methods. SPEER-SERVER is available at http://www.hpppi.iicb.res.in/ss/. PMID:22689646

  14. Direct determination of the redox status of cysteine residues in proteins in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Hara, Satoshi [Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503 (Japan); Tatenaka, Yuki; Ohuchi, Yuya [Dojindo Laboratories, 2025-5 Tabaru, Mashiki-machi, Kumamoto 861-2202 (Japan); Hisabori, Toru, E-mail: thisabor@res.titech.ac.jp [Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259-R1-8, Midori-ku, Yokohama 226-8503 (Japan); Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo 102-0075 (Japan)

    2015-01-02

    Highlights: • A new DNA-maleimide which is cleaved by UV irradiation, DNA-PCMal, was developed. • DNA-PCMal can be used like DNA-Mal to analyze the redox state of cysteine residues. • It is useful for detecting the thiol redox status of a protein in vivo by Western blotting method. • Thus, DNA-PCMal can be a powerful tool for redox proteomics analysis. - Abstract: The redox states of proteins in cells are key factors in many cellular processes. To determine the redox status of cysteinyl thiol groups in proteins in vivo, we developed a new maleimide reagent, a photocleavable maleimide-conjugated single stranded DNA (DNA-PCMal). The DNA moiety of DNA-PCMal is easily removed by UV-irradiation, allowing DNA-PCMal to be used in Western blotting applications. Thereby the state of thiol groups in intracellular proteins can be directly evaluated. This new maleimide compound can provide information concerning redox proteins in vivo, which is important for our understanding of redox networks in the cell.

  15. [Determination of total protein content in soya-bean milk via visual moving reaction boundary titration].

    Science.gov (United States)

    Guo, Chengye; Wang, Houyu; Zhang, Lei; Fan, Liuyin; Cao, Chengxi

    2013-11-01

    A visual, rapid and accurate moving reaction boundary titration (MRBT) method was used for the determination of the total protein in soya-bean milk. During the process, moving reaction boundary (MRB) was formed by hydroxyl ions in the catholyte and soya-bean milk proteins immobilized in polyacrylamide gel (PAG), and an acid-base indicator was used to denote the boundary motion. The velocity of MRB has a relationship with protein concentration, which was used to obtain a standard curve. By paired t-test, there was no significant difference of the protein content between MRBT and Kjeldahl method at 95% confidence interval. The procedure of MRBT method required about 10 min, and it had linearity in the range of 2.0-14.0 g/L, low limit of detection (0.05 g/L), good precision (RSD of intra-day protein nitrogen (NPN) such as melamine added into the soya-bean milk had weak influence on MRBT results.

  16. Ruthenium-catalyzed olefin metathesis accelerated by the steric effect of the backbone substituent in cyclic (alkyl)(amino) carbenes.

    Science.gov (United States)

    Zhang, Jun; Song, Shangfei; Wang, Xiao; Jiao, Jiajun; Shi, Min

    2013-10-21

    Three ruthenium complexes bearing backbone-monosubstituted CAACs were prepared and displayed dramatic improvement in catalytic efficiency not only in RCM reaction but also in the ethenolysis of methyl oleate, compared to those bearing backbone-disubstituted CAACs.

  17. Requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by NMR.

    Science.gov (United States)

    Gottstein, Daniel; Reckel, Sina; Dötsch, Volker; Güntert, Peter

    2012-06-06

    Nuclear magnetic resonance (NMR) structure calculations of the α-helical integral membrane proteins DsbB, GlpG, and halorhodopsin show that distance restraints from paramagnetic relaxation enhancement (PRE) can provide sufficient structural information to determine their structure with an accuracy of about 1.5 Å in the absence of other long-range conformational restraints. Our systematic study with simulated NMR data shows that about one spin label per transmembrane helix is necessary for obtaining enough PRE distance restraints to exclude wrong topologies, such as pseudo mirror images, if only limited other NMR restraints are available. Consequently, an experimentally realistic amount of PRE data enables α-helical membrane protein structure determinations that would not be feasible with the very limited amount of conventional NOESY data normally available for these systems. These findings are in line with our recent first de novo NMR structure determination of a heptahelical integral membrane protein, proteorhodopsin, that relied extensively on PRE data.

  18. Frequency Assignment for Joint Aerial Layer Network High-Capacity Backbone

    Science.gov (United States)

    2017-08-11

    ARL-TR-8093•AUG 2017 US Army Research Laboratory Frequency Assignment for Joint Aerial Layer Network High-Capacity Backbone by Peng Wang and Brian...2017 US Army Research Laboratory Frequency Assignment for Joint Aerial Layer Network High-Capacity Backbone by Peng Wang and Brian Henz Computational...Rev. 8/98)    Prescribed by ANSI Std. Z39.18 August 2017 Technical Report Frequency Assignment for Joint Aerial Layer Network High-Capacity Backbone

  19. Technological advances in site-directed spin labeling of proteins.

    Science.gov (United States)

    Hubbell, Wayne L; López, Carlos J; Altenbach, Christian; Yang, Zhongyu

    2013-10-01

    Molecular flexibility over a wide time range is of central importance to the function of many proteins, both soluble and membrane. Revealing the modes of flexibility, their amplitudes, and time scales under physiological conditions is the challenge for spectroscopic methods, one of which is site-directed spin labeling EPR (SDSL-EPR). Here we provide an overview of some recent technological advances in SDSL-EPR related to investigation of structure, structural heterogeneity, and dynamics of proteins. These include new classes of spin labels, advances in measurement of long range distances and distance distributions, methods for identifying backbone and conformational fluctuations, and new strategies for determining the kinetics of protein motion.

  20. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, Mitsuhiro [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan); Ono, Akira M.; Terauchi, Tsutomu [SAIL Technologies Co., Inc. (Japan); Kainosho, Masatsune, E-mail: kainosho@nmr.chem.metro-u.ac.j [Nagoya University, Structural Biology Research Center, Graduate School of Science (Japan)

    2010-01-15

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines ({epsilon}- and {zeta}-SAIL Phe) and tyrosine ({epsilon}-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized {delta}-SAIL Phe and {delta}-SAIL Tyr, which allow us to observe and assign {delta}-{sup 13}C/{sup 1}H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the {delta}-, {epsilon}- or {zeta}-{sup 13}C/{sup 1}H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the {delta}-, {epsilon}-, and {zeta}-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly {sup 13}C, {sup 15}N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of {zeta}-SAIL Phe and {epsilon}-SAIL Tyr would be practically the best choice for protein structural determinations.

  1. Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions

    Science.gov (United States)

    Greenfield, Norma J.

    2009-01-01

    Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy (ΔH) and entropy (ΔS) of unfolding, the midpoint of the unfolding transition (TM) and the free energy (ΔG) of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength takes approximately eight hours. PMID:17406506

  2. Molecular determinants for ATP-binding in proteins: a data mining and quantum chemical analysis.

    Science.gov (United States)

    Mao, Lisong; Wang, Yanli; Liu, Yuemin; Hu, Xiche

    2004-02-20

    Adenosine 5'-triphosphate (ATP) plays an essential role in all forms of life. Molecular recognition of ATP in proteins is a subject of great importance for understanding enzymatic mechanism and for drug design. We have carried out a large-scale data mining of the Protein Data Bank (PDB) to analyze molecular determinants for recognition of the adenine moiety of ATP by proteins. Non-bonded intermolecular interactions (hydrogen bonding, pi-pi stacking interactions, and cation-pi interactions) between adenine base and surrounding residues in its binding pockets are systematically analyzed for 68 non-redundant, high-resolution crystal structures of adenylate-binding proteins. In addition to confirming the importance of the widely known hydrogen bonding, we found out that cation-pi interactions between adenine base and positively charged residues (Lys and Arg) and pi-pi stacking interactions between adenine base and surrounding aromatic residues (Phe, Tyr, Trp) are also crucial for adenine binding in proteins. On average, there exist 2.7 hydrogen bonding interactions, 1.0 pi-pi stacking interactions, and 0.8 cation-pi interactions in each adenylate-binding protein complex. Furthermore, a high-level quantum chemical analysis was performed to analyze contributions of each of the three forms of intermolecular interactions (i.e. hydrogen bonding, pi-pi stacking interactions, and cation-pi interactions) to the overall binding force of the adenine moiety of ATP in proteins. Intermolecular interaction energies for representative configurations of intermolecular complexes were analyzed using the supermolecular approach at the MP2/6-311 + G* level, which resulted in substantial interaction strengths for all the three forms of intermolecular interactions. This work represents a timely undertaking at a historical moment when a large number of X-ray crystallographic structures of proteins with bound ATP ligands have become available, and when high-level quantum chemical analysis of

  3. Protein secondary structure determination by constrained single-particle cryo-electron tomography.

    Science.gov (United States)

    Bartesaghi, Alberto; Lecumberry, Federico; Sapiro, Guillermo; Subramaniam, Sriram

    2012-12-05

    Cryo-electron microscopy (cryo-EM) is a powerful technique for 3D structure determination of protein complexes by averaging information from individual molecular images. The resolutions that can be achieved with single-particle cryo-EM are frequently limited by inaccuracies in assigning molecular orientations based solely on 2D projection images. Tomographic data collection schemes, however, provide powerful constraints that can be used to more accurately determine molecular orientations necessary for 3D reconstruction. Here, we propose "constrained single-particle tomography" as a general strategy for 3D structure determination in cryo-EM. A key component of our approach is the effective use of images recorded in tilt series to extract high-resolution information and correct for the contrast transfer function. By incorporating geometric constraints into the refinement to improve orientational accuracy of images, we reduce model bias and overrefinement artifacts and demonstrate that protein structures can be determined at resolutions of ∼8 Å starting from low-dose tomographic tilt series.

  4. Determination of ring-necked pheasant (Phasianus colchicus) serum protein concentrations by refractometry and the biuret method

    OpenAIRE

    2008-01-01

    The purpose of this study was to evaluate the accuracy of hand-held refractometer in determining serum protein concentrations in ring-necked pheasants (Phasianus colchicus) as compared with the standard biuret method. The results indicated that serum protein values may be accurately determined in ring-necked pheasants with a hand-held refractometer. © Asian Network for Scientific Information, 2008.

  5. Distributions of amino acids suggest that certain residue types more effectively determine protein secondary structure

    Science.gov (United States)

    Battelle, S. Saraswathi; Fernández-Martínez, J. L.; Koliński, A.; Jernigan, R. L.; Battelle, A. Kloczkowski

    2013-01-01

    Exponential growth in the number of available protein sequences is unmatched by the slower growth in the number of structures. As a result, the development of efficient and fast protein secondary structure prediction methods is essential for the broad comprehension of protein structures. Computational methods that can efficiently determine secondary structure can in turn facilitate protein tertiary structure prediction, since most methods rely initially on secondary structure predictions. Recently, we have developed a fast learning optimized prediction methodology (FLOPRED) for predicting protein secondary structure (S. Saraswathi, et al., [1]). Data are generated by using knowledge-based potentials combined with structure information from the CATH database. A neural network-based extreme learning machine (ELM) and advanced particle swarm optimization (PSO) are used with this data to obtain better and faster convergence to more accurate secondary structure predicted results. A five-fold cross-validated testing accuracy of 83.8 % and a segment overlap (SOV) score of 78.3 % are obtained in this study. Secondary structure predictions and their accuracy are usually presented for three secondary structure elements: α-helix, β-strand and coil but rarely have the results been analyzed with respect to their constituent amino acids. In this paper, we use the results obtained with FLOPRED to provide detailed behaviors for different amino acid types in the secondary structure prediction. We investigate the influence of the composition, physico-chemical properties and position specific occurrence preferences of amino acids within secondary structure elements. In addition, we identify the correlation between these properties and prediction accuracy. The present detailed results suggest several important ways that secondary structure predictions can be improved in the future that might lead to improved protein design and engineering. PMID:23907551

  6. Backbone resonance assignment and order tensor estimation using residual dipolar couplings

    Science.gov (United States)

    Shealy, Paul; Liu, Yizhou; Simin, Mikhail

    2014-01-01

    An NMR investigation of proteins with known X-ray structures is of interest in a number of endeavors. Performing these studies through nuclear magnetic resonance (NMR) requires the costly step of resonance assignment. The prevalent assignment strategy does not make use of existing structural information and requires uniform isotope labeling. Here we present a rapid and cost-effective method of assigning NMR data to an existing structure—either an X-ray or computationally modeled structure. The presented method, Exhaustively Permuted Assignment of RDCs (EPAR), utilizes unassigned residual dipolar coupling (RDC) data that can easily be obtained by NMR spectroscopy. The algorithm uses only the backbone N–H RDCs from multiple alignment media along with the amino acid type of the RDCs. It is inspired by previous work from Zweckstetter and provides several extensions. We present results on 13 synthetic and experimental datasets from 8 different structures, including two homodimers. Using just two alignment media, EPAR achieves an average assignment accuracy greater than 80%. With three media, the average accuracy is higher than 94%. The algorithm also outputs a prediction of the assignment accuracy, which has a correlation of 0.77 to the true accuracy. This prediction score can be used to establish the needed confidence in assignment accuracy. PMID:21667298

  7. STARD6 on steroids: solution structure, multiple timescale backbone dynamics and ligand binding mechanism

    Science.gov (United States)

    Létourneau, Danny; Bédard, Mikaël; Cabana, Jérôme; Lefebvre, Andrée; Lehoux, Jean-Guy; Lavigne, Pierre

    2016-06-01

    START domain proteins are conserved α/β helix-grip fold that play a role in the non-vesicular and intracellular transport of lipids and sterols. The mechanism and conformational changes permitting the entry of the ligand into their buried binding sites is not well understood. Moreover, their functions and the identification of cognate ligands is still an active area of research. Here, we report the solution structure of STARD6 and the characterization of its backbone dynamics on multiple time-scales through 15N spin-relaxation and amide exchange studies. We reveal for the first time the presence of concerted fluctuations in the Ω1 loop and the C-terminal helix on the microsecond-millisecond time-scale that allows for the opening of the binding site and ligand entry. We also report that STARD6 binds specifically testosterone. Our work represents a milestone for the study of ligand binding mechanism by other START domains and the elucidation of the biological function of STARD6.

  8. Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism

    Science.gov (United States)

    Greenfield, Norma J.

    2009-01-01

    Circular dichroism (CD) is an excellent tool for examining the interactions and stability of proteins. This protocol covers methods to obtain and analyze circular dichroism spectra to measure changes in the folding of proteins as a function of denaturants, osmolytes or ligands. Applications include determination of the free energy of folding of a protein, the effects of mutations on protein stability and the estimation of binding constants for the interactions of proteins with other proteins, DNA or ligands, such as substrates or inhibitors. The experiments take 2-5 h. PMID:17406529

  9. Potential Contributions of Heat Shock Proteins to Temperature-Dependent Sex Determination in the American Alligator

    Science.gov (United States)

    Kohno, S.; Katsu, Y.; Urushitani, H.; Ohta, Y.; Iguchi, T.; Guillette, L. J.

    2010-01-01

    Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be ‘reversed’ by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90α, HSP90β, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed. PMID:19940440

  10. The role of the Drosophila LAMMER protein kinase DOA in somatic sex determination

    Indian Academy of Sciences (India)

    Leonard Rabinow; Marie-Laure Samson

    2010-09-01

    DOA kinase, the Drosophila member of the LAMMER/Clk protein kinase family, phosphorylates SR and SR-like proteins, including TRA, TRA2 and RBP1, which are responsible for the alternative splicing of transcripts encoding the key regulator of sex-specific expression in somatic cells of the fly, DOUBLESEX. Specific Doa alleles induce somatic female-to-male sex transformations, which can be enhanced when combined with mutations in loci encoding SR and SR-like proteins. The Doa locus encodes six different kinases, of which a 69-kDa isoform is expressed solely in females. Expression of this isoform is itself under the regulation of the somatic sex determination regulatory network, thus forming a putative positive autoregulatory loop which would reinforce the choice of the female cell-fate. We speculate that this loop is part of the evolutionary ancestral sex-determination machinery, based upon evidence demonstrating the existence of an autoregulatory loop involving TRA and TRA2 in several other insect species.

  11. Viral protein determinants of Lassa virus entry and release from polarized epithelial cells.

    Science.gov (United States)

    Schlie, Katrin; Maisa, Anna; Freiberg, Fabian; Groseth, Allison; Strecker, Thomas; Garten, Wolfgang

    2010-04-01

    The epithelium plays a key role in the spread of Lassa virus. Transmission from rodents to humans occurs mainly via inhalation or ingestion of droplets, dust, or food contaminated with rodent urine. Here, we investigated Lassa virus infection in cultured epithelial cells and subsequent release of progeny viruses. We show that Lassa virus enters polarized Madin-Darby canine kidney (MDCK) cells mainly via the basolateral route, consistent with the basolateral localization of the cellular Lassa virus receptor alpha-dystroglycan. In contrast, progeny virus was efficiently released from the apical cell surface. Further, we determined the roles of the glycoprotein, matrix protein, and nucleoprotein in directed release of nascent virus. To do this, a virus-like-particle assay was developed in polarized MDCK cells based on the finding that, when expressed individually, both the glycoprotein GP and matrix protein Z form virus-like particles. We show that GP determines the apical release of Lassa virus from epithelial cells, presumably by recruiting the matrix protein Z to the site of virus assembly, which is in turn essential for nucleocapsid incorporation into virions.

  12. Differences between antigenic determinants of pig and cat zona pellucida proteins.

    Science.gov (United States)

    Jewgenow, K; Rohleder, M; Wegner, I

    2000-05-01

    Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

  13. Conserved differences in protein sequence determine the human pathogenicity of Ebolaviruses

    Science.gov (United States)

    Pappalardo, Morena; Juliá, Miguel; Howard, Mark J.; Rossman, Jeremy S.; Michaelis, Martin; Wass, Mark N.

    2016-01-01

    Reston viruses are the only Ebolaviruses that are not pathogenic in humans. We analyzed 196 Ebolavirus genomes and identified specificity determining positions (SDPs) in all nine Ebolavirus proteins that distinguish Reston viruses from the four human pathogenic Ebolaviruses. A subset of these SDPs will explain the differences in human pathogenicity between Reston and the other four ebolavirus species. Structural analysis was performed to identify those SDPs that are likely to have a functional effect. This analysis revealed novel functional insights in particular for Ebolavirus proteins VP40 and VP24. The VP40 SDP P85T interferes with VP40 function by altering octamer formation. The VP40 SDP Q245P affects the structure and hydrophobic core of the protein and consequently protein function. Three VP24 SDPs (T131S, M136L, Q139R) are likely to impair VP24 binding to human karyopherin alpha5 (KPNA5) and therefore inhibition of interferon signaling. Since VP24 is critical for Ebolavirus adaptation to novel hosts, and only a few SDPs distinguish Reston virus VP24 from VP24 of other Ebolaviruses, human pathogenic Reston viruses may emerge. This is of concern since Reston viruses circulate in domestic pigs and can infect humans, possibly via airborne transmission. PMID:27009368

  14. Method for determining the three-dimensional structure of a protein

    Science.gov (United States)

    Morrison, Dennis R. (Inventor); Mosier, Benjamin (Inventor)

    2004-01-01

    Microcapsules prepared by encapsulating an aqueous solution of a protein, drug or other bioactive substance inside a semi-permeable membrane by are disclosed. The microcapsules are formed by interfacial coacervation under conditions where the shear forces are limited to 0-100 dynes/cm.sup.2 at the interface. By placing the microcapsules in a high osmotic dewatering solution, the protein solution is gradually made saturated and then supersaturated, and the controlled nucleation and crystallization of the protein is achieved. The crystal-filled microcapsules prepared by this method can be conveniently harvested and stored while keeping the encapsulated crystals in essentially pristine condition due to the rugged, protective membrane. Because the membrane components themselves are x-ray transparent, large crystal-containing microcapsules can be individually selected, mounted in x-ray capillary tubes and subjected to high energy x-ray diffraction studies to determine the 3-D structure of the protein molecules. Certain embodiments of the microcapsules of the invention have composite polymeric outer membranes which are somewhat elastic, water insoluble, permeable only to water, salts, and low molecular weight molecules and are structurally stable in fluid shear forces typically encountered in the human vascular system.

  15. Dependence of crystallite formation and preferential backbone orientations on the side chain pattern in PBDTTPD polymers

    KAUST Repository

    El Labban, Abdulrahman

    2014-11-26

    (Figure Presented) Alkyl substituents appended to the π-conjugated main chain account for the solution-processability and film-forming properties of most π-conjugated polymers for organic electronic device applications, including field-effect transistors (FETs) and bulk-heterojunction (BHJ) solar cells. Beyond film-forming properties, recent work has emphasized the determining role that side-chain substituents play on polymer self-assembly and thin-film nanostructural order, and, in turn, on device performance. However, the factors that determine polymer crystallite orientation in thin-films, implying preferential backbone orientation relative to the device substrate, are a matter of some debate, and these structural changes remain difficult to anticipate. In this report, we show how systematic changes in the side-chain pattern of poly(benzo[1,2-b:4,5-b′]dithiophene-alt-thieno[3,4-c]pyrrole-4,6-dione) (PBDTTPD) polymers can (i) influence the propensity of the polymer to order in the π-stacking direction, and (ii) direct the preferential orientation of the polymer crystallites in thin films (e.g., "face-on" vs "edge-on"). Oriented crystallites, specifically crystallites that are well-ordered in the π-stacking direction, are believed to be a key contributor to improved thin-film device performance in both FETs and BHJ solar cells.

  16. Dependence of crystallite formation and preferential backbone orientations on the side chain pattern in PBDTTPD polymers.

    Science.gov (United States)

    El Labban, Abdulrahman; Warnan, Julien; Cabanetos, Clément; Ratel, Olivier; Tassone, Christopher; Toney, Michael F; Beaujuge, Pierre M

    2014-11-26

    Alkyl substituents appended to the π-conjugated main chain account for the solution-processability and film-forming properties of most π-conjugated polymers for organic electronic device applications, including field-effect transistors (FETs) and bulk-heterojunction (BHJ) solar cells. Beyond film-forming properties, recent work has emphasized the determining role that side-chain substituents play on polymer self-assembly and thin-film nanostructural order, and, in turn, on device performance. However, the factors that determine polymer crystallite orientation in thin-films, implying preferential backbone orientation relative to the device substrate, are a matter of some debate, and these structural changes remain difficult to anticipate. In this report, we show how systematic changes in the side-chain pattern of poly(benzo[1,2-b:4,5-b']dithiophene-alt-thieno[3,4-c]pyrrole-4,6-dione) (PBDTTPD) polymers can (i) influence the propensity of the polymer to order in the π-stacking direction, and (ii) direct the preferential orientation of the polymer crystallites in thin films (e.g., "face-on" vs "edge-on"). Oriented crystallites, specifically crystallites that are well-ordered in the π-stacking direction, are believed to be a key contributor to improved thin-film device performance in both FETs and BHJ solar cells.

  17. A Distributed Virtual Backbone Formation for Wireless Ad Hoc and Sensor Networks

    Institute of Scientific and Technical Information of China (English)

    CAO Yong-tao; HE Chen; JIANG Ling-ge

    2007-01-01

    The virtual backbone is an approach for solving routing problems in wireless ad hoc and sensor networks. A connected dominating set (CDS) was proposed as a virtual backbone to improve the performance of wireless networks. The quality of a virtual backbone is measured not only by approximation factor, which is the ratio of its size to that of minimum CDS, but also time complexity and message complexity. In this paper, a distributed algorithm is presented to construct a minimum CDS for ad hoc and sensor networks. By destroying triangular loops in the virtual backbone, the proposed algorithm can effectively construct a CDS with smaller size. Moreover, our algorithm, which is fully localized, has a constant approximation ratio, linear message and time complexity, and low implementation complexity. The simulation results and theoretical analysis show that our algorithm has better efficiency and performance than conventional approaches.

  18. Integration of XAS and NMR techniques for the structure determination of metalloproteins. Examples from the study of copper transport proteins.

    Science.gov (United States)

    Banci, Lucia; Bertini, Ivano; Mangani, Stefano

    2005-01-01

    Nuclear magnetic resonance (NMR) is a powerful technique for protein structure determination in solution. However, when dealing with metalloproteins, NMR methods are unable to directly determine the structure of the metal site and its coordination geometry. The capability of X-ray absorption spectroscopy (XAS) to provide the structure of a metal ion bound to a protein is then perfectly suited to complement the process of the structure determination. This aspect is particularly relevant in structural genomic projects where high throughput of structural results is the main goal. The synergism of the two techniques has been exploited in the structure determination of bacterial copper transport proteins.

  19. Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states.

    Science.gov (United States)

    Kendrick, B S; Kerwin, B A; Chang, B S; Philo, J S

    2001-12-15

    Characterizing the solution structure of protein-polymer conjugates and protein-ligand interactions is important in fields such as biotechnology and biochemistry. Size-exclusion high-performance liquid chromatography with online classical light scattering (LS), refractive index (RI), and UV detection offers a powerful tool in such characterization. Novel methods are presented utilizing LS, RI, and UV signals to rapidly determine the degree of conjugation and the molecular mass of the protein conjugate. Baseline resolution of the chromatographic peaks is not required; peaks need only be sufficiently separated to represent relatively pure fractions. An improved technique for determining the polypeptide-only mass of protein conjugates is also described. These techniques are applied to determining the degree of erythropoietin glycosylation, the degree of polyethylene glycol conjugation to RNase A and brain-derived neurotrophic factor, and the solution association states of these molecules. Calibration methods for the RI, UV, and LS detectors will also be addressed, as well as online methods to determine protein extinction coefficients and dn/dc values both unconjugated and conjugated protein molecules. (c)2001 Elsevier Science.

  20. Dosimetry Determines the Initial OH Radical Concentration in Fast Photochemical Oxidation of Proteins (FPOP)

    Science.gov (United States)

    Niu, Ben; Zhang, Hao; Giblin, Daryl; Rempel, Don L.; Gross, Michael L.

    2015-05-01

    Fast photochemical oxidation of proteins (FPOP) employs laser photolysis of hydrogen peroxide to give OH radicals that label amino acid side-chains of proteins on the microsecond time scale. A method for quantitation of hydroxyl radicals after laser photolysis is of importance to FPOP because it establishes a means to adjust the yield of •OH, offers the opportunity of tunable modifications, and provides a basis for kinetic measurements. The initial concentration of OH radicals has yet to be measured experimentally. We report here an approach using isotope dilution gas chromatography/mass spectrometry (GC/MS) to determine quantitatively the initial •OH concentration (we found ~0.95 mM from 15 mM H2O2) from laser photolysis and to investigate the quenching efficiencies for various •OH scavengers.

  1. A cell-free assay to determine the stoichiometry of plasma membrane proteins.

    Science.gov (United States)

    Trigo, Cesar; Vivar, Juan P; Gonzalez, Carlos B; Brauchi, Sebastian

    2013-04-01

    Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.

  2. Retinoblastoma protein functions as a molecular switch determining white versus brown adipocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Jacob B; Jørgensen, Claus; Petersen, Rasmus K

    2004-01-01

    Adipocyte precursor cells give raise to two major cell populations with different physiological roles: white and brown adipocytes. Here we demonstrate that the retinoblastoma protein (pRB) regulates white vs. brown adipocyte differentiation. Functional inactivation of pRB in wild-type mouse embryo...... into adipocytes with a gene expression pattern and mitochondria content resembling brown adipose tissue. pRB-deficient MEFs exhibit an increased expression of the Forkhead transcription factor Foxc2 and its target gene cAMP-dependent protein kinase regulatory subunit RIalpha, resulting in increased c...... transdifferentiation of white into brown adipocytes in response to beta3-adrenergic receptor agonist treatment. We propose that pRB acts as a molecular switch determining white vs. brown adipogenesis, suggesting a previously uncharacterized function of this key cell cycle regulator in adipocyte lineage commitment...

  3. Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

    KAUST Repository

    Li, Jianping

    2013-06-05

    Magic angle spinning solid-state NMR is a unique technique to study atomic-resolution structure of biomacromolecules which resist crystallization or are too large to study by solution NMR techniques. However, difficulties in obtaining sufficient number of long-range distance restraints using dipolar coupling based spectra hamper the process of structure determination of proteins in solid-state NMR. In this study it is shown that high-resolution structure of proteins in solid phase can be determined without the use of traditional dipolar-dipolar coupling based distance restraints by combining the measurements of pseudocontact shifts (PCSs) with Rosetta calculations. The PCSs were generated by chelating exogenous paramagnetic metal ions to a tag 4-mercaptomethyl-dipicolinic acid, which is covalently attached to different residue sites in a 56-residue immunoglobulin-binding domain of protein G (GB1). The long-range structural restraints with metal-nucleus distance of up to ∼20 Å are quantitatively extracted from experimentally observed PCSs, and these are in good agreement with the distances back-calculated using an X-ray structure model. Moreover, we demonstrate that using several paramagnetic ions with varied paramagnetic susceptibilities as well as the introduction of paramagnetic labels at different sites can dramatically increase the number of long-range restraints and cover different regions of the protein. The structure generated from solid-state NMR PCSs restraints combined with Rosetta calculations has 0.7 Å root-mean-square deviation relative to X-ray structure. © 2013 American Chemical Society.

  4. Spectrophotometric Determination of Phenolic Antioxidants in the Presence of Thiols and Proteins

    Directory of Open Access Journals (Sweden)

    Aslı Neslihan Avan

    2016-08-01

    Full Text Available Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET-based total antioxidant capacity (TAC assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC, and ferric reducing antioxidant power (FRAP, were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol and (phenol + protein mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II compounds were added to stabilize the thiol components in the form of Hg(II-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols mixtures.

  5. ALMOST: an all atom molecular simulation toolkit for protein structure determination.

    Science.gov (United States)

    Fu, Biao; Sahakyan, Aleksandr B; Camilloni, Carlo; Tartaglia, Gian Gaetano; Paci, Emanuele; Caflisch, Amedeo; Vendruscolo, Michele; Cavalli, Andrea

    2014-05-30

    Almost (all atom molecular simulation toolkit) is an open source computational package for structure determination and analysis of complex molecular systems including proteins, and nucleic acids. Almost has been designed with two primary goals: to provide tools for molecular structure determination using various types of experimental measurements as conformational restraints, and to provide methods for the analysis and assessment of structural and dynamical properties of complex molecular systems. The methods incorporated in Almost include the determination of structural and dynamical features of proteins using distance restraints derived from nuclear Overhauser effect measurements, orientational restraints obtained from residual dipolar couplings and the structural restraints from chemical shifts. Here, we present the first public release of Almost, highlight the key aspects of its computational design and discuss the main features currently implemented. Almost is available for the most common Unix-based operating systems, including Linux and Mac OS X. Almost is distributed free of charge under the GNU Public License, and is available both as a source code and as a binary executable from the project web site at http://www.open-almost.org. Interested users can follow and contribute to the further development of Almost on http://sourceforge.net/projects/almost.

  6. Spectrophotometric Determination of Phenolic Antioxidants in the Presence of Thiols and Proteins

    Science.gov (United States)

    Avan, Aslı Neslihan; Demirci Çekiç, Sema; Uzunboy, Seda; Apak, Reşat

    2016-01-01

    Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET)-based total antioxidant capacity (TAC) assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC), and ferric reducing antioxidant power (FRAP), were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols) mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol) and (phenol + protein) mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II) compounds were added to stabilize the thiol components in the form of Hg(II)-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols) mixtures. PMID:27529232

  7. Accurate determination of the diffusion coefficient of proteins by Fourier analysis with whole column imaging detection.

    Science.gov (United States)

    Zarabadi, Atefeh S; Pawliszyn, Janusz

    2015-02-17

    Analysis in the frequency domain is considered a powerful tool to elicit precise information from spectroscopic signals. In this study, the Fourier transformation technique is employed to determine the diffusion coefficient (D) of a number of proteins in the frequency domain. Analytical approaches are investigated for determination of D from both experimental and data treatment viewpoints. The diffusion process is modeled to calculate diffusion coefficients based on the Fourier transformation solution to Fick's law equation, and its results are compared to time domain results. The simulations characterize optimum spatial and temporal conditions and demonstrate the noise tolerance of the method. The proposed model is validated by its application for the electropherograms from the diffusion path of a set of proteins. Real-time dynamic scanning is conducted to monitor dispersion by employing whole column imaging detection technology in combination with capillary isoelectric focusing (CIEF) and the imaging plug flow (iPF) experiment. These experimental techniques provide different peak shapes, which are utilized to demonstrate the Fourier transformation ability in extracting diffusion coefficients out of irregular shape signals. Experimental results confirmed that the Fourier transformation procedure substantially enhanced the accuracy of the determined values compared to those obtained in the time domain.

  8. Molecular recognition of the neurotransmitter acetylcholine by an acetylcholine binding protein reveals determinants of binding to nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Jeppe A Olsen

    Full Text Available Despite extensive studies on nicotinic acetylcholine receptors (nAChRs and homologues, details of acetylcholine binding are not completely resolved. Here, we report the crystal structure of acetylcholine bound to the receptor homologue acetylcholine binding protein from Lymnaea stagnalis. This is the first structure of acetylcholine in a binding pocket containing all five aromatic residues conserved in all mammalian nAChRs. The ligand-protein interactions are characterized by contacts to the aromatic box formed primarily by residues on the principal side of the intersubunit binding interface (residues Tyr89, Trp143 and Tyr185. Besides these interactions on the principal side, we observe a cation-π interaction between acetylcholine and Trp53 on the complementary side and a water-mediated hydrogen bond from acetylcholine to backbone atoms of Leu102 and Met114, both of importance for anchoring acetylcholine to the complementary side. To further study the role of Trp53, we mutated the corresponding tryptophan in the two different acetylcholine-binding interfaces of the widespread α4β2 nAChR, i.e. the interfaces α4(+β2(- and α4(+α4(-. Mutation to alanine (W82A on the β2 subunit or W88A on the α4 subunit significantly altered the response to acetylcholine measured by oocyte voltage-clamp electrophysiology in both interfaces. This shows that the conserved tryptophan residue is important for the effects of ACh at α4β2 nAChRs, as also indicated by the crystal structure. The results add important details to the understanding of how this neurotransmitter exerts its action and improves the foundation for rational drug design targeting these receptors.

  9. An algebraic geometry approach to protein structure determination from NMR data.

    Science.gov (United States)

    Wang, Lincong; Mettu, Ramgopal R; Donald, Bruce Randall

    2005-01-01

    Our paper describes the first provably-efficient algorithm for determining protein structures de novo, solely from experimental data. We show how the global nature of a certain kind of NMR data provides quantifiable complexity-theoretic benefits, allowing us to classify our algorithm as running in polynomial time. While our algorithm uses NMR data as input, it is the first polynomial-time algorithm to compute high-resolution structures de novo using any experimentally-recorded data, from either NMR spectroscopy or X-Ray crystallography. Improved algorithms for protein structure determination are needed, because currently, the process is expensive and time-consuming. For example, an area of intense research in NMR methodology is automated assignment of nuclear Overhauser effect (NOE) restraints, in which structure determination sits in a tight inner-loop (cycle) of assignment/refinement. These algorithms are very time-consuming, and typically require a large cluster. Thus, algorithms for protein structure determination that are known to run in polynomial time and provide guarantees on solution accuracy are likely to have great impact in the long-term. Methods stemming from a technique called "distance geometry embedding" do come with provable guarantees, but the NP-hardness of these problem formulations implies that in the worst case these techniques cannot run in polynomial time. We are able to avoid the NP-hardness by (a) some mild assumptions about the protein being studied, (b) the use of residual dipolar couplings (RDCs) instead of a dense network of NOEs, and (c) novel algorithms and proofs that exploit the biophysical geometry of (a) and (b), drawing on a variety of computer science, computational geometry, and computational algebra techniques. In our algorithm, RDC data, which gives global restraints on the orientation of internuclear bond vectors, is used in conjunction with very sparse NOE data to obtain a polynomial-time algorithm for protein structure

  10. Determination of energy and protein requirements for crossbred Holstein × Gyr preweaned dairy calves.

    Science.gov (United States)

    Silva, A L; Marcondes, M I; Detmann, E; Campos, M M; Machado, F S; Filho, S C Valadares; Castro, M M D; Dijkstra, J

    2017-02-01

    The objective was to quantify the energy and protein nutritional requirements of Holstein × Gyr crossbred preweaned dairy calves until 64 d of age. Thirty-nine Holstein × Gyr crossbred male calves with an average initial live weight (mean ± SEM; for all next values) of 36 ± 1.0 kg were used. Five calves were slaughtered at 4 d of life to estimate the animals' initial body composition (reference group). The remaining 34 calves were distributed in a completely randomized design in a 3 × 2 factorial arrangement consisting of 3 levels of milk (2, 4, or 8 L/d) and 2 levels of starter feed (presence or absence in diet). At 15 and 45 d of life, 4 animals from each treatment were subjected to digestibility trials with total collection of feces (for 72 h) and urine (for 24 h). At 64 d of age, all animals were slaughtered, their gastro-intestinal tract was washed to determine the empty body weight (EBW; kg), and their body tissues were sampled for subsequent analyses. The net energy requirement for maintenance was estimated using an exponential regression between metabolizable energy intake and heat production (both in Mcal/EBW(0.75) per d) and was 74.3 ± 5.7 kcal/EBW(0.75) per d, and was not affected by inclusion of starter feed in the diet. The metabolizable energy requirement for maintenance was determined at the point of zero energy retention in the body and was 105.2 ± 5.8 kcal/EBW(0.75) per d. The net energy for gain was estimated using the EBW and the empty body gain (EBG; kg/d) as 0.0882 ± 0.0028 × EBW(0.75) × EBG(0.9050±0.0706). The metabolizable energy efficiency for gain (kg) of the milk was 57.4 ± 3.45%, and the kg of the starter feed was 39.3 ± 2.09%. The metabolizable protein requirement for maintenance was 3.52 ± 0.34 g/BW(0.75) per d. The net protein required for each kilogram gained was estimated as 119.1 ± 32.9 × EBW(0.0663±0.059). The metabolizable protein efficiency for gain was 77 ± 8.5% and was not affected by inclusion of starter feed

  11. Protein folding and wring resonances

    DEFF Research Database (Denmark)

    Bohr, Jakob; Bohr, Henrik; Brunak, Søren

    1997-01-01

    The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested that prot......The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested...... that protein folding takes place when the amplitude of a wring excitation becomes so large that it is energetically favorable to bend the protein backbone. The condition under which such structural transformations can occur is found, and it is shown that both cold and hot denaturation (the unfolding...

  12. Outer membrane protein DsrA is the major fibronectin-binding determinant of Haemophilus ducreyi.

    Science.gov (United States)

    Leduc, Isabelle; White, C Dinitra; Nepluev, Igor; Throm, Robert E; Spinola, Stanley M; Elkins, Christopher

    2008-04-01

    The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains.

  13. Determining protein biomarkers for DLBCL using FFPE tissues from HIV negative and HIV positive patients.

    Science.gov (United States)

    Magangane, Pumza; Sookhayi, Raveendra; Govender, Dhirendra; Naidoo, Richard

    2016-12-01

    DLBCL is the most common lymphoma subtype occurring in older populations as well as in younger HIV infected patients. The current treatment options for DLBCL are effective for most patients yet the relapse rate is high. While many biomarkers for DLBCL exist, they are not in clinical use due to low sensitivity and specificity. In addition, these biomarkers have not been studied in the HIV context. Therefore, the identification of new biomarkers for HIV negative and HIV positive DLBCL, may lead to a better understanding of the disease pathology and better therapeutic design. Protein biomarkers for DLBCL were determined using MALDI imaging mass spectrometry (IMS) and characterised using LC-MS. The expression of one of the biomarkers, heat shock protein (Hsp) 70, was confirmed on a separate cohort of samples using immunohistochemistry. The biomarkers identified in the study consisted of four protein clusters including glycolytic enzymes, ribosomal proteins, histones and collagen. These proteins could differentiate between control and tumour tissue, and the DLBCL immunohistochemical subtypes in both cohorts. The majority (41/52) of samples in the confirmation cohort were negative for Hsp70 expression. The HIV positive DLBCL cases had a higher percentage of cases expressing Hsp70 than their HIV negative counterparts. The non-GC subtype also frequently overexpressed Hsp70, confirming MALDI IMS data. The expression of Hsp70 did not correlate with survival in both the HIV negative and HIV positive cohort. This study identified potential biomarkers for HIV negative and HIV positive DLBCL from FFPE tissue sections. These may be used as diagnostic and prognostic markers complementary to current clinical management programmes for DLBCL.

  14. Exploring Structure, Dynamics, and Topology of Nitroxide Spin-Labeled Proteins Using Continuous-Wave Electron Paramagnetic Resonance Spectroscopy.

    Science.gov (United States)

    Altenbach, Christian; López, Carlos J; Hideg, Kálmán; Hubbell, Wayne L

    2015-01-01

    Structural and dynamical characterization of proteins is of central importance in understanding the mechanisms underlying their biological functions. Site-directed spin labeling (SDSL) combined with continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy has shown the capability of providing this information with site-specific resolution under physiological conditions for proteins of any degree of complexity, including those associated with membranes. This chapter introduces methods commonly employed for SDSL and describes selected CW EPR-based methods that can be applied to (1) map secondary and tertiary protein structure, (2) determine membrane protein topology, (3) measure protein backbone flexibility, and (4) reveal the existence of conformational exchange at equilibrium.

  15. Extracytoplasmic proteases determining the cleavage and release of secreted proteins, lipoproteins, and membrane proteins in Bacillus subtilis

    NARCIS (Netherlands)

    Krishnappa, Laxmi; Dreisbach, Annette; Otto, Andreas; Goosens, Vivianne J; Cranenburgh, Rocky M; Harwood, Colin R; Becher, Dörte; van Dijl, Jan Maarten

    2013-01-01

    Gram-positive bacteria are known to export many proteins to the cell wall and growth medium, and accordingly, many studies have addressed the respective protein export mechanisms. In contrast, very little is known about the subsequent fate of these proteins. The present studies were therefore aimed

  16. Longitudinal determination of serum placental protein 13 during development of preeclampsia

    OpenAIRE

    Huppertz, Berthold; Sammar, Marei; Chefetz, Ilana; Neumaier-Wagner, Peruka; Bartz, Clemens; Meiri, Hamutal

    2008-01-01

    Objective: To determine maternal serum placental protein 13 (PP13) in normal pregnancy and preeclampsia. Methods: A prospective, longitudinal study with 41 normal pregnant women, 18 cases with preterm delivery or cervix insufficiency and 4 with developing late-onset preeclampsia. Six hundred and sixty-six maternal blood samples were obtained every 2-4 weeks starting at 5-8 weeks gestation (10-12 samples/patient) and tested for serum PP13 by ELISA. Results: In normal pregnant women delivering ...

  17. Determination of the amino acid requirements for a protein hinge in triosephosphate isomerase.

    OpenAIRE

    Sun, J.; Sampson, N. S.

    1998-01-01

    We have determined the sequence requirements for a protein hinge in triosephosphate isomerase. The codons encoding the hinge at the C-terminus of the active-site lid of triosephosphate isomerase were replaced with a genetic library of all possible 8,000 amino acid combinations. The most active of these 8,000 mutants were selected using in vivo complementation of a triosephosphate isomerase deficient strain of E. coli, DF502. Approximately 3% of the mutants complement DF502 with an activity th...

  18. Comparison of LECO FP-228 "nitrogen determinator" with AOAC copper catalyst Kjeldahl method for crude protein.

    Science.gov (United States)

    Sweeney, R A; Rexroad, P R

    1987-01-01

    The LECO FP-228 "Nitrogen Determinator" was compared with the AOAC copper catalyst Kjeldahl method, 7.033-7.037, for the determination of crude protein in feed materials. The completely microprocessor-controlled instrument determines nitrogen by measuring the nitrogen gas following combustion of the sample; it was easy to operate and broadly applicable. A wide variety of feed materials of various nitrogen levels were analyzed in one mixed sequence. Results were precise, accurate, and rapid. Analysis time for one sample was approximately 3 min. Fourteen samples containing 2.5-15.5% N were selected for study and consisted of meals, grains, forages, and standard organic materials. The overall mean for the 14 samples by the LECO combustion method was 8.61% N compared with an overall mean of 8.58% N for the AOAC Kjeldahl method. Within-sample standard deviations for the LECO combustion method ranged from 0.013 to 0.052% N with a pooled standard deviation (SD) of 0.033% N for the 14 samples. Standard deviations for the AOAC Kjeldahl method ranged from 0.006 to 0.035% N with a pooled SD of 0.022% N. Combined average recovery of nitrogen from tryptophan, lysine-HCl, and EDTA determined by the LECO combustion method was 99.94% compared to 99.88% determined by the AOAC Kjeldahl method.

  19. Spectrophotometric determination of total proteins in blood plasma: a comparative study among dye-binding methods

    Directory of Open Access Journals (Sweden)

    Dimas Augusto Morozin Zaia

    2005-05-01

    Full Text Available A comparative study between the biuret method (standard method for total proteins and spectrophotometric methods using dyes (Bradford, 3',3",5',5"-tetrabromophenolphthalein ethyl ester-TBPEE, and erythrosin-B was carried out for the determination of total proteins in blood plasma from rats. Bradford method showed the highest sensitivity for proteins and biuret method showed the lowest. For all the methods, the absorbance for different proteins (BSA, casein, and egg albumin was measured and Bradford method showed the lowest variation of absorbance. The concentration of total protein obtained by using Bradford method was not statistically different (p>0.05 from concentration of total protein obtained by the biuret method. But in regard to erythrosin-B and TBPEE methods the concentrations of total protein were statistically different (pA determinação de proteínas totais em plasma sangüíneo é importante em diversas áreas de pesquisa. Um estudo comparativo entre o método de biureto (método padrão para proteínas totais e diversos métodos que utilizam corantes (Bradford, tetrabromofenolftaleína etil éster-TBPEE, e eritrosina-B foi realizado para a determinação de proteínas totais em plasma sangüíneo de ratos. O método de Bradford mostrou a maior sensibilidade para proteínas e o de biureto a menor. Para todos os métodos, as absorbâncias para diferentes proteínas (BSA, caseína, e ovoalbumina foram medidas e o método de Bradford mostrou a menor variação da absorbância. Utilizando o método de Bradford a concentração de proteínas totais obtida não foi estatisticamente diferente (p>0.05 daquela obtida pelo método do biureto. Porém, para os métodos da eritrosina-B e TBPEE as concentrações de proteínas totais foram estatisticamente diferentes (p<0.05 da obtida pelo método de biureto. Portanto o método de Bradford pode ser utilizado no lugar do método de biureto para a determinação de proteínas totais em plasma sangüíneo.

  20. N-H stretching modes around 3300 wavenumber from peptide backbones observed by chiral sum frequency generation vibrational spectroscopy.

    Science.gov (United States)

    Fu, Li; Wang, Zhuguang; Yan, Elsa C Y

    2014-09-01

    We present a detailed analysis of the molecular origin of the chiral sum frequency generation (SFG) signals of proteins and peptides at interfaces in the N-H stretching vibrational region. The N-H stretching can be a probe for investigating structural and functional properties of proteins, but remains technically difficult to analyze due to the overlapping with the O-H stretching of water molecules. Chiral SFG spectroscopy offers unique tools to study the N-H stretching from proteins at interfaces without interference from the water background. However, the molecular origin of the N-H stretching signals of proteins is still unclear. This work provides a justification of the origin of chiral N-H signals by analyzing the vibrational frequencies, examining chiral SFG theory, studying proton (hydrogen/deuterium) exchange kinetics, and performing optical control experiments. The results demonstrate that the chiral N-H stretching signals at ~3300 cm(-1) originate from the amide group of the protein backbones. This chiral N-H stretching signal offers an in situ, real-time, and background-free probe for interrogating the protein structures and dynamics at interfaces at the molecular level. © 2014 Wiley Periodicals, Inc.

  1. A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

    Science.gov (United States)

    Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

    2017-09-15

    Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Fast spatiotemporal correlation spectroscopy to determine protein lateral diffusion laws in live cell membranes.

    Science.gov (United States)

    Di Rienzo, Carmine; Gratton, Enrico; Beltram, Fabio; Cardarelli, Francesco

    2013-07-23

    Spatial distribution and dynamics of plasma-membrane proteins are thought to be modulated by lipid composition and by the underlying cytoskeleton, which forms transient barriers to diffusion. So far this idea was probed by single-particle tracking of membrane components in which gold particles or antibodies were used to individually monitor the molecules of interest. Unfortunately, the relatively large particles needed for single-particle tracking can in principle alter the very dynamics under study. Here, we use a method that makes it possible to investigate plasma-membrane proteins by means of small molecular labels, specifically single GFP constructs. First, fast imaging of the region of interest on the membrane is performed. For each time delay in the resulting stack of images the average spatial correlation function is calculated. We show that by fitting the series of correlation functions, the actual protein "diffusion law" can be obtained directly from imaging, in the form of a mean-square displacement vs. time-delay plot, with no need for interpretative models. This approach is tested with several simulated 2D diffusion conditions and in live Chinese hamster ovary cells with a GFP-tagged transmembrane transferrin receptor, a well-known benchmark of membrane-skeleton-dependent transiently confined diffusion. This approach does not require extraction of the individual trajectories and can be used also with dim and dense molecules. We argue that it represents a powerful tool for the determination of kinetic and thermodynamic parameters over very wide spatial and temporal scales.

  3. Determination of Protein and Starch Content in Whole Maize Kernel by Near Infrared Reflectance Spectroscopy

    Institute of Scientific and Technical Information of China (English)

    WEI Liang-ming; YAN Yan-lu; DAI Jing-rui

    2004-01-01

    Using 128 bulk-kernel samples of inbred lines and hybrids,a study was conducted to investigate the feasibility and method of measuring protein and starch contents in intact seeds of maize by near infrared reflectance spectroscopy(NIRS).The chemometric algorithms of partial least square(PLS)regression was used.The results indicated that the calibration models developed by the spectral data pretreatment of first derivative+multivariate scattering correction within the spectral region of 10 000-4 000 cm-1,and first derivative + straight line subtraction in 9 000-4 000 cm-1 were the best for protein and starch,respectively.All these models yielded coefficients of determination of calibration(R2ca1)above 0.97,while R2cv and R2va1 of cross and external validation ranged from 0.92 to 0.95,respectively; however,the root of mean square errors of calibration,cross and external validation(RMSEE,RMSECV and RMSEP)were below l(ranged 0.3-0.7),respectively.This study demonstrated that it is feasible to use NIRS as a rapid,accurate,and none-destructive technique to predict protein and starch contents of whole kernel in the maize quality improvement program.

  4. Backbone exponents of the two-dimensional q-state Potts model: a Monte Carlo investigation.

    Science.gov (United States)

    Deng, Youjin; Blöte, Henk W J; Nienhuis, Bernard

    2004-02-01

    We determine the backbone exponent X(b) of several critical and tricritical q-state Potts models in two dimensions. The critical systems include the bond percolation, the Ising, the q=2-sqrt[3], 3, and 4 state Potts, and the Baxter-Wu model, and the tricritical ones include the q=1 Potts model and the Blume-Capel model. For this purpose, we formulate several efficient Monte Carlo methods and sample the probability P2 of a pair of points connected via at least two independent paths. Finite-size-scaling analysis of P2 yields X(b) as 0.3566(2), 0.2696(3), 0.2105(3), and 0.127(4) for the critical q=2-sqrt[3], 1,2, 3, and 4 state Potts model, respectively. At tricriticality, we obtain X(b)=0.0520(3) and 0.0753(6) for the q=1 and 2 Potts model, respectively. For the critical q-->0 Potts model it is derived that X(b)=3/4. From a scaling argument, we find that, at tricriticality, X(b) reduces to the magnetic exponent, as confirmed by the numerical results.

  5. Toward structural dynamics: protein motions viewed by chemical shift modulations and direct detection of C'N multiple-quantum relaxation.

    Science.gov (United States)

    Mori, Mirko; Kateb, Fatiha; Bodenhausen, Geoffrey; Piccioli, Mario; Abergel, Daniel

    2010-03-17

    Multiple quantum relaxation in proteins reveals unexpected relationships between correlated or anti-correlated conformational backbone dynamics in alpha-helices or beta-sheets. The contributions of conformational exchange to the relaxation rates of C'N coherences (i.e., double- and zero-quantum coherences involving backbone carbonyl (13)C' and neighboring amide (15)N nuclei) depend on the kinetics of slow exchange processes, as well as on the populations of the conformations and chemical shift differences of (13)C' and (15)N nuclei. The relaxation rates of C'N coherences, which reflect concerted fluctuations due to slow chemical shift modulations (CSMs), were determined by direct (13)C detection in diamagnetic and paramagnetic proteins. In well-folded proteins such as lanthanide-substituted calbindin (CaLnCb), copper,zinc superoxide dismutase (Cu,Zn SOD), and matrix metalloproteinase (MMP12), slow conformational exchange occurs along the entire backbone. Our observations demonstrate that relaxation rates of C'N coherences arising from slow backbone dynamics have positive signs (characteristic of correlated fluctuations) in beta-sheets and negative signs (characteristic of anti-correlated fluctuations) in alpha-helices. This extends the prospects of structure-dynamics relationships to slow time scales that are relevant for protein function and enzymatic activity.

  6. Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein*

    Science.gov (United States)

    Rahman, M. Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-01-01

    Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

  7. Calcite formation in soft coral sclerites is determined by a single reactive extracellular protein.

    Science.gov (United States)

    Rahman, M Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-09-09

    Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called "calcite and aragonite seas." Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that -OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature.

  8. Determination of multivalent protein-ligand binding kinetics by second-harmonic correlation spectroscopy.

    Science.gov (United States)

    Sly, Krystal L; Conboy, John C

    2014-11-18

    Binding kinetics of the multivalent proteins peanut agglutinin (PnA) and cholera toxin B subunit (CTB) to a GM1-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer were investigated by both second-harmonic correlation spectroscopy (SHCS) and a traditional equilibrium binding isotherm. Adsorption and desorption rates, as well as binding affinity and binding free energy, for three bulk protein concentrations were determined by SHCS. For PnA binding to GM1, the measured adsorption rate decreased with increasing bulk PnA concentration from (3.7 ± 0.3) × 10(6) M(-1)·s(-1) at 0.43 μM PnA to (1.1 ± 0.1) × 10(5) M(-1)·s(-1) at 12 μM PnA. CTB-GM1 exhibited a similar trend, decreasing from (1.0 ± 0.1) × 10(9) M(-1)·s(-1) at 0.5 nM CTB to (3.5 ± 0.2) × 10(6) M(-1)·s(-1) at 240 nM CTB. The measured desorption rates in both studies did not exhibit any dependence on initial protein concentration. As such, 0.43 μM PnA and 0.5 nM CTB had the strongest measured binding affinities, (3.7 ± 0.8) × 10(9) M(-1) and (2.8 ± 0.5) × 10(13) M(-1), respectively. Analysis of the binding isotherm data suggests there is electrostatic repulsion between protein molecules when PnA binds GM1, while CTB-GM1 demonstrates positive ligand-ligand cooperativity. This study provides additional insight into the complex interactions between multivalent proteins and their ligands and showcases SHCS for examining these complex yet technologically important protein-ligand complexes used in biosensors, immunoassays, and other biomedical diagnostics.

  9. Turnover of amyloid precursor protein family members determines their nuclear signaling capability.

    Science.gov (United States)

    Gersbacher, Manuel T; Goodger, Zoë V; Trutzel, Annette; Bundschuh, Diana; Nitsch, Roger M; Konietzko, Uwe

    2013-01-01

    The amyloid precursor protein (APP) as well as its homologues, APP-like protein 1 and 2 (APLP1 and APLP2), are cleaved by α-, β-, and γ-secretases, resulting in the release of their intracellular domains (ICDs). We have shown that the APP intracellular domain (AICD) is transported to the nucleus by Fe65 where they jointly bind the histone acetyltransferase Tip60 and localize to spherical nuclear complexes (AFT complexes), which are thought to be sites of transcription. We have now analyzed the subcellular localization and turnover of the APP family members. Similarly to AICD, the ICD of APLP2 localizes to spherical nuclear complexes together with Fe65 and Tip60. In contrast, the ICD of APLP1, despite binding to Fe65, does not translocate to the nucleus. In addition, APLP1 predominantly localizes to the plasma membrane, whereas APP and APLP2 are detected in vesicular structures. APLP1 also demonstrates a much slower turnover of the full-length protein compared to APP and APLP2. We further show that the ICDs of all APP family members are degraded by the proteasome and that the N-terminal amino acids of ICDs determine ICD degradation rate. Together, our results suggest that different nuclear signaling capabilities of APP family members are due to different rates of full-length protein processing and ICD proteasomal degradation. Our results provide evidence in support of a common nuclear signaling function for APP and APLP2 that is absent in APLP1, but suggest that APLP1 has a regulatory role in the nuclear translocation of APP family ICDs due to the sequestration of Fe65.

  10. Combustion method for determination of crude protein in meat and meat products: collaborative study.

    Science.gov (United States)

    King-Brink, M; Sebranek, J G

    1993-01-01

    Twelve laboratories participated in a collaborative study to compare a combustion method with the AOAC mercury catalyst Kjeldahl method (928.08) for the determination of crude protein in meat and meat products. Three different combustion instruments were used; consequently, the combustion method for this study is written in generic terms describing the principle, the apparatus specifications, and the performance requirements needed. Fifteen sample pairs were used for the study; each pair consisted of the same commercial meat product from each of 2 different manufacturers. Protein content of all samples ranged from about 10 to 20%. In addition, nicotinic acid and lysine monohydrochloride were used as standards to assess combustion equipment performance. All laboratories and all instruments performed the combustion method satisfactorily on the basis of results for the standards. For the meat samples, repeatability standard deviations (Sr) ranged from 0.11 to 0.40 for the Kjeldahl method and from 0.12 to 0.41 for the combustion method; the repeatability relative standard deviations (RSDr) ranged from 0.82 to 2.41% and from 0.60 to 2.23% for the Kjeldahl and combustion methods, respectively. Reproducibility standard deviations (SR) ranged from 0.20 to 0.49 for the Kjeldahl method and from 0.18 to 0.46 for the combustion method, whereas the reproducibility relative standard deviations (RSDR) ranged from 1.59 to 2.84% for the Kjeldahl method and from 1.32 to 3.35% for the combustion method. Overall grand means were 15.59% protein for the Kjeldahl method and 15.75% protein for the combustion method. The combustion method was adopted first action by AOAC International.

  11. Determinants of protein elution rates from preparative ion-exchange adsorbents.

    Science.gov (United States)

    Angelo, James M; Lenhoff, Abraham M

    2016-04-01

    The rate processes involved in elution in preparative chromatography can affect both peak resolution and hence selectivity as well as practical factors such as facility fit. These processes depend on the physical structure of the adsorbent particles, the amount of bound solute, the solution conditions for operation or some combination of these factors. Ion-exchange adsorbents modified with covalently attached or grafted polymer layers have become widely used in preparative chromatography. Their often easily accessible microstructures offer substantial binding capacities for biomolecules, but elution has sometimes been observed to be undesirably slow. In order to determine which physicochemical phenomena control elution behavior, commercially available cellulosic, dextran-grafted and unmodified agarose materials were characterized here by their elution profiles at various conditions, including different degrees of loading. Elution data were analyzed under the assumption of purely diffusion-limited control, including the role of pore structure properties such as porosity and tortuosity. In general, effective elution rates decreased with the reduction of accessible pore volume, but differences among different proteins indicated the roles of additional factors. Additional measurements and analysis, including the use of confocal laser scanning microscopy to observe elution within single chromatographic particles, indicated the importance of protein association within the particle during elution. The use of protein stabilizing agents was explored in systems presenting atypical elution behavior, and l-arginine and disaccharide excipients were shown to alleviate the effects for one protein, lysozyme, in the presence of sodium chloride. Incorporation of these excipients into eluent buffer gave rise to faster elution and significantly lower pool volumes in elution from polymer-modified adsorbents.

  12. Orientation determination of protein helical secondary structures using linear and nonlinear vibrational spectroscopy.

    Science.gov (United States)

    Nguyen, Khoi Tan; Le Clair, Stéphanie V; Ye, Shuji; Chen, Zhan

    2009-09-10

    In this paper, we systematically presented the orientation determination of protein helical secondary structures using vibrational spectroscopic methods, particularly, nonlinear sum frequency generation (SFG) vibrational spectroscopy, along with linear vibrational spectroscopic techniques such as infrared spectroscopy and Raman scattering. SFG amide I signals can be collected using different polarization combinations of the input laser beams and output signal beam to measure the second-order nonlinear optical susceptibility components of the helical amide I modes, which are related to their molecular hyperpolarizability elements through the orientation distribution of these helices. The molecular hyperpolarizability elements of amide I modes of a helix can be calculated based on the infrared transition dipole moment and Raman polarizability tensor of the helix; these quantities are determined by using the bond additivity model to sum over the individual infrared transition dipole moments and Raman polarizability tensors, respectively, of the peptide units (or the amino acid residues). The computed overall infrared transition dipole moment and Raman polarizability tensor of a helix can be validated by experimental data using polarized infrared and polarized Raman spectroscopy on samples with well-aligned helical structures. From the deduced SFG hyperpolarizability elements and measured SFG second-order nonlinear susceptibility components, orientation information regarding helical structures can be determined. Even though such orientation information can also be measured using polarized infrared or polarized Raman amide I signals, SFG has a much lower detection limit, which can be used to study the orientation of a helix when its surface coverage is much lower than a monolayer. In addition, the combination of different vibrational spectroscopic techniques, for example, SFG and attenuated total reflectance Fourier transform infrared spectroscopy, provides more

  13. 22 Protein-Protein Interactions Determine IgE Reactivity to Polygalacturonase From Cupressus sempervirens Pollen

    OpenAIRE

    Shahali, Youcef; Sutra, Jean-Pierre; Chollet-Martin, Sylvie; Haddad, Iman; Vinh, Joëlle; Mari, Adriano; Charpin, Denis; Sénéchal, Hélène; Poncet, Pascal

    2012-01-01

    Background In a recent proteomic study, we identified in Italian cypress (Cupressus sempervirens, Cups) pollen grains, 2 proteins at 43 and 60 kDa, homologous to already known Cupressaceae polygalacturonase (PG) proteins. The 60-kDa PG is suspected to be a multi-protein complex including the 43-kDa PG and one or more proteins with lectin-like properties Objective In the present study, cypress pollen PGs were further characterized and the molecular basis of their allergenicity including the pr...

  14. The Synthesis and Structural Characterization of Graft Copolymers Composed of γ-PGA Backbone and Oligoesters Pendant Chains

    Science.gov (United States)

    Kwiecień, Iwona; Radecka, Iza; Kowalczuk, Marek; Jelonek, Katarzyna; Orchel, Arkadiusz; Adamus, Grażyna

    2017-07-01

    The novel copolymers composed of poly-γ-glutamic acid (γ-PGA) and oligoesters have been developed. The structures of the obtained copolymers including variety of end groups were determined at the molecular level with the aid of electrospray ionization multistage mass spectrometry (ESI-MSn). The fragmentation experiment performed for the selected sodium adducts of the copolymers confirmed that the developed methods lead to the formation of graft copolymers composed of poly-γ-glutamic acid (γ-PGA) backbone and oligoesters pendant chains. Moreover, it was established that fragmentation of selected sodium adducts of graft copolymers proceeded via random breakage of amide bonds along the backbone and ester bonds of the oligoesters pendant chains. Considering potential applications of the synthesized copolymers in the area of biomaterials, the hydrolytic degradation under laboratory conditions and in vitro cytotoxicity tests were performed. The ESI-MSn technique applied in this study has been proven to be a useful tool in structural studies of novel graft copolymers as well as their degradation products.

  15. Purification, crystallization and preliminary crystallographic analysis of the SpaA backbone-pilin subunit from probiotic Lactobacillus rhamnosus GG.

    Science.gov (United States)

    Singh, Deepak; von Ossowski, Ingemar; Palva, Airi; Krishnan, Vengadesan

    2013-10-01

    Lactobacillus rhamnosus GG, a widely used Gram-positive probiotic strain, is clinically well known for its perceived health-promoting effects. It has recently been shown to display proteinaceous pilus fibres (called SpaCBA) on its cell surface. Structurally, SpaCBA pili possess a characteristic three-pilin polymerized architecture, with repeating SpaA major pilins that form the backbone and two types of minor subunits (SpaB and SpaC). In this study, recombinant SpaA protein was purified, characterized and crystallized. The crystals diffracted to a resolution of 2.0 Å and belonged to space group C2, with unit-cell parameters a=227.9, b=63.2, c=104.3 Å, β=95.1°.

  16. A rapid and sensitive method for the determination of hypusine in proteins and its distribution and developmental changes.

    Science.gov (United States)

    Sano, A; Miyake, M; Kakimoto, Y

    1984-07-30

    A simple and sensitive method for determining hypusine in proteins was developed. A greater part of amino acids in the acid hydrolysate of proteins was separated from hypusine by treatment with an ion-exchange resin. The sample containing partially purified hypusine was then analyzed by high-performance liquid chromatography using the post-column derivatization method with o-phthalaldehyde. The recovery rate of hypusine through the overall procedure was more than 95%. Using this method, the distribution and developmental changes of hypusine in proteins were determined. The amino acid was found in proteins of all examined organs of rat. Its concentration was 5-40 nmol/g protein. The subcellular distribution in rat liver was also determined. About 60% of total amount of hypusine was present in the proteins of cytoplasmic and microsomal fractions and its relative concentration was high in the proteins of microsome and lysosome and low in mitochondria. In developing rat, the concentration of hypusine in the brain proteins was relatively high during the first 2 or 3 weeks of postnatal life and then decreased until adulthood. Its concentration in the liver proteins was highest at birth and then decreased continuously to the adult level.

  17. Creation and structure determination of an artificial protein with three complete sequence repeats

    Energy Technology Data Exchange (ETDEWEB)

    Adachi, Motoyasu, E-mail: adachi.motoyasu@jaea.go.jp; Shimizu, Rumi; Kuroki, Ryota [Japan Atomic Energy Agency, Shirakatashirane 2-4, Nakagun Tokaimura, Ibaraki 319-1195 (Japan); Blaber, Michael [Japan Atomic Energy Agency, Shirakatashirane 2-4, Nakagun Tokaimura, Ibaraki 319-1195 (Japan); Florida State University, Tallahassee, FL 32306-4300 (United States)

    2013-11-01

    An artificial protein with three complete sequence repeats was created and the structure was determined by X-ray crystallography. The structure showed threefold symmetry even though there is an amino- and carboxy-terminal. The artificial protein with threefold symmetry may be useful as a scaffold to capture small materials with C3 symmetry. Symfoil-4P is a de novo protein exhibiting the threefold symmetrical β-trefoil fold designed based on the human acidic fibroblast growth factor. First three asparagine–glycine sequences of Symfoil-4P are replaced with glutamine–glycine (Symfoil-QG) or serine–glycine (Symfoil-SG) sequences protecting from deamidation, and His-Symfoil-II was prepared by introducing a protease digestion site into Symfoil-QG so that Symfoil-II has three complete repeats after removal of the N-terminal histidine tag. The Symfoil-QG and SG and His-Symfoil-II proteins were expressed in Eschericha coli as soluble protein, and purified by nickel affinity chromatography. Symfoil-II was further purified by anion-exchange chromatography after removing the HisTag by proteolysis. Both Symfoil-QG and Symfoil-II were crystallized in 0.1 M Tris-HCl buffer (pH 7.0) containing 1.8 M ammonium sulfate as precipitant at 293 K; several crystal forms were observed for Symfoil-QG and II. The maximum diffraction of Symfoil-QG and II crystals were 1.5 and 1.1 Å resolution, respectively. The Symfoil-II without histidine tag diffracted better than Symfoil-QG with N-terminal histidine tag. Although the crystal packing of Symfoil-II is slightly different from Symfoil-QG and other crystals of Symfoil derivatives having the N-terminal histidine tag, the refined crystal structure of Symfoil-II showed pseudo-threefold symmetry as expected from other Symfoils. Since the removal of the unstructured N-terminal histidine tag did not affect the threefold structure of Symfoil, the improvement of diffraction quality of Symfoil-II may be caused by molecular characteristics of

  18. Gender features of functional condition of backbone of teenagers with scoliotic posture

    Directory of Open Access Journals (Sweden)

    Sergiy Afanasiev

    2016-10-01

    Full Text Available Purpose: to study mobility of backbone, endurance of muscles of a trunk and to define gender features of functional condition of backbone at children of the middle school age with scoliotic posture depending on the direction of the top of arch of curvature of spine. Material & Methods: 40 girls and 40 boys, including 18 girls and 18 boys with the right-side deformation of backbone in the thoracic department, the left-side – 22 girls and 22 boys are examined. Results: features of changes of indicators, depending on sex of children and frontage of the top of arch of curvature of spine column, are revealed when studying the level of flexibility of backbone and endurance of muscles of a trunk at children of the middle school age with scoliotic posture. Conclusions: it is established that the level of decrease in flexibility of backbone is higher at boys, than at girls, whereas indicators of contractile ability and tone of muscles of "muscular corset" are higher at boys.

  19. Identification of Immunogenic Determinants of the Spike Protein of SARS-like Coronavirus

    Institute of Scientific and Technical Information of China (English)

    Peng Zhou; Zhenggang Han; Lin-Fa Wang; Zhengli Shi

    2013-01-01

    Bat SARS-like coronavirus (SL-CoV) has a genome organization almost identical to that of SARS-CoV,but the N-terminus of the Spike (S) proteins,which interacts with host receptor and is a major target of neutralizing antibodies against CoVs,of the two viruses has only 63-64% sequence identity.Although there have been reports studying the overall immunogenicity of SSL,knowledge on the precise location of immunodominant determinants for SSL is still lacking.In this study,using a series of truncated expressed SSL fragments and SsL specific mouse sera,we identified two immunogenic determinants for SSL.Importantly,one of the two regions seems to be located in a region not shared by known immunogenic determinants of the SSARS.This finding will be of potential use in future monitoring of SL-CoV infection in bats and spillover animals and in development of more effective vaccine to cover broad protection against this new group of coronaviruses.

  20. Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

    Science.gov (United States)

    Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-15

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

  1. Detection and use of pseudo-translation in determination of protein structures.

    Science.gov (United States)

    Chook, Y M; Lipscomb, W N; Ke, H

    1998-09-01

    Two types of pseudo-translation symmetry, pseudo-twofold translational symmetry and pseudo-body-centered symmetry, have been found in protein crystals of chorismate mutase and cyclophilin C. Statistics on diffraction intensity from these two crystals showed that the presence of pseudo-translations in atomic space yielded a distribution of systematically strong and weak reflections at low resolution. The diffraction pattern resulting from pseudo-translational symmetry was apparently similar to that from true crystallographic symmetry at 4 A resolution, but was distinct at high resolution. Pseudo-translation can be detected by comparing the average magnitudes of certain parity groups of reflections in three-dimensional hkl space. Based on the structures of chorismate mutase and cyclophilin C, the ratio of >1.2 for the average magnitudes of parity groups is sufficient to indicate the existence of pseudo-translation. Although pseudo-translation often makes structure determination more difficult, the additional information of pseudo-translation has been used successfully in the structure determination of chorismate mutase by multiple isomorphous replacement and of cyclophilin C by molecular replacement. Thus, examination of pseudo-translation is recommended at an early stage of structure determination.

  2. Protein crystallography for aspiring crystallographers or how to avoid pitfalls and traps in macromolecular structure determination.

    Science.gov (United States)

    Wlodawer, Alexander; Minor, Wladek; Dauter, Zbigniew; Jaskolski, Mariusz

    2013-11-01

    The number of macromolecular structures deposited in the Protein Data Bank now approaches 100,000, with the vast majority of them determined by crystallographic methods. Thousands of papers describing such structures have been published in the scientific literature, and 20 Nobel Prizes in chemistry or medicine have been awarded for discoveries based on macromolecular crystallography. New hardware and software tools have made crystallography appear to be an almost routine (but still far from being analytical) technique and many structures are now being determined by scientists with very limited experience in the practical aspects of the field. However, this apparent ease is sometimes illusory and proper procedures need to be followed to maintain high standards of structure quality. In addition, many noncrystallographers may have problems with the critical evaluation and interpretation of structural results published in the scientific literature. The present review provides an outline of the technical aspects of crystallography for less experienced practitioners, as well as information that might be useful for users of macromolecular structures, aiming to show them how to interpret (but not overinterpret) the information present in the coordinate files and in their description. A discussion of the extent of information that can be gleaned from the atomic coordinates of structures solved at different resolution is provided, as well as problems and pitfalls encountered in structure determination and interpretation.

  3. Assessment of 7.5% NaCl /6% Dextran-70 (HSD) Effects on Serum or Plasma Protein Determinations

    Science.gov (United States)

    1990-12-26

    determined by modified Lowry, dye-binding, and an automated biuret method, as well as by refractometry , before, and at various times following HSD...protein concentrations determined by the biuret assay or refractometry when dextran serum concentrations exceeded 1.2 g/dl. The in vivo studies... refractometry , before, and at various times following HSD infusion in both euvolemic and hemorrhaged animals. Other studies analyzed plasma protein

  4. Soluble and Membrane-Bound β-Glucosidases Are Involved in Trimming the Xyloglucan Backbone.

    Science.gov (United States)

    Sampedro, Javier; Valdivia, Elene R; Fraga, Patricia; Iglesias, Natalia; Revilla, Gloria; Zarra, Ignacio

    2017-02-01

    In many flowering plants, xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Xyloglucan can be degraded by a suite of exoglycosidases that remove specific sugars. In this work, we show that the xyloglucan backbone, formed by (1→4)-linked β-d-glucopyranosyl residues, can be attacked by two different Arabidopsis (Arabidopsis thaliana) β-glucosidases from glycoside hydrolase family 3. While BGLC1 (At5g20950; for β-glucosidase active against xyloglucan 1) is responsible for all or most of the soluble activity, BGLC3 (At5g04885) is usually a membrane-anchored protein. Mutations in these two genes, whether on their own or combined with mutations in other exoglycosidase genes, resulted in the accumulation of partially digested xyloglucan subunits, such as GXXG, GXLG, or GXFG. While a mutation in BGLC1 had significant effects on its own, lack of BGLC3 had only minor effects. On the other hand, double bglc1 bglc3 mutants revealed a synergistic interaction that supports a role for membrane-bound BGLC3 in xyloglucan metabolism. In addition, bglc1 bglc3 was complemented by overexpression of either BGLC1 or BGLC3 In overexpression lines, BGLC3 activity was concentrated in a microsome-enriched fraction but also was present in soluble form. Finally, both genes were generally expressed in the same cell types, although, in some cases, BGLC3 was expressed at earlier stages than BGLC1 We propose that functional specialization could explain the separate localization of both enzymes, as a membrane-bound β-glucosidase could specifically digest soluble xyloglucan without affecting the wall-bound polymer.

  5. Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β-N-acetylhexosaminidases.

    Science.gov (United States)

    Nyffenegger, Christian; Nordvang, Rune Thorbjørn; Zeuner, Birgitte; Łężyk, Mateusz; Difilippo, Elisabetta; Logtenberg, Madelon J; Schols, Henk A; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

    2015-10-01

    This paper describes the discovery and characterization of two novel β-N-acetylhexosaminidases HEX1 and HEX2, capable of catalyzing the synthesis of human milk oligosaccharides (HMO) backbone structures with fair yields using chitin oligomers as β-N-acetylglucosamine (GlcNAc) donor. The enzyme-encoding genes were identified by functional screening of a soil-derived metagenomic library. The β-N-acetylhexosaminidases were expressed in Escherichia coli with an N-terminal His6-tag and were purified by nickel affinity chromatography. The sequence similarities of the enzymes with their respective closest homologues are 59 % for HEX1 and 51 % for HEX2 on the protein level. Both β-N-acetylhexosaminidases are classified into glycosyl hydrolase family 20 (GH 20) are able to hydrolyze para-nitrophenyl-β-N-acetylglucosamine (pNP-GlcNAc) as well as para-nitrophenyl-β-N-acetylgalactosamine (pNP-GalNAc) and exhibit pH optima of 8 and 6 for HEX1 and HEX2, respectively. The enzymes are able to hydrolyze N-acetylchitooligosaccharides with a degree of polymerization of two, three, and four. The major findings were, that HEX1 and HEX2 catalyze trans-glycosylation reactions with lactose as acceptor, giving rise to the human milk oligosaccharide precursor lacto-N-triose II (LNT2) with yields of 2 and 8 % based on the donor substrate. In total, trans-glycosylation reactions were tested with the disaccharide acceptors β-lactose, sucrose, and maltose, as well as with the monosaccharides galactose and glucose resulting in the successful attachment of GlcNAc to the acceptor in all cases.

  6. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins: Focus on sample preparation and derivatization conditions.

    Science.gov (United States)

    Weber, Daniela; Davies, Michael J; Grune, Tilman

    2015-08-01

    Protein oxidation is involved in regulatory physiological events as well as in damage to tissues and is thought to play a key role in the pathophysiology of diseases and in the aging process. Protein-bound carbonyls represent a marker of global protein oxidation, as they are generated by multiple different reactive oxygen species in blood, tissues and cells. Sample preparation and stabilization are key steps in the accurate quantification of oxidation-related products and examination of physiological/pathological processes. This review therefore focuses on the sample preparation processes used in the most relevant methods to detect protein carbonyls after derivatization with 2,4-dinitrophenylhydrazine with an emphasis on measurement in plasma, cells, organ homogenates, isolated proteins and organelles. Sample preparation, derivatization conditions and protein handling are presented for the spectrophotometric and HPLC method as well as for immunoblotting and ELISA. An extensive overview covering these methods in previously published articles is given for researchers who plan to measure protein carbonyls in different samples.

  7. Protein intake as a determinant factor of physical activity in older persons

    Directory of Open Access Journals (Sweden)

    Rina K. Kusumaratna

    2016-02-01

    Full Text Available Worldwide, the proportion of people aged 60 and over is growing faster than any other age group. It has been well-established that the aging process can be associated with increased susceptibility to chronic conditions, disability, and co-morbidity, which however may be minimized or even partially reversed by physical activity. The assessment of physical activity is becoming an increasingly important component in the evaluation of elderly persons. Nutritional intake and status play an essential role in determining the physical activity level potentially capable of minimizing the health burden of older persons. The objective of this study was to find out whether nutritional intake and status were correlated with physical activity in community-dwelling older persons. The study population included 219 aged 60 to 69 years, of whom complete measures of socio-demographic characteristics, nutritional status, nutritional intake and physical activity were obtained. Serum total protein, albumin, globulin and hemoglobin concentration were measured as nutritional indicators (biomarkers. The nutrient content of food intakes was analyzed and calculated by “Nutrisurvey” software. Analysis indicated that there was a significant correlation between nutritional biomarkers [total protein (r=-0.211; p=0.002 and globulin (r=-0.247; p=0.000] and physical activity. Compared to other food intakes, intakes of carbohydrate (r=0.241; p=0.001 and animal protein (r=0.149; p=0.027 were significantly correlated with physical activity. Our findings lend support to the existence among healthy older persons of a relationship between nutrional intake and status and physical activity.

  8. Determination of contact maps in proteins: A combination of structural and chemical approaches

    Energy Technology Data Exchange (ETDEWEB)

    Wołek, Karol; Cieplak, Marek, E-mail: mc@ifpan.edu.pl [Institute of Physics, Polish Academy of Science, Al. Lotników 32/46, 02-668 Warsaw (Poland); Gómez-Sicilia, Àngel [Instituto Cajal, Consejo Superior de Investigaciones Cientificas (CSIC), Av. Doctor Arce, 37, 28002 Madrid (Spain); Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA-Nanociencia), C/Faraday 9, 28049 Cantoblanco (Madrid) (Spain)

    2015-12-28

    Contact map selection is a crucial step in structure-based molecular dynamics modelling of proteins. The map can be determined in many different ways. We focus on the methods in which residues are represented as clusters of effective spheres. One contact map, denoted as overlap (OV), is based on the overlap of such spheres. Another contact map, named Contacts of Structural Units (CSU), involves the geometry in a different way and, in addition, brings chemical considerations into account. We develop a variant of the CSU approach in which we also incorporate Coulombic effects such as formation of the ionic bridges and destabilization of possible links through repulsion. In this way, the most essential and well defined contacts are identified. The resulting residue-residue contact map, dubbed repulsive CSU (rCSU), is more sound in its physico-chemical justification than CSU. It also provides a clear prescription for validity of an inter-residual contact: the number of attractive atomic contacts should be larger than the number of repulsive ones — a feature that is not present in CSU. However, both of these maps do not correlate well with the experimental data on protein stretching. Thus, we propose to use rCSU together with the OV map. We find that the combined map, denoted as OV+rCSU, performs better than OV. In most situations, OV and OV+rCSU yield comparable folding properties but for some proteins rCSU provides contacts which improve folding in a substantial way. We discuss the likely residue-specificity of the rCSU contacts. Finally, we make comparisons to the recently proposed shadow contact map, which is derived from different principles.

  9. Protein intake as a determinant factor of physical activity in older persons

    Directory of Open Access Journals (Sweden)

    Rina K. Kusumaratna

    2011-04-01

    Full Text Available Worldwide, the proportion of people aged 60 and over is growing faster than any other age group. It has been well-established that the aging process can be associated with increased susceptibility to chronic conditions, disability, and co-morbidity, which however may be minimized or even partially reversed by physical activity. The assessment of physical activity is becoming an increasingly important component in the evaluation of elderly persons. Nutritional intake and status play an essential role in determining the physical activity level potentially capable of minimizing the health burden of older persons. The objective of this study was to find out whether nutritional intake and status were correlated with physical activity in community-dwelling older persons. The study population included 219 aged 60 to 69 years, of whom complete measures of socio-demographic characteristics, nutritional status, nutritional intake and physical activity were obtained. Serum total protein, albumin, globulin and hemoglobin concentration were measured as nutritional indicators (biomarkers. The nutrient content of food intakes was analyzed and calculated by “Nutrisurvey” software. Analysis indicated that there was a significant correlation between nutritional biomarkers [total protein (r=-0.211; p=0.002 and globulin (r=-0.247; p=0.000] and physical activity. Compared to other food intakes, intakes of carbohydrate (r=0.241; p=0.001 and animal protein (r=0.149; p=0.027 were significantly correlated with physical activity. Our findings lend support to the existence among healthy older persons of a relationship between nutrional intake and status and physical activity.

  10. Active machine learning-driven experimentation to determine compound effects on protein patterns.

    Science.gov (United States)

    Naik, Armaghan W; Kangas, Joshua D; Sullivan, Devin P; Murphy, Robert F

    2016-02-03

    High throughput screening determines the effects of many conditions on a given biological target. Currently, to estimate the effects of those conditions on other targets requires either strong modeling assumptions (e.g. similarities among targets) or separate screens. Ideally, data-driven experimentation could be used to learn accurate models for many conditions and targets without doing all possible experiments. We have previously described an active machine learning algorithm that can iteratively choose small sets of experiments to learn models of multiple effects. We now show that, with no prior knowledge and with liquid handling robotics and automated microscopy under its control, this learner accurately learned the effects of 48 chemical compounds on the subcellular localization of 48 proteins while performing only 29% of all possible experiments. The results represent the first practical demonstration of the utility of active learning-driven biological experimentation in which the set of possible phenotypes is unknown in advance.

  11. On-chip determination of C-reactive protein using magnetic particles in continuous flow.

    Science.gov (United States)

    Phurimsak, Chayakom; Tarn, Mark D; Peyman, Sally A; Greenman, John; Pamme, Nicole

    2014-11-01

    We demonstrate the application of a multilaminar flow platform, in which functionalized magnetic particles are deflected through alternating laminar flow streams of reagents and washing solutions via an external magnet, for the rapid detection of the inflammatory biomarker, C-reactive protein (CRP). The two-step sandwich immunoassay was accomplished in less than 60 s, a vast improvement on the 80-300 min time frame required for enzyme-linked immunosorbent assays (ELISA) and the 50 min necessary for off-chip magnetic particle-based assays. The combination of continuous flow and a stationary magnet enables a degree of autonomy in the system, while a detection limit of 0.87 μg mL(-1) makes it suitable for the determination of CRP concentrations in clinical diagnostics. Its applicability was further proven by assaying real human serum samples and comparing those results to values obtained using standard ELISA tests.