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Sample records for determining protein backbone

  1. Determination of protein global folds using backbone residual dipolar coupling and long-range NOE restraints

    International Nuclear Information System (INIS)

    Giesen, Alexander W.; Homans, Steve W.; Brown, Jonathan Miles

    2003-01-01

    We report the determination of the global fold of human ubiquitin using protein backbone NMR residual dipolar coupling and long-range nuclear Overhauser effect (NOE) data as conformational restraints. Specifically, by use of a maximum of three backbone residual dipolar couplings per residue (N i -H N i , N i -C' i-1 , H N i - C' i-1 ) in two tensor frames and only backbone H N -H N NOEs, a global fold of ubiquitin can be derived with a backbone root-mean-square deviation of 1.4 A with respect to the crystal structure. This degree of accuracy is more than adequate for use in databases of structural motifs, and suggests a general approach for the determination of protein global folds using conformational restraints derived only from backbone atoms

  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. © 2015 Wiley Periodicals, Inc.

  3. Exact Solutions for Internuclear Vectors and Backbone Dihedral Angles from NH Residual Dipolar Couplings in Two Media, and their Application in a Systematic Search Algorithm for Determining Protein Backbone Structure

    International Nuclear Information System (INIS)

    Wang Lincong; Donald, Bruce Randall

    2004-01-01

    We have derived a quartic equation for computing the direction of an internuclear vector from residual dipolar couplings (RDCs) measured in two aligning media, and two simple trigonometric equations for computing the backbone (φ,ψ) angles from two backbone vectors in consecutive peptide planes. These equations make it possible to compute, exactly and in constant time, the backbone (φ,ψ) angles for a residue from RDCs in two media on any single backbone vector type. Building upon these exact solutions we have designed a novel algorithm for determining a protein backbone substructure consisting of α-helices and β-sheets. Our algorithm employs a systematic search technique to refine the conformation of both α-helices and β-sheets and to determine their orientations using exclusively the angular restraints from RDCs. The algorithm computes the backbone substructure employing very sparse distance restraints between pairs of α-helices and β-sheets refined by the systematic search. The algorithm has been demonstrated on the protein human ubiquitin using only backbone NH RDCs, plus twelve hydrogen bonds and four NOE distance restraints. Further, our results show that both the global orientations and the conformations of α-helices and β-strands can be determined with high accuracy using only two RDCs per residue. The algorithm requires, as its input, backbone resonance assignments, the identification of α-helices and β-sheets as well as sparse NOE distance and hydrogen bond restraints.Abbreviations: NMR - nuclear magnetic resonance; RDC - residual dipolar coupling; NOE - nuclear Overhauser effect; SVD - singular value decomposition; DFS - depth-first search; RMSD - root mean square deviation; POF - principal order frame; PDB - protein data bank; SA - simulated annealing; MD - molecular dynamics

  4. 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

  5. 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.

  6. Mars - robust automatic backbone assignment of proteins

    International Nuclear Information System (INIS)

    Jung, Young-Sang; Zweckstetter, Markus

    2004-01-01

    MARS a program for robust automatic backbone assignment of 13 C/ 15 N labeled proteins is presented. MARS does not require tight thresholds for establishing sequential connectivity or detailed adjustment of these thresholds and it can work with a wide variety of NMR experiments. Using only 13 C α / 13 C β connectivity information, MARS allows automatic, error-free assignment of 96% of the 370-residue maltose-binding protein. MARS can successfully be used when data are missing for a substantial portion of residues or for proteins with very high chemical shift degeneracy such as partially or fully unfolded proteins. Other sources of information, such as residue specific information or known assignments from a homologues protein, can be included into the assignment process. MARS exports its result in SPARKY format. This allows visual validation and integration of automated and manual assignment

  7. Local Backbone Flexibility as a Determinant of the Apparent pKa Values of Buried Ionizable Groups in Proteins.

    Science.gov (United States)

    Peck, Meredith T; Ortega, Gabriel; De Luca-Johnson, Javier N; Schlessman, Jamie L; Robinson, Aaron C; García-Moreno E, Bertrand

    2017-10-10

    Ionizable groups buried in the hydrophobic interior of proteins are essential for energy transduction. These groups can have highly anomalous pK a values that reflect the incompatibility between charges and dehydrated environments. A systematic study of pK a values of buried ionizable groups in staphylococcal nuclease (SNase) suggests that these pK a values are determined in part by conformational reorganization of the protein. Lys-66 is one of the most deeply buried residues in SNase. We show that its apparent pK a of 5.7 reflects the average of the pK a values of Lys-66 in different conformational states of the protein. In the fully folded state, Lys-66 is deeply buried in the hydrophobic core of SNase and must titrate with a pK a of ≪5.7. In other states, the side chain of Lys-66 is fully solvent-exposed and has a normal pK a of ≈10.4. We show that the pK a of Lys-66 can be shifted from 5.7 toward a more normal value of 7.1 via the insertion of flanking Gly residues at positions 64 and 67 to promote an "open" conformation of SNase. Crystal structures and nuclear magnetic resonance spectroscopy show that in these Gly-containing variants Lys-66 can access bulk water as a consequence of overwinding of the C-terminal end of helix 1. These data illustrate that the apparent pK a values of buried groups in proteins are governed in part by the difference in free energy between different conformational states of the protein and by differences in the pK a values of the buried groups in the different conformations.

  8. Optimized set of two-dimensional experiments for fast sequential assignment, secondary structure determination, and backbone fold validation of 13C/15N-labelled proteins

    International Nuclear Information System (INIS)

    Bersch, Beate; Rossy, Emmanuel; Coves, Jacques; Brutscher, Bernhard

    2003-01-01

    NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional 1 H- 15 N HSQC-like spectra. The 1 H- 15 N correlation peaks are frequency shifted by an amount ± ω X along the 15 N dimension, where ω X is the C α , C β , or H α frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces

  9. Pairwise NMR experiments for the determination of protein backbone dihedral angle Φ based on cross-correlated spin relaxation

    International Nuclear Information System (INIS)

    Takahashi, Hideo; Shimada, Ichio

    2007-01-01

    Novel cross-correlated spin relaxation (CCR) experiments are described, which measure pairwise CCR rates for obtaining peptide dihedral angles Φ. The experiments utilize intra-HNCA type coherence transfer to refocus 2-bond J NCα coupling evolution and generate the N (i)-C α (i) or C'(i-1)-C α (i) multiple quantum coherences which are required for measuring the desired CCR rates. The contribution from other coherences is also discussed and an appropriate setting of the evolution delays is presented. These CCR experiments were applied to 15 N- and 13 C-labeled human ubiquitin. The relevant CCR rates showed a high degree of correlation with the Φ angles observed in the X-ray structure. By utilizing these CCR experiments in combination with those previously established for obtaining dihedral angle Ψ, we can determine high resolution structures of peptides that bind weakly to large target molecules

  10. High-resolution protein design with backbone freedom.

    Science.gov (United States)

    Harbury, P B; Plecs, J J; Tidor, B; Alber, T; Kim, P S

    1998-11-20

    Recent advances in computational techniques have allowed the design of precise side-chain packing in proteins with predetermined, naturally occurring backbone structures. Because these methods do not model protein main-chain flexibility, they lack the breadth to explore novel backbone conformations. Here the de novo design of a family of alpha-helical bundle proteins with a right-handed superhelical twist is described. In the design, the overall protein fold was specified by hydrophobic-polar residue patterning, whereas the bundle oligomerization state, detailed main-chain conformation, and interior side-chain rotamers were engineered by computational enumerations of packing in alternate backbone structures. Main-chain flexibility was incorporated through an algebraic parameterization of the backbone. The designed peptides form alpha-helical dimers, trimers, and tetramers in accord with the design goals. The crystal structure of the tetramer matches the designed structure in atomic detail.

  11. Underestimated Halogen Bonds Forming with Protein Backbone in Protein Data Bank.

    Science.gov (United States)

    Zhang, Qian; Xu, Zhijian; Shi, Jiye; Zhu, Weiliang

    2017-07-24

    Halogen bonds (XBs) are attracting increasing attention in biological systems. Protein Data Bank (PDB) archives experimentally determined XBs in biological macromolecules. However, no software for structure refinement in X-ray crystallography takes into account XBs, which might result in the weakening or even vanishing of experimentally determined XBs in PDB. In our previous study, we showed that side-chain XBs forming with protein side chains are underestimated in PDB on the basis of the phenomenon that the proportion of side-chain XBs to overall XBs decreases as structural resolution becomes lower and lower. However, whether the dominant backbone XBs forming with protein backbone are overlooked is still a mystery. Here, with the help of the ratio (R F ) of the observed XBs' frequency of occurrence to their frequency expected at random, we demonstrated that backbone XBs are largely overlooked in PDB, too. Furthermore, three cases were discovered possessing backbone XBs in high resolution structures while losing the XBs in low resolution structures. In the last two cases, even at 1.80 Å resolution, the backbone XBs were lost, manifesting the urgent need to consider XBs in the refinement process during X-ray crystallography study.

  12. 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.

  13. Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy

    International Nuclear Information System (INIS)

    Lopez del Amo, Juan-Miguel; Fink, Uwe; Reif, Bernd

    2010-01-01

    We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual 15 N-T 1 timescales). We observed chemical exchange for 6 residues with HDX exchange rates in the range from 0.2 to 5 s -1 . Backbone amide 15 N longitudinal relaxation times that we determined previously are not significantly affected for most residues, yielding no systematic artifacts upon quantification of backbone dynamics (Chevelkov et al. 2008b). Significant exchange was observed for the backbone amides of R21, S36 and K60, as well as for the sidechain amides of N38, N35 and for W41ε. These residues could not be fit in our previous motional analysis, demonstrating that amide proton chemical exchange needs to be considered in the analysis of protein dynamics in the solid-state, in case D 2 O is employed as a solvent for sample preparation. Due to the intrinsically long 15 N relaxation times in the solid-state, the approach proposed here can expand the range of accessible HDX rates in the intermediate regime that is not accessible so far with exchange quench and MEXICO type experiments.

  14. On the relationship between NMR-derived amide order parameters and protein backbone entropy changes.

    Science.gov (United States)

    Sharp, Kim A; O'Brien, Evan; Kasinath, Vignesh; Wand, A Joshua

    2015-05-01

    Molecular dynamics simulations are used to analyze the relationship between NMR-derived squared generalized order parameters of amide NH groups and backbone entropy. Amide order parameters (O(2) NH ) are largely determined by the secondary structure and average values appear unrelated to the overall flexibility of the protein. However, analysis of the more flexible subset (O(2) NH  entropy than that reported by the side chain methyl axis order parameters, O(2) axis . A calibration curve for backbone entropy vs. O(2) NH is developed, which accounts for both correlations between amide group motions of different residues, and correlations between backbone and side chain motions. This calibration curve can be used with experimental values of O(2) NH changes obtained by NMR relaxation measurements to extract backbone entropy changes, for example, upon ligand binding. In conjunction with our previous calibration for side chain entropy derived from measured O(2) axis values this provides a prescription for determination of the total protein conformational entropy changes from NMR relaxation measurements. © 2015 Wiley Periodicals, Inc.

  15. Automated backbone assignment of labeled proteins using the threshold accepting algorithm

    International Nuclear Information System (INIS)

    Leutner, Michael; Gschwind, Ruth M.; Liermann, Jens; Schwarz, Christian; Gemmecker, Gerd; Kessler, Horst

    1998-01-01

    The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus C β information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, noise, spectral overlap and missing peaks is shown by repeating the assignment with reduced sequential information and increased chemical shift tolerances. The performance of the program on real data is finally demonstrated with automatically picked peak lists of human nonpancreatic synovial phospholipase A 2 , a protein with 124 residues

  16. Structural test of the parameterized-backbone method for protein design.

    Science.gov (United States)

    Plecs, Joseph J; Harbury, Pehr B; Kim, Peter S; Alber, Tom

    2004-09-03

    Designing new protein folds requires a method for simultaneously optimizing the conformation of the backbone and the side-chains. One approach to this problem is the use of a parameterized backbone, which allows the systematic exploration of families of structures. We report the crystal structure of RH3, a right-handed, three-helix coiled coil that was designed using a parameterized backbone and detailed modeling of core packing. This crystal structure was determined using another rationally designed feature, a metal-binding site that permitted experimental phasing of the X-ray data. RH3 adopted the intended fold, which has not been observed previously in biological proteins. Unanticipated structural asymmetry in the trimer was a principal source of variation within the RH3 structure. The sequence of RH3 differs from that of a previously characterized right-handed tetramer, RH4, at only one position in each 11 amino acid sequence repeat. This close similarity indicates that the design method is sensitive to the core packing interactions that specify the protein structure. Comparison of the structures of RH3 and RH4 indicates that both steric overlap and cavity formation provide strong driving forces for oligomer specificity.

  17. Backbone dynamics of the EIAV-Tat protein from 15N relaxation studies

    International Nuclear Information System (INIS)

    Ejchart, A.; Herrmann, F.; Roesch, P.; Sticht, H.; Willbold, D.

    1994-01-01

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

  18. Annotating the protein-RNA interaction sites in proteins using evolutionary information and protein backbone structure.

    Science.gov (United States)

    Li, Tao; Li, Qian-Zhong

    2012-11-07

    RNA-protein interactions play important roles in various biological processes. The precise detection of RNA-protein interaction sites is very important for understanding essential biological processes and annotating the function of the proteins. In this study, based on various features from amino acid sequence and structure, including evolutionary information, solvent accessible surface area and torsion angles (φ, ψ) in the backbone structure of the polypeptide chain, a computational method for predicting RNA-binding sites in proteins is proposed. When the method is applied to predict RNA-binding sites in three datasets: RBP86 containing 86 protein chains, RBP107 containing 107 proteins chains and RBP109 containing 109 proteins chains, better sensitivities and specificities are obtained compared to previously published methods in five-fold cross-validation tests. In order to make further examination for the efficiency of our method, the RBP107 dataset is used as training set, RBP86 and RBP109 datasets are used as the independent test sets. In addition, as examples of our prediction, RNA-binding sites in a few proteins are presented. The annotated results are consistent with the PDB annotation. These results show that our method is useful for annotating RNA binding sites of novel proteins.

  19. Determination of backbone chain direction of PDA using FFM

    Science.gov (United States)

    Jo, Sadaharu; Okamoto, Kentaro; Takenaga, Mitsuru

    2010-01-01

    The effect of backbone chains on friction force was investigated on both Langmuir-Blodgett (LB) films of 10,12-heptacosadiynoic acid and the (0 1 0) surfaces of single crystals of 2,4-hexadiene-1,6-diol using friction force microscopy (FFM). It was observed that friction force decreased when the scanning direction was parallel to the [0 0 1] direction in both samples. Moreover, friction force decreased when the scanning direction was parallel to the crystallographic [1 0 2], [1 0 1], [1 0 0] and [1 0 1¯] directions in only the single crystals. For the LB films, the [0 0 1] direction corresponds to the backbone chain direction of 10,12-heptacosadiynoic acid. For the single crystals, both the [0 0 1] and [1 0 1] directions correspond to the backbone chain direction, and the [1 0 2], [1 0 0] and [1 0 1¯] directions correspond to the low-index crystallographic direction. In both the LB films and single crystals, the friction force was minimized when the directions of scanning and the backbone chain were parallel.

  20. Selective backbone labelling of ILV methyl labelled proteins

    International Nuclear Information System (INIS)

    Sibille, Nathalie; Hanoulle, Xavier; Bonachera, Fanny; Verdegem, Dries; Landrieu, Isabelle; Wieruszeski, Jean-Michel; Lippens, Guy

    2009-01-01

    Adding the 13 C labelled 2-keto-isovalerate and 2-oxobutanoate precursors to a minimal medium composed of 12 C labelled glucose instead of the commonly used ( 2 D, 13 C) glucose leads not only to the 13 C labelling of (I, L, V) methyls but also to the selective 13 C labelling of the backbone C α and CO carbons of the Ile and Val residues. As a result, the backbone ( 1 H, 15 N) correlations of the Ile and Val residues and their next neighbours in the (i + 1) position can be selectively identified in HN(CA) and HN(CO) planes. The availability of a selective HSQC spectrum corresponding to the sole amide resonances of the Ile and Val residues allows connecting them to their corresponding methyls by the intra-residue NOE effect, and should therefore be applicable to larger systems

  1. 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. © 2015 Wiley Periodicals, Inc.

  2. 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.

  3. Protein backbone angle restraints from searching a database for chemical shift and sequence homology

    Energy Technology Data Exchange (ETDEWEB)

    Cornilescu, Gabriel; Delaglio, Frank; Bax, Ad [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)

    1999-03-15

    Chemical shifts of backbone atoms in proteins are exquisitely sensitive to local conformation, and homologous proteins show quite similar patterns of secondary chemical shifts. The inverse of this relation is used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence similarity which provide the best match to the query triplet of interest. The database contains 13C{alpha}, 13C{beta}, 13C', 1H{alpha} and 15N chemical shifts for 20 proteins for which a high resolution X-ray structure is available. The computer program TALOS was developed to search this database for strings of residues with chemical shift and residue type homology. The relative importance of the weighting factors attached to the secondary chemical shifts of the five types of resonances relative to that of sequence similarity was optimized empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar {phi} and {psi} backbone angles, their averages can reliably be used as angular restraints for the protein whose structure is being studied. Tests carried out for proteins of known structure indicate that the root-mean-square difference (rmsd) between the output of TALOS and the X-ray derived backbone angles is about 15 deg. Approximately 3% of the predictions made by TALOS are found to be in error.

  4. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-07-15

    A new program, TALOS-N, is introduced for predicting protein backbone torsion angles from NMR chemical shifts. The program relies far more extensively on the use of trained artificial neural networks than its predecessor, TALOS+. Validation on an independent set of proteins indicates that backbone torsion angles can be predicted for a larger, {>=}90 % fraction of the residues, with an error rate smaller than ca 3.5 %, using an acceptance criterion that is nearly two-fold tighter than that used previously, and a root mean square difference between predicted and crystallographically observed ({phi}, {psi}) torsion angles of ca 12 Masculine-Ordinal-Indicator . TALOS-N also reports sidechain {chi}{sup 1} rotameric states for about 50 % of the residues, and a consistency with reference structures of 89 %. The program includes a neural network trained to identify secondary structure from residue sequence and chemical shifts.

  5. 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/.

  6. Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology

    International Nuclear Information System (INIS)

    Shen Yang; Bax, Ad

    2007-01-01

    Chemical shifts of nuclei in or attached to a protein backbone are exquisitely sensitive to their local environment. A computer program, SPARTA, is described that uses this correlation with local structure to predict protein backbone chemical shifts, given an input three-dimensional structure, by searching a newly generated database for triplets of adjacent residues that provide the best match in φ/ψ/χ 1 torsion angles and sequence similarity to the query triplet of interest. The database contains 15 N, 1 H N , 1 H α , 13 C α , 13 C β and 13 C' chemical shifts for 200 proteins for which a high resolution X-ray (≤2.4 A) structure is available. The relative importance of the weighting factors for the φ/ψ/χ 1 angles and sequence similarity was optimized empirically. The weighted, average secondary shifts of the central residues in the 20 best-matching triplets, after inclusion of nearest neighbor, ring current, and hydrogen bonding effects, are used to predict chemical shifts for the protein of known structure. Validation shows good agreement between the SPARTA-predicted and experimental shifts, with standard deviations of 2.52, 0.51, 0.27, 0.98, 1.07 and 1.08 ppm for 15 N, 1 H N , 1 H α , 13 C α , 13 C β and 13 C', respectively, including outliers

  7. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-08-15

    NMR chemical shifts in proteins depend strongly on local structure. The program TALOS establishes an empirical relation between {sup 13}C, {sup 15}N and {sup 1}H chemical shifts and backbone torsion angles {phi} and {psi} (Cornilescu et al. J Biomol NMR 13 289-302, 1999). Extension of the original 20-protein database to 200 proteins increased the fraction of residues for which backbone angles could be predicted from 65 to 74%, while reducing the error rate from 3 to 2.5%. Addition of a two-layer neural network filter to the database fragment selection process forms the basis for a new program, TALOS+, which further enhances the prediction rate to 88.5%, without increasing the error rate. Excluding the 2.5% of residues for which TALOS+ makes predictions that strongly differ from those observed in the crystalline state, the accuracy of predicted {phi} and {psi} angles, equals {+-}13{sup o}. Large discrepancies between predictions and crystal structures are primarily limited to loop regions, and for the few cases where multiple X-ray structures are available such residues are often found in different states in the different structures. The TALOS+ output includes predictions for individual residues with missing chemical shifts, and the neural network component of the program also predicts secondary structure with good accuracy.

  8. Correlation between protein secondary structure, backbone bond angles, and side-chain orientations

    Science.gov (United States)

    Lundgren, Martin; Niemi, Antti J.

    2012-08-01

    We investigate the fine structure of the sp3 hybridized covalent bond geometry that governs the tetrahedral architecture around the central Cα carbon of a protein backbone, and for this we develop new visualization techniques to analyze high-resolution x-ray structures in the 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 Cβ carbon orientations. The energy function can model the side-chain geometry with a subatomic precision. As an example we construct the Cα-Cβ structure of HP35 chicken villin headpiece. We obtain a configuration that deviates less than 0.4 Å in root-mean-square distance from the experimental x-ray structure.

  9. Assignment of protein backbone resonances using connectivity, torsion angles and 13Cα chemical shifts

    International Nuclear Information System (INIS)

    Morris, Laura C.; Valafar, Homayoun; Prestegard, James H.

    2004-01-01

    A program is presented which will return the most probable sequence location for a short connected set of residues in a protein given just 13 C α chemical shifts (δ( 13 C α )) and data restricting the φ and ψ backbone angles. Data taken from both the BioMagResBank and the Protein Data Bank were used to create a probability density function (PDF) using a multivariate normal distribution in δ( 13 C α ), φ, and ψ space for each amino acid residue. Extracting and combining probabilities for particular amino acid residues in a short proposed sequence yields a score indicative of the correctness of the proposed assignment. The program is illustrated using several proteins for which structure and 13 C α chemical shift data are available

  10. Hidden Markov model approach for identifying the modular framework of the protein backbone.

    Science.gov (United States)

    Camproux, A C; Tuffery, P; Chevrolat, J P; Boisvieux, J F; Hazout, S

    1999-12-01

    The hidden Markov model (HMM) was used to identify recurrent short 3D structural building blocks (SBBs) describing protein backbones, independently of any a priori knowledge. Polypeptide chains are decomposed into a series of short segments defined by their inter-alpha-carbon distances. Basically, the model takes into account the sequentiality of the observed segments and assumes that each one corresponds to one of several possible SBBs. Fitting the model to a database of non-redundant proteins allowed us to decode proteins in terms of 12 distinct SBBs with different roles in protein structure. Some SBBs correspond to classical regular secondary structures. Others correspond to a significant subdivision of their bounding regions previously considered to be a single pattern. The major contribution of the HMM is that this model implicitly takes into account the sequential connections between SBBs and thus describes the most probable pathways by which the blocks are connected to form the framework of the protein structures. Validation of the SBBs code was performed by extracting SBB series repeated in recoding proteins and examining their structural similarities. Preliminary results on the sequence specificity of SBBs suggest promising perspectives for the prediction of SBBs or series of SBBs from the protein sequences.

  11. 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

    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...... 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...... as an established fact. We hope the current paper helps counter this new situation and leads to a reassessment of those papers that make this erroneous claim....

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

    International Nuclear Information System (INIS)

    Marassi, Francesca M.; Ding, Yi; Schwieters, Charles D.; Tian, Ye; Yao, Yong

    2015-01-01

    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

  13. Protein backbone motions viewed by intraresidue and sequential H{sup N}-H{sup {alpha}} residual dipolar couplings

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-05-15

    Triple resonance E.COSY-based techniques were used to measure intra-residue and sequential H{sup N}-H{sup {alpha}} residual dipolar couplings (RDCs) for the third IgG-binding domain of protein G (GB3), aligned in Pf1 medium. Measurements closely correlate with values predicted on the basis of an NMR structure, previously determined on the basis of a large number of one-bond backbone RDCs measured in five alignment media. However, in particular the sequential H{sup N}-H{sup {alpha}} RDCs are smaller than predicted for a static structure, suggesting a degree of motion for these internuclear vectors that exceeds that of the backbone amide N-H vectors. Of all experimentally determined GB3 structures available, the best correlation between experimental {sup 1}H-{sup 1}H couplings is observed for a GB3 ensemble, previously derived to generate a realistic picture of the conformational space sampled by GB3 (Clore and Schwieters, J Mol Biol 355:879-886, 2006). However, for both NMR and X-ray-derived structures the {sup 1}H-{sup 1}H couplings are found to be systematically smaller than expected on the basis of alignment tensors derived from {sup 15}N-{sup 1}H amide RDCs, assuming librationally corrected N-H bond lengths of 1.041 A.

  14. 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...

  15. Chemical synthesis of membrane proteins by the removable backbone modification method.

    Science.gov (United States)

    Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

    2017-12-01

    Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

  16. Cross-correlated relaxation rates between protein backbone H–X dipolar interactions

    International Nuclear Information System (INIS)

    Vögeli, Beat

    2017-01-01

    The relaxation interference between dipole–dipole interactions of two separate spin pairs carries structural and dynamics information. In particular, when compared to individual dynamic behavior of those spin pairs, such cross-correlated relaxation (CCR) rates report on the correlation between the spin pairs. We have recently mapped out correlated motion along the backbone of the protein GB3, using CCR rates among and between consecutive H N –N and H α –C α dipole–dipole interactions. Here, we provide a detailed account of the measurement of the four types of CCR rates. All rates were obtained from at least two different pulse sequences, of which the yet unpublished ones are presented. Detailed comparisons between the different methods and corrections for unwanted pathways demonstrate that the averaged CCR rates are highly accurate and precise with errors of 1.5–3% of the entire value ranges.

  17. Cross-correlated relaxation rates between protein backbone H–X dipolar interactions

    Energy Technology Data Exchange (ETDEWEB)

    Vögeli, Beat, E-mail: beat.vogeli@ucdenver.edu [University of Colorado Denver, Department of Biochemistry and Molecular Genetics (United States)

    2017-03-15

    The relaxation interference between dipole–dipole interactions of two separate spin pairs carries structural and dynamics information. In particular, when compared to individual dynamic behavior of those spin pairs, such cross-correlated relaxation (CCR) rates report on the correlation between the spin pairs. We have recently mapped out correlated motion along the backbone of the protein GB3, using CCR rates among and between consecutive H{sup N}–N and H{sup α}–C{sup α} dipole–dipole interactions. Here, we provide a detailed account of the measurement of the four types of CCR rates. All rates were obtained from at least two different pulse sequences, of which the yet unpublished ones are presented. Detailed comparisons between the different methods and corrections for unwanted pathways demonstrate that the averaged CCR rates are highly accurate and precise with errors of 1.5–3% of the entire value ranges.

  18. A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment

    International Nuclear Information System (INIS)

    Pervushin, Konstantin; Eletsky, Alexander

    2003-01-01

    A new strategy of backbone resonance assignment is proposed based on a combination of the most sensitive TROSY-type triple resonance experiments such as TROSY-HNCA and TROSY-HNCO with a new 3D multiple-quantum HACACO experiment. The favourable relaxation properties of the multiple-quantum coherences and signal detection using the 13 C' antiphase coherences optimize the performance of the proposed experiment for application to larger proteins. In addition to the 1 H N , 15 N, 13 C α and 13 C' chemical shifts the 3D multiple-quantum HACACO experiment provides assignment for the 1 H α resonances in contrast to previously proposed experiments for large proteins. The strategy is demonstrated with the 44 kDa uniformly 15 N, 13 C-labeled and fractionally 35% deuterated trimeric B. subtilis Chorismate Mutase measured at 20 deg. C and 9 deg. C. Measurements at the lower temperature indicate that the new strategy can be applied to even larger proteins with molecular weights up to 80 kDa

  19. 4D experiments measured with APSY for automated backbone resonance assignments of large proteins

    International Nuclear Information System (INIS)

    Krähenbühl, Barbara; Boudet, Julien; Wider, Gerhard

    2013-01-01

    Detailed structural and functional characterization of proteins by solution NMR requires sequence-specific resonance assignment. We present a set of transverse relaxation optimization (TROSY) based four-dimensional automated projection spectroscopy (APSY) experiments which are designed for resonance assignments of proteins with a size up to 40 kDa, namely HNCACO, HNCOCA, HNCACB and HN(CO)CACB. These higher-dimensional experiments include several sensitivity-optimizing features such as multiple quantum parallel evolution in a ‘just-in-time’ manner, aliased off-resonance evolution, evolution-time optimized APSY acquisition, selective water-handling and TROSY. The experiments were acquired within the concept of APSY, but they can also be used within the framework of sparsely sampled experiments. The multidimensional peak lists derived with APSY provided chemical shifts with an approximately 20 times higher precision than conventional methods usually do, and allowed the assignment of 90 % of the backbone resonances of the perdeuterated primase-polymerase ORF904, which contains 331 amino acid residues and has a molecular weight of 38.4 kDa.

  20. PASA - A Program for Automated Protein NMR Backbone Signal Assignment by Pattern-Filtering Approach

    International Nuclear Information System (INIS)

    Xu Yizhuang; Wang Xiaoxia; Yang Jun; Vaynberg, Julia; Qin Jun

    2006-01-01

    We present a new program, PASA (Program for Automated Sequential Assignment), for assigning protein backbone resonances based on multidimensional heteronuclear NMR data. Distinct from existing programs, PASA emphasizes a per-residue-based pattern-filtering approach during the initial stage of the automated 13 C α and/or 13 C β chemical shift matching. The pattern filter employs one or multiple constraints such as 13 C α /C β chemical shift ranges for different amino acid types and side-chain spin systems, which helps to rule out, in a stepwise fashion, improbable assignments as resulted from resonance degeneracy or missing signals. Such stepwise filtering approach substantially minimizes early false linkage problems that often propagate, amplify, and ultimately cause complication or combinatorial explosion of the automation process. Our program (http://www.lerner.ccf.org/moleccard/qin/) was tested on four representative small-large sized proteins with various degrees of resonance degeneracy and missing signals, and we show that PASA achieved the assignments efficiently and rapidly that are fully consistent with those obtained by laborious manual protocols. The results demonstrate that PASA may be a valuable tool for NMR-based structural analyses, genomics, and proteomics

  1. 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.

  2. Five and four dimensional experiments for robust backbone resonance assignment of large intrinsically disordered proteins: application to Tau3x protein

    International Nuclear Information System (INIS)

    Żerko, Szymon; Byrski, Piotr; Włodarczyk-Pruszyński, Paweł; Górka, Michał; Ledolter, Karin; Masliah, Eliezer; Konrat, Robert; Koźmiński, Wiktor

    2016-01-01

    New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.

  3. Five and four dimensional experiments for robust backbone resonance assignment of large intrinsically disordered proteins: application to Tau3x protein

    Energy Technology Data Exchange (ETDEWEB)

    Żerko, Szymon; Byrski, Piotr; Włodarczyk-Pruszyński, Paweł; Górka, Michał [University of Warsaw, Faculty of Chemistry, Biological and Chemical Research Centre (Poland); Ledolter, Karin [University of Vienna, Department of Computational and Structural Biology, Max F. Perutz Laboratories (Austria); Masliah, Eliezer [University of California, San Diego, Departments of Neuroscience and Pathology (United States); Konrat, Robert [University of Vienna, Department of Computational and Structural Biology, Max F. Perutz Laboratories (Austria); Koźmiński, Wiktor, E-mail: kozmin@chem.uw.edu.pl [University of Warsaw, Faculty of Chemistry, Biological and Chemical Research Centre (Poland)

    2016-08-15

    New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.

  4. Prediction of mutational tolerance in HIV-1 protease and reverse transcriptase using flexible backbone protein design.

    Directory of Open Access Journals (Sweden)

    Elisabeth Humphris-Narayanan

    Full Text Available Predicting which mutations proteins tolerate while maintaining their structure and function has important applications for modeling fundamental properties of proteins and their evolution; it also drives progress in protein design. Here we develop a computational model to predict the tolerated sequence space of HIV-1 protease reachable by single mutations. We assess the model by comparison to the observed variability in more than 50,000 HIV-1 protease sequences, one of the most comprehensive datasets on tolerated sequence space. We then extend the model to a second protein, reverse transcriptase. The model integrates multiple structural and functional constraints acting on a protein and uses ensembles of protein conformations. We find the model correctly captures a considerable fraction of protease and reverse-transcriptase mutational tolerance and shows comparable accuracy using either experimentally determined or computationally generated structural ensembles. Predictions of tolerated sequence space afforded by the model provide insights into stability-function tradeoffs in the emergence of resistance mutations and into strengths and limitations of the computational model.

  5. 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.

  6. 5D {sup 13}C-detected experiments for backbone assignment of unstructured proteins with a very low signal dispersion

    Energy Technology Data Exchange (ETDEWEB)

    Novacek, Jiri [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic); Zawadzka-Kazimierczuk, Anna [University of Warsaw, Faculty of Chemistry (Poland); Papouskova, Veronika; Zidek, Lukas, E-mail: lzidek@chemi.muni.cz [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic); Sanderova, Hana; Krasny, Libor [Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Molecular Genetics of Bacteria and Department of Bacteriology (Czech Republic); Kozminski, Wiktor [University of Warsaw, Faculty of Chemistry (Poland); Sklenar, Vladimir [Masaryk University, Faculty of Science, NCBR, and CEITEC (Czech Republic)

    2011-05-15

    Two novel 5D NMR experiments (CACONCACO, NCOCANCO) for backbone assignment of disordered proteins are presented. The pulse sequences exploit relaxation properties of the unstructured proteins and combine the advantages of {sup 13}C-direct detection, non-uniform sampling, and longitudinal relaxation optimization to maximize the achievable resolution and minimize the experimental time. The pulse sequences were successfully tested on the sample of partially disordered delta subunit from RNA polymerase from Bacillus subtilis. The unstructured part of this 20 kDa protein consists of 81 amino acids with frequent sequential repeats. A collection of 0.0003% of the data needed for a conventional experiment with linear sampling was sufficient to perform an unambiguous assignment of the disordered part of the protein from a single 5D spectrum.

  7. 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

  8. Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data.

    Science.gov (United States)

    Hamuro, Yoshitomo; E, Sook Yen

    2018-05-01

    The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. Graphical Abstract ᅟ.

  9. Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data

    Science.gov (United States)

    Hamuro, Yoshitomo; E, Sook Yen

    2018-05-01

    The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. [Figure not available: see fulltext.

  10. Determination of Backbone Amide Hydrogen Exchange Rates of Cytochrome c Using Partially Scrambled Electron Transfer Dissociation Data

    Science.gov (United States)

    Hamuro, Yoshitomo; E, Sook Yen

    2018-03-01

    The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR. [Figure not available: see fulltext.

  11. Solution structure and backbone dynamics of recombinant Cucurbita maxima trypsin inhibitor-V determined by NMR spectroscopy.

    Science.gov (United States)

    Liu, J; Prakash, O; Cai, M; Gong, Y; Huang, Y; Wen, L; Wen, J J; Huang, J K; Krishnamoorthi, R

    1996-02-06

    The solution structure of recombinant Cucurbita maxima trypsin inhibitor-V (rCMTI-V), whose N-terminal is unacetylated and carries an extra glycine residue, was determined by means of two-dimensional (2D) homo and 3D hetero NMR experiments in combination with a distance geometry and simulated annealing algorithm. A total of 927 interproton distances and 123 torsion angle constraints were utilized to generate 18 structures. The root mean squared deviation (RMSD) of the mean structure is 0.53 A for main-chain atoms and 0.95 A for all the non-hydrogen atoms of residues 3-40 and 49-67. The average structure of rCMTI-V is found to be almost the same as that of the native protein [Cai, M., Gong, Y., Kao, J.-L., & Krishnamoorthi, R. (1995) Biochemistry 34, 5201-5211]. The backbone dynamics of uniformly 15N-labeled rCMTI-V were characterized by 2D 1H-15N NMR methods. 15N spin-lattice and spin-spin relaxation rate constants (R1 and R2, respectively) and [1H]-15N steady-state heteronuclear Overhauser effect enhancements were measured for the peptide NH units and, using the model-free formalism [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559, 4559-4570], the following parameters were determined: overall tumbling correlation time for the protein molecule (tau m), generalized order parameters for the individual N-H vectors (S2), effective correlation times for their internal motions (tau e), and terms to account for motions on a slower time scale (second) due to chemical exchange and/or conformational averaging (R(ex)). Most of the backbone NH groups of rCMTI-V are found to be highly constrained ((S2) = 0.83) with the exception of those in the binding loop (residues 41-48, (S2) = 0.71) and the N-terminal region ((S2) = 0.73). Main-chain atoms in these regions show large RMSD values in the average NMR structure. Residues involved in turns also appear to have more mobility ((S2) = 0.80). Dynamical properties of rCMTI-V were compared with those of two other

  12. A Bayesian-probability-based method for assigning protein backbone dihedral angles based on chemical shifts and local sequences

    Energy Technology Data Exchange (ETDEWEB)

    Wang Jun; Liu Haiyan [University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at the Microscale, and Key Laboratory of Structural Biology, School of Life Sciences (China)], E-mail: hyliu@ustc.edu.cn

    2007-01-15

    Chemical shifts contain substantial information about protein local conformations. We present a method to assign individual protein backbone dihedral angles into specific regions on the Ramachandran map based on the amino acid sequences and the chemical shifts of backbone atoms of tripeptide segments. The method uses a scoring function derived from the Bayesian probability for the central residue of a query tripeptide segment to have a particular conformation. The Ramachandran map is partitioned into representative regions at two levels of resolution. The lower resolution partitioning is equivalent to the conventional definitions of different secondary structure regions on the map. At the higher resolution level, the {alpha} and {beta} regions are further divided into subregions. Predictions are attempted at both levels of resolution. We compared our method with TALOS using the original TALOS database, and obtained comparable results. Although TALOS may produce the best results with currently available databases which are much enlarged, the Bayesian-probability-based approach can provide a quantitative measure for the reliability of predictions.

  13. CSSI-PRO: a method for secondary structure type editing, assignment and estimation in proteins using linear combination of backbone chemical shifts

    International Nuclear Information System (INIS)

    Swain, Monalisa; Atreya, Hanudatta S.

    2009-01-01

    Estimation of secondary structure in polypeptides is important for studying their structure, folding and dynamics. In NMR spectroscopy, such information is generally obtained after sequence specific resonance assignments are completed. We present here a new methodology for assignment of secondary structure type to spin systems in proteins directly from NMR spectra, without prior knowledge of resonance assignments. The methodology, named Combination of Shifts for Secondary Structure Identification in Proteins (CSSI-PRO), involves detection of specific linear combination of backbone 1 H α and 13 C' chemical shifts in a two-dimensional (2D) NMR experiment based on G-matrix Fourier transform (GFT) NMR spectroscopy. Such linear combinations of shifts facilitate editing of residues belonging to α-helical/β-strand regions into distinct spectral regions nearly independent of the amino acid type, thereby allowing the estimation of overall secondary structure content of the protein. Comparison of the predicted secondary structure content with those estimated based on their respective 3D structures and/or the method of Chemical Shift Index for 237 proteins gives a correlation of more than 90% and an overall rmsd of 7.0%, which is comparable to other biophysical techniques used for structural characterization of proteins. Taken together, this methodology has a wide range of applications in NMR spectroscopy such as rapid protein structure determination, monitoring conformational changes in protein-folding/ligand-binding studies and automated resonance assignment

  14. Predicting backbone Cα angles and dihedrals from protein sequences by stacked sparse auto-encoder deep neural network.

    Science.gov (United States)

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

    2014-10-30

    Because a nearly constant distance between two neighbouring Cα atoms, local backbone structure of proteins can be represented accurately by the angle between C(αi-1)-C(αi)-C(αi+1) (θ) and a dihedral angle rotated about the C(αi)-C(αi+1) bond (τ). θ and τ angles, as the representative of structural properties of three to four amino-acid residues, offer a description of backbone conformations that is complementary to φ and ψ angles (single residue) and secondary structures (>3 residues). Here, we report the first machine-learning technique for sequence-based prediction of θ and τ angles. Predicted angles based on an independent test have a mean absolute error of 9° for θ and 34° for τ with a distribution on the θ-τ plane close to that of native values. The average root-mean-square distance of 10-residue fragment structures constructed from predicted θ and τ angles is only 1.9Å from their corresponding native structures. Predicted θ and τ angles are expected to be complementary to predicted ϕ and ψ angles and secondary structures for using in model validation and template-based as well as template-free structure prediction. The deep neural network learning technique is available as an on-line server called Structural Property prediction with Integrated DEep neuRal network (SPIDER) at http://sparks-lab.org. Copyright © 2014 Wiley Periodicals, Inc.

  15. Empirical correlation between protein backbone {sup 15}N and {sup 13}C secondary chemical shifts and its application to nitrogen chemical shift re-referencing

    Energy Technology Data Exchange (ETDEWEB)

    Wang Liya [Cold Spring Harbor Laboratory (United States); Markley, John L. [University of Wisconsin, Biochemistry Department (United States)], E-mail: markley@nmrfam.wisc.edu

    2009-06-15

    The linear analysis of chemical shifts (LACS) has provided a robust method for identifying and correcting {sup 13}C chemical shift referencing problems in data from protein NMR spectroscopy. Unlike other approaches, LACS does not require prior knowledge of the three-dimensional structure or inference of the secondary structure of the protein. It also does not require extensive assignment of the NMR data. We report here a way of extending the LACS approach to {sup 15}N NMR data from proteins, so as to enable the detection and correction of inconsistencies in chemical shift referencing for this nucleus. The approach is based on our finding that the secondary {sup 15}N chemical shift of the backbone nitrogen atom of residue i is strongly correlated with the secondary chemical shift difference (experimental minus random coil) between the alpha and beta carbons of residue i - 1. Thus once alpha and beta {sup 13}C chemical shifts are available (their difference is referencing error-free), the {sup 15}N referencing can be validated, and an appropriate offset correction can be derived. This approach can be implemented prior to a structure determination and can be used to analyze potential referencing problems in database data not associated with three-dimensional structure. Application of the LACS algorithm to the current BMRB protein chemical shift database, revealed that nearly 35% of the BMRB entries have {delta}{sup 15}N values mis-referenced by over 0.7 ppm and over 25% of them have {delta}{sup 1}H{sup N} values mis-referenced by over 0.12 ppm. One implication of the findings reported here is that a backbone {sup 15}N chemical shift provides a better indicator of the conformation of the preceding residue than of the residue itself.

  16. 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.

  17. 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.

  18. Backbone assignment and secondary structure of the PsbQ protein from Photosystem II

    Czech Academy of Sciences Publication Activity Database

    Horničáková, M.; Kohoutová, Jaroslava; Schlagnitweit, J.; Wohlschlager, Ch.; Ettrich, Rüdiger; Fiala, R.; Schoefberger, W.; Müller, N.

    2011-01-01

    Roč. 5, č. 2 (2011), s. 169-175 ISSN 1874-2718 R&D Projects: GA MŠk(CZ) LC06010 Institutional research plan: CEZ:AV0Z60870520 Keywords : Photosystem II * PsbQ * Missing link * NMR resonance assignment * Protein-protein interaction Subject RIV: BO - Biophysics Impact factor: 0.720, year: 2011 http://www.springerlink.com/content/3n38075w5h1l1082/fulltext.pdf

  19. Density functional calculations of backbone 15N shielding tensors in beta-sheet and turn residues of protein G

    International Nuclear Information System (INIS)

    Cai Ling; Kosov, Daniel S.; Fushman, David

    2011-01-01

    We performed density functional calculations of backbone 15 N shielding tensors in the regions of beta-sheet and turns of protein G. The calculations were carried out for all twenty-four beta-sheet residues and eight beta-turn residues in the protein GB3 and the results were compared with the available experimental data from solid-state and solution NMR measurements. Together with the alpha-helix data, our calculations cover 39 out of the 55 residues (or 71%) in GB3. The applicability of several computational models developed previously (Cai et al. in J Biomol NMR 45:245–253, 2009) to compute 15 N shielding tensors of alpha-helical residues is assessed. We show that the proposed quantum chemical computational model is capable of predicting isotropic 15 N chemical shifts for an entire protein that are in good correlation with experimental data. However, the individual components of the predicted 15 N shielding tensor agree with experiment less well: the computed values show much larger spread than the experimental data, and there is a profound difference in the behavior of the tensor components for alpha-helix/turns and beta-sheet residues. We discuss possible reasons for this.

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

    KAUST Repository

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

    2012-01-01

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

  1. 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.

  2. High dimensional and high resolution pulse sequences for backbone resonance assignment of intrinsically disordered proteins

    Energy Technology Data Exchange (ETDEWEB)

    Zawadzka-Kazimierczuk, Anna; Kozminski, Wiktor, E-mail: kozmin@chem.uw.edu.pl [University of Warsaw, Faculty of Chemistry (Poland); Sanderova, Hana; Krasny, Libor [Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Molecular Genetics of Bacteria, Department of Bacteriology (Czech Republic)

    2012-04-15

    Four novel 5D (HACA(N)CONH, HNCOCACB, (HACA)CON(CA)CONH, (H)NCO(NCA)CONH), and one 6D ((H)NCO(N)CACONH) NMR pulse sequences are proposed. The new experiments employ non-uniform sampling that enables achieving high resolution in indirectly detected dimensions. The experiments facilitate resonance assignment of intrinsically disordered proteins. The novel pulse sequences were successfully tested using {delta} subunit (20 kDa) of Bacillus subtilis RNA polymerase that has an 81-amino acid disordered part containing various repetitive sequences.

  3. Backbone and sidechain methyl Ile (δ1), Leu and Val chemical shift assignments of RDE-4 (1-243), an RNA interference initiation protein in C. elegans.

    Science.gov (United States)

    Chiliveri, Sai Chaitanya; Kumar, Sonu; Marelli, Udaya Kiran; Deshmukh, Mandar V

    2012-10-01

    The RNAi pathway of several organisms requires presence of double stranded RNA binding proteins for functioning of Dicer in gene regulation. In C. elegans, a double stranded RNA binding protein, RDE-4 (385 aa, 44 kDa) recognizes long exogenous dsRNA and initiates the RNAi pathway. We have achieved complete backbone and stereospecific methyl sidechain Ile (δ1), Leu and Val chemical shifts of first 243 amino acids of RDE-4, namely RDE-4ΔC.

  4. 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. Graphical Abstract ᅟ.

  5. Beta-scission of alkoxyl radicals on peptides and proteins can give rise to backbone cleavage and loss of side-chains

    International Nuclear Information System (INIS)

    Headlam, H.A.; Davies, M.J.; Mortimer, A.; Easton, C.J.

    2000-01-01

    Full text: Exposure of proteins to radicals in the presence of O 2 brings about multiple changes including side-chain oxidation, backbone fragmentation, cross-linking, unfolding, changes in hydrophobicity and conformation, altered susceptibility to proteolytic enzymes and formation of new reactive groups (e.g. hydroperoxides and 3,4-dihydroxyphenylalanine). All of these processes can result in loss of structural or enzymatic activity. The mechanisms that give rise to backbone cleavage are only partly understood. Whilst it is known that direct hydrogen atom abstraction at a-carbon sites gives backbone cleavages it has also been proposed that initial attack at side-chain sites might also give rise to backbone cleavage. In this study we have examined whether initial attack at the β- (C-3) position can give rise to α-carbon radicals (and hence backbone cleavage) via the formation, and subsequent β- scission, of C-3 alkoxyl radicals. This process has been observed previously with protected amino acids in organic solvents (J. Chem. Soc. Perkin Trans. 2, 1997, 503-507) but the occurrence of such reactions with proteins in aqueous solution has not been explored. Alkoxyl radicals were generated at the C-3 position of a variety of protected amino acids and small peptides by two methods: metal-ion catalysed decomposition of hydroperoxides formed as a result of γ-radiolysis in the presence of O 2 , and UV photolysis of nitrate esters. In most cases radicals have been detected by EPR spectroscopy using nitroso and nitrone spin traps, which can be assigned by comparison with literature data to α-carbon radicals; in some case assignments were confirmed by the generation of the putative species by other routes. With Ala peptide hydroperoxides and nitrate esters, and MNP as the spin trap, the major radical detected in each case has been assigned to the adduct of an α-carbon radical with partial structure - NH- . CH-C(O) - consistent with the rapid occurrence of the above

  6. Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

    International Nuclear Information System (INIS)

    Hefke, Frederik; Bagaria, Anurag; Reckel, Sina; Ullrich, Sandra Johanna; Dötsch, Volker; Glaubitz, Clemens; Güntert, Peter

    2011-01-01

    We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with 13 C or/and 15 N such that cross peaks between 13 CO(i – 1) and 15 NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with 13 C and the second with 15 N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B 2 R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.

  7. Backbone dynamics of free barnase and its complex with barstar determined by 15N NMR relaxation study

    International Nuclear Information System (INIS)

    Sahu, Sarata C.; Bhuyan, Abani K.; Udgaonkar, Jayant B.; Hosur, R.V.

    2000-01-01

    Backbone dynamics of uniformly 15 N-labeled free barnase and its complex with unlabelled barstar have been studied at 40 deg. C, pH 6.6, using 15 N relaxation data obtained from proton-detected 2D { 1 H}- 15 N NMR spectroscopy. 15 N spin-lattice relaxation rate constants (R 1 ), spin-spin relaxation rate constants (R 2 ), and steady-state heteronuclear { 1 H}- 15 N NOEs have been measured at a magnetic field strength of 14.1 Tesla for 91 residues of free barnase and for 90 residues out of a total of 106 in the complex (excluding three prolines and the N-terminal residue) backbone amide 15 N sites of barnase. The primary relaxation data for both the cases have been analyzed in the framework of the model-free formalism using both isotropic and axially symmetric models of the rotational diffusion tensor. As per the latter, the overall rotational correlation times (τ m ) are 5.0 and 9.5 ns for the free and complexed barnase, respectively. The average order parameter is found to be 0.80 for free barnase and 0.86 for the complex. However, the changes are not uniform along the backbone and for about 5 residues near the binding interface there is actually a significant decrease in the order parameters on complex formation. These residues are not involved in the actual binding. For the residues where the order parameter increases, the magnitudes vary significantly. It is observed that the complex has much less internal mobility, compared to free barnase. From the changes in the order parameters, the entropic contribution of NH bond vector motion to the free energy of complex formation has been calculated. It is apparent that these motions cause significant unfavorable contributions and therefore must be compensated by many other favorable contributions to effect tight complex formation. The observed variations in the motion and their different locations with regard to the binding interface may have important implications for remote effects and regulation of the enzyme

  8. 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.

  9. Backbone amide linker strategy

    DEFF Research Database (Denmark)

    Shelton, Anne Pernille Tofteng; Jensen, Knud Jørgen

    2013-01-01

    In the backbone amide linker (BAL) strategy, the peptide is anchored not at the C-terminus but through a backbone amide, which leaves the C-terminal available for various modifications. This is thus a very general strategy for the introduction of C-terminal modifications. The BAL strategy...

  10. 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

  11. 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

  12. 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

    . Molecular dynamics (MD) simulation provides a complementary approach to the study of protein dynamics on similar time scales. Comparisons between NMR spectroscopy and MD simulations can be used to interpret experimental results and to improve the quality of simulation-related force fields and integration......A molecular-level understanding of the function of a protein requires knowledge of both its structural and dynamic properties. NMR spectroscopy allows the measurement of generalized order parameters that provide an atomistic description of picosecond and nanosecond fluctuations in protein structure...... 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...

  13. 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.

  14. A ‘just-in-time’ HN(CA)CO experiment for the backbone assignment of large proteins with high sensitivity

    Science.gov (United States)

    Werner-Allen, Jon W.; Jiang, Ling; Zhou, Pei

    2006-07-01

    Among the suite of commonly used backbone experiments, HNCACO presents an unresolved sensitivity limitation due to fast 13CO transverse relaxation and passive 13Cα-13Cβ coupling. Here, we present a high-sensitivity 'just-in-time' (JIT) HN(CA)CO pulse sequence that uniformly refocuses 13Cα-13Cβ coupling while collecting 13CO shifts in real time. Sensitivity comparisons of the 3-D JIT HN(CA)CO, a CT-HMQC-based control, and a HSQC-based control with selective 13Cα inversion pulses were performed using a 2H/13C/15N labeled sample of the 29 kDa HCA II protein at 15 °C. The JIT experiment shows a 42% signal enhancement over the CT-HMQC-based experiment. Compared to the HSQC-based experiment, the JIT experiment is 16% less sensitive for residues experiencing proper 13Cα refocusing and 13Cα-13Cβ decoupling. However, for the remaining residues, the JIT spectrum shows a 106% average sensitivity gain over the HSQC-based experiment. The high-sensitivity JIT HNCACO experiment should be particularly beneficial for studies of large proteins to provide 13CO resonance information regardless of residue type.

  15. Easy and unambiguous sequential assignments of intrinsically disordered proteins by correlating the backbone 15N or 13C′ chemical shifts of multiple contiguous residues in highly resolved 3D spectra

    International Nuclear Information System (INIS)

    Yoshimura, Yuichi; Kulminskaya, Natalia V.; Mulder, Frans A. A.

    2015-01-01

    Sequential resonance assignment strategies are typically based on matching one or two chemical shifts of adjacent residues. However, resonance overlap often leads to ambiguity in resonance assignments in particular for intrinsically disordered proteins. We investigated the potential of establishing connectivity through the three-bond couplings between sequentially adjoining backbone carbonyl carbon nuclei, combined with semi-constant time chemical shift evolution, for resonance assignments of small folded and larger unfolded proteins. Extended sequential connectivity strongly lifts chemical shift degeneracy of the backbone nuclei in disordered proteins. We show here that 3D (H)N(COCO)NH and (HN)CO(CO)NH experiments with relaxation-optimized multiple pulse mixing correlate up to seven adjacent backbone amide nitrogen or carbonyl carbon nuclei, respectively, and connections across proline residues are also obtained straightforwardly. Multiple, recurrent long-range correlations with ultra-high resolution allow backbone 1 H N , 15 N H , and 13 C′ resonance assignments to be completed from a single pair of 3D experiments

  16. Protein determination in single corns

    International Nuclear Information System (INIS)

    Knorr, J.; Schiekel, M.; Franke, W.; Focke, F.

    1994-01-01

    Determination of protein content in food materials is usually done by analyzing the nitrogen amount by wet chemical Kjeldahl method. An improved accuracy accompanied by smaller analyzing intervals can be achieved using nondestructive neutron activation. Analyses have been performed using 14 MeV neutrons to determine the content of N and P in single wheat corns. Irradiation parameters have been optimized to prevent serious radiation damage in grains. About 200 single corns have been investigated with total net weights ranging from 30 to 70 mg. The tested arrangement allows determination of nitrogen amount in a single corn down to 0.3 mg with an accuracy of better than 4 %. Mean nitrogen concentrations in the range from 9 to 19% per corn have been detected. (author) 5 refs.; 6 figs

  17. 1990s: High Capacity Backbones

    Indian Academy of Sciences (India)

    First page Back Continue Last page Overview Graphics. 1990s: High Capacity Backbones. Backbone capacities increased from 2.5 Gb/s to 100s of Gb/s during the 1990's. Wavelength division multiplexing with 160 waves of 10 Gb/s was commercially available. Several high-capacity backbones built in the US and Europe.

  18. Quantitative assessments of the distinct contributions of polypeptide backbone amides versus sidechain groups to chain expansion via chemical denaturation

    Science.gov (United States)

    Holehouse, Alex S.; Garai, Kanchan; Lyle, Nicholas; Vitalis, Andreas; Pappu, Rohit V.

    2015-01-01

    In aqueous solutions with high concentrations of chemical denaturants such as urea and guanidinium chloride (GdmCl) proteins expand to populate heterogeneous conformational ensembles. These denaturing environments are thought to be good solvents for generic protein sequences because properties of conformational distributions align with those of canonical random coils. Previous studies showed that water is a poor solvent for polypeptide backbones and therefore backbones form collapsed globular structures in aqueous solvents. Here, we ask if polypeptide backbones can intrinsically undergo the requisite chain expansion in aqueous solutions with high concentrations of urea and GdmCl. We answer this question using a combination of molecular dynamics simulations and fluorescence correlation spectroscopy. We find that the degree of backbone expansion is minimal in aqueous solutions with high concentrations denaturants. Instead, polypeptide backbones sample conformations that are denaturant-specific mixtures of coils and globules, with a persistent preference for globules. Therefore, typical denaturing environments cannot be classified as good solvents for polypeptide backbones. How then do generic protein sequences expand in denaturing environments? To answer this question, we investigated the effects of sidechains using simulations of two archetypal sequences with amino acid compositions that are mixtures of charged, hydrophobic, and polar groups. We find that sidechains lower the effective concentration of backbone amides in water leading to an intrinsic expansion of polypeptide backbones in the absence of denaturants. Additional dilution of the effective concentration of backbone amides is achieved through preferential interactions with denaturants. These effects lead to conformational statistics in denaturing environments that are congruent with those of canonical random coils. Our results highlight the role of sidechain-mediated interactions as determinants of the

  19. An approach for high-throughput structure determination of proteins by NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Medek, Ales; Olejniczak, Edward T.; Meadows, Robert P.; Fesik, Stephen W. [Abbott Laboratories, Pharmaceutical Discovery Division (United States)

    2000-11-15

    An approach is described for rapidly determining protein structures by NMR that utilizes proteins containing {sup 13}C-methyl labeled Val, Leu, and Ile ({delta}1) and protonated Phe and Tyr in a deuterated background. Using this strategy, the key NOEs that define the hydrophobic core and overall fold of the protein are easily obtained. NMR data are acquired using cryogenic probe technology which markedly reduces the spectrometer time needed for data acquisition. The approach is demonstrated by determining the overall fold of the antiapoptotic protein, Bcl-xL, from data collected in only 4 days. Refinement of the Bcl-xL structure to a backbone rmsd of 0.95 A was accomplished with data collected in an additional 3 days. A distance analysis of 180 different proteins and structure calculations using simulated data suggests that our method will allow the global folds of a wide variety of proteins to be determined.

  20. 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

    RNA, and to investigate how this interaction is influenced by other ribosomal components. Complexes were characterized in both naked 23 S rRNA and ribosomes from an E. coli L11-minus strain, before and after reconstitution with L11. The protein protects 17 riboses between positions 1058 and 1085 in the naked 23 S r......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 r......)4 and other proteins within the ribosome. The antibiotics thiostrepton and micrococcin inhibit the catalytic functions of this region by slotting in between the accessible loops and interacting with nucleotides there....

  1. AbDesign: An algorithm for combinatorial backbone design guided by natural conformations and sequences.

    Science.gov (United States)

    Lapidoth, Gideon D; Baran, Dror; Pszolla, Gabriele M; Norn, Christoffer; Alon, Assaf; Tyka, Michael D; Fleishman, Sarel J

    2015-08-01

    Computational design of protein function has made substantial progress, generating new enzymes, binders, inhibitors, and nanomaterials not previously seen in nature. However, the ability to design new protein backbones for function--essential to exert control over all polypeptide degrees of freedom--remains a critical challenge. Most previous attempts to design new backbones computed the mainchain from scratch. Here, instead, we describe a combinatorial backbone and sequence optimization algorithm called AbDesign, which leverages the large number of sequences and experimentally determined molecular structures of antibodies to construct new antibody models, dock them against target surfaces and optimize their sequence and backbone conformation for high stability and binding affinity. We used the algorithm to produce antibody designs that target the same molecular surfaces as nine natural, high-affinity antibodies; in five cases interface sequence identity is above 30%, and in four of those the backbone conformation at the core of the antibody binding surface is within 1 Å root-mean square deviation from the natural antibodies. Designs recapitulate polar interaction networks observed in natural complexes, and amino acid sidechain rigidity at the designed binding surface, which is likely important for affinity and specificity, is high compared to previous design studies. In designed anti-lysozyme antibodies, complementarity-determining regions (CDRs) at the periphery of the interface, such as L1 and H2, show greater backbone conformation diversity than the CDRs at the core of the interface, and increase the binding surface area compared to the natural antibody, potentially enhancing affinity and specificity. © 2015 Wiley Periodicals, Inc.

  2. Temperature-dependent spectral density analysis applied to monitoring backbone dynamics of major urinary protein-I complexed with the pheromone 2-sec-butyl-4,5-dihydrothiazole

    International Nuclear Information System (INIS)

    Krizova, Hana; Zidek, Lukas; Stone, Martin J.; Novotny, Milos V.; Sklenar, Vladimir

    2004-01-01

    Backbone dynamics of mouse major urinary protein I (MUP-I) was studied by 15 N NMR relaxation. Data were collected at multiple temperatures for a complex of MUP-I with its natural pheromonal ligand, 2-sec-4,5-dihydrothiazole, and for the free protein. The measured relaxation rates were analyzed using the reduced spectral density mapping. Graphical analysis of the spectral density values provided an unbiased qualitative picture of the internal motions. Varying temperature greatly increased the range of analyzed spectral density values and therefore improved reliability of the analysis. Quantitative parameters describing the dynamics on picosecond to nanosecond time scale were obtained using a novel method of simultaneous data fitting at multiple temperatures. Both methods showed that the backbone flexibility on the fast time scale is slightly increased upon pheromone binding, in accordance with the previously reported results. Zero-frequency spectral density values revealed conformational changes on the microsecond to millisecond time scale. Measurements at different temperatures allowed to monitor temperature depencence of the motional parameters

  3. Solution NMR structure determination of proteins revisited

    International Nuclear Information System (INIS)

    Billeter, Martin; Wagner, Gerhard; Wuethrich, Kurt

    2008-01-01

    This 'Perspective' bears on the present state of protein structure determination by NMR in solution. The focus is on a comparison of the infrastructure available for NMR structure determination when compared to protein crystal structure determination by X-ray diffraction. The main conclusion emerges that the unique potential of NMR to generate high resolution data also on dynamics, interactions and conformational equilibria has contributed to a lack of standard procedures for structure determination which would be readily amenable to improved efficiency by automation. To spark renewed discussion on the topic of NMR structure determination of proteins, procedural steps with high potential for improvement are identified

  4. 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...

  5. Determination of soluble protein contents from RVNRL

    International Nuclear Information System (INIS)

    Wan Manshol Wan Zin; Nurulhuda Othman

    1996-01-01

    This project was carried out to determine the soluble protein contents on RVNRL film vulcanisates, with respect to the RVNRL storage time, gamma irradiation dose absorbed by the latex and the effect of different leaching time and leaching conditions. These three factors are important in the hope to determine the best possible mean of minimizing the soluble protein contents in products made from RVNRL. Within the nine months storage period employed in the study, the results show that, the longer the storage period the less the soluble protein extracted from the film samples. Gamma irradiation dose absorbed by the samples, between 5.3 kGy to 25.2 kGy seems to influence the soluble protein contents of the RVNRL films vulcanisates. The higher the dose the more was the soluble protein extracted from the film samples. At an absorbed dose of 5.3 kGy and 25.2 kGy, the soluble contents were 0. 198 mg/ml and 0.247 mg/ml respectively. At a fixed leaching temperature, the soluble proteins increases with leaching time and at a fixed leaching time, the soluble proteins increases with leaching temperature. ne highest extractable protein contents was determined at a leaching time of 10 minutes and leaching temperature of 90'C The protein analysis were done by using Modified Lowry Method

  6. 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....

  7. Simultaneous determination of protein structure and dynamics

    DEFF Research Database (Denmark)

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

    2005-01-01

    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......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...... 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...

  8. Measurement of backbone hydrogen-deuterium exchange in the type III secretion system needle protein PrgI by solid-state NMR

    Science.gov (United States)

    Chevelkov, Veniamin; Giller, Karin; Becker, Stefan; Lange, Adam

    2017-10-01

    In this report we present site-specific measurements of amide hydrogen-deuterium exchange rates in a protein in the solid state phase by MAS NMR. Employing perdeuteration, proton detection and a high external magnetic field we could adopt the highly efficient Relax-EXSY protocol previously developed for liquid state NMR. According to this method, we measured the contribution of hydrogen exchange on apparent 15N longitudinal relaxation rates in samples with differing D2O buffer content. Differences in the apparent T1 times allowed us to derive exchange rates for multiple residues in the type III secretion system needle protein.

  9. Inferential backbone assignment for sparse data

    International Nuclear Information System (INIS)

    Vitek, Olga; Bailey-Kellogg, Chris; Craig, Bruce; Vitek, Jan

    2006-01-01

    This paper develops an approach to protein backbone NMR assignment that effectively assigns large proteins while using limited sets of triple-resonance experiments. Our approach handles proteins with large fractions of missing data and many ambiguous pairs of pseudoresidues, and provides a statistical assessment of confidence in global and position-specific assignments. The approach is tested on an extensive set of experimental and synthetic data of up to 723 residues, with match tolerances of up to 0.5 ppm for C α and C β resonance types. The tests show that the approach is particularly helpful when data contain experimental noise and require large match tolerances. The keys to the approach are an empirical Bayesian probability model that rigorously accounts for uncertainty in the data at all stages in the analysis, and a hybrid stochastic tree-based search algorithm that effectively explores the large space of possible assignments

  10. Deuterium isotope shifts for backbone {sup 1}H, {sup 15}N and {sup 13}C nuclei in intrinsically disordered protein {alpha}-synuclein

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-10-15

    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 {sup 2}H isotope shift measurements for {sup 13}C{sup {alpha}}, {sup 13}C{sup {beta}}, {sup 13}C Prime , {sup 15}N, and {sup 1}H{sup N} have been obtained by using a mixed sample of protonated and uniformly perdeuterated {alpha}-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 {sup 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.

  11. Protein Determinants of Meiotic DNA Break Hotspots

    Science.gov (United States)

    Fowler, Kyle R.; Gutiérrez-Velasco, Susana

    2013-01-01

    SUMMARY Meiotic recombination, crucial for proper chromosome segregation and genome evolution, is initiated by programmed DNA double-strand breaks (DSBs) in yeasts and likely all sexually reproducing species. In fission yeast, DSBs occur up to hundreds of times more frequently at special sites, called hotspots, than in other regions of the genome. What distinguishes hotspots from cold regions is an unsolved problem, although transcription factors determine some hotspots. We report the discovery that three coiled-coil proteins – Rec25, Rec27, and Mug20 – bind essentially all hotspots with unprecedented specificity even without DSB formation. These small proteins are components of linear elements, are related to synaptonemal complex proteins, and are essential for nearly all DSBs at most hotspots. Our results indicate these hotspot determinants activate or stabilize the DSB-forming protein Rec12 (Spo11 homolog) rather than promote its binding to hotspots. We propose a new paradigm for hotspot determination and crossover control by linear element proteins. PMID:23395004

  12. 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

  13. Protein Structure Determination Using Chemical Shifts

    DEFF Research Database (Denmark)

    Christensen, Anders Steen

    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...

  14. Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

    International Nuclear Information System (INIS)

    Henry, G.D.; Weiner, J.H.; Sykes, B.D.

    1987-01-01

    Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13 C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D 2 O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H 2 O solutions; in 1:1 H 2 O/D 2 O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13 C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects, the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule. A model of the detergent-solubilized coat protein is constructed from these H-exchange data which is consistent with circular dichroism and other NMR results

  15. Solution Structure and Backbone Dynamics of the Pleckstrin Homology Domain of the Human Protein Kinase B (PKB/Akt). Interaction with Inositol Phosphates

    International Nuclear Information System (INIS)

    Auguin, Daniel; Barthe, Philippe; Auge-Senegas, Marie-Therese; Stern, Marc-Henri; Noguchi, Masayuki; Roumestand, Christian

    2004-01-01

    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 β). PKBβ-PH is an electrostatically polarized molecule that adopts the same fold and topology as other PH-domains, consisting of a β-sandwich of seven strands capped on one top by an α-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 from a

  16. Backbone upgrades and DEC equipment replacement

    Science.gov (United States)

    Vancamp, Warren

    1991-01-01

    The NASA Science Internet (NSI) dual protocol backbone is outlined. It includes DECnet link upgrades to match TCP/IP link performance. It also includes the integration of backbone resources and central management. The phase 1 transition process is outlined.

  17. DNA-to-protein crosslinks and backbone breaks caused by far- and near-ultraviolet, and visible light radiations in mammalian cells

    International Nuclear Information System (INIS)

    Peak, M.J.; Peak, J.G.

    1986-01-01

    Spectral responses for DNA damages caused by far-uv, near-uv, and visible light radiations have been studied. The near congruence of the spectra for far-uv damages and the spectrum of DNA is good evidence that the mechanism is the same for the induction of breaks, crosslinks, and pyrimidine dimers. For near-uv, the different spectra imply that at least several nonDNA sensitizer molecules act as primary chromophores, but that DNA damage eventually results. With the understanding that near-uv and visible radiations produce a variety of chemically potent reactive oxygen species within the cell, we recognize the possibility for many types of DNA damage. If we assume that SSBs and DNA-to-protein crosslinks are random single events along the genome, it is possible to compute the number of events per cell genome per lethal event caused by the different energies used. In the near-uv and visible region, many more breaks and crosslinks are formed per lethal event than by far-uv. About 20 times more SSBs per lethal event are caused by 365-nm radiation than by x-rays, strong evidence that these breaks are effectively repaired. It is therefore likely that SSBs are not a serious event with regard to cellular lethality. The role of crosslinks and their repair in lethal events is less clear. The lack of any correlation at all between the action spectra for SSBs, or crosslinks, and lethality and mutagenesis in the same cells is evidence that another lesion or lesions are involved in these events. The multitude of chemical events that can be caused in cellular metabolites by the reactive species generated by these long wavelengths of radiation means that death is attributable to the total spectrum of changed chemicals delivered by a lethal dose, only some of which are DNA changes leading to SSBs and crosslinks. 43 refs., 3 figs., 2 tabs

  18. 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.

    2015-01-01

    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. PMID:25741692

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

    International Nuclear Information System (INIS)

    Guerry, Paul; Duong, Viet Dung; Herrmann, Torsten

    2015-01-01

    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

  20. PONDEROSA, an automated 3D-NOESY peak picking program, enables automated protein structure determination.

    Science.gov (United States)

    Lee, Woonghee; Kim, Jin Hae; Westler, William M; Markley, John L

    2011-06-15

    PONDEROSA (Peak-picking Of Noe Data Enabled by Restriction of Shift Assignments) accepts input information consisting of a protein sequence, backbone and sidechain NMR resonance assignments, and 3D-NOESY ((13)C-edited and/or (15)N-edited) spectra, and returns assignments of NOESY crosspeaks, distance and angle constraints, and a reliable NMR structure represented by a family of conformers. PONDEROSA incorporates and integrates external software packages (TALOS+, STRIDE and CYANA) to carry out different steps in the structure determination. PONDEROSA implements internal functions that identify and validate NOESY peak assignments and assess the quality of the calculated three-dimensional structure of the protein. The robustness of the analysis results from PONDEROSA's hierarchical processing steps that involve iterative interaction among the internal and external modules. PONDEROSA supports a variety of input formats: SPARKY assignment table (.shifts) and spectrum file formats (.ucsf), XEASY proton file format (.prot), and NMR-STAR format (.star). To demonstrate the utility of PONDEROSA, we used the package to determine 3D structures of two proteins: human ubiquitin and Escherichia coli iron-sulfur scaffold protein variant IscU(D39A). The automatically generated structural constraints and ensembles of conformers were as good as or better than those determined previously by much less automated means. The program, in the form of binary code along with tutorials and reference manuals, is available at http://ponderosa.nmrfam.wisc.edu/.

  1. Capturing non-local interactions by long short-term memory bidirectional recurrent neural networks for improving prediction of protein secondary structure, backbone angles, contact numbers and solvent accessibility.

    Science.gov (United States)

    Heffernan, Rhys; Yang, Yuedong; Paliwal, Kuldip; Zhou, Yaoqi

    2017-09-15

    The accuracy of predicting protein local and global structural properties such as secondary structure and solvent accessible surface area has been stagnant for many years because of the challenge of accounting for non-local interactions between amino acid residues that are close in three-dimensional structural space but far from each other in their sequence positions. All existing machine-learning techniques relied on a sliding window of 10-20 amino acid residues to capture some 'short to intermediate' non-local interactions. Here, we employed Long Short-Term Memory (LSTM) Bidirectional Recurrent Neural Networks (BRNNs) which are capable of capturing long range interactions without using a window. We showed that the application of LSTM-BRNN to the prediction of protein structural properties makes the most significant improvement for residues with the most long-range contacts (|i-j| >19) over a previous window-based, deep-learning method SPIDER2. Capturing long-range interactions allows the accuracy of three-state secondary structure prediction to reach 84% and the correlation coefficient between predicted and actual solvent accessible surface areas to reach 0.80, plus a reduction of 5%, 10%, 5% and 10% in the mean absolute error for backbone ϕ , ψ , θ and τ angles, respectively, from SPIDER2. More significantly, 27% of 182724 40-residue models directly constructed from predicted C α atom-based θ and τ have similar structures to their corresponding native structures (6Å RMSD or less), which is 3% better than models built by ϕ and ψ angles. We expect the method to be useful for assisting protein structure and function prediction. The method is available as a SPIDER3 server and standalone package at http://sparks-lab.org . yaoqi.zhou@griffith.edu.au or yuedong.yang@griffith.edu.au. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email

  2. Future High Capacity Backbone Networks

    DEFF Research Database (Denmark)

    Wang, Jiayuan

    are proposed. The work focuses on energy efficient routing algorithms in a dynamic optical core network environment, with Generalized MultiProtocol Label Switching (GMPLS) as the control plane. Energy ef- ficient routing algorithms for energy savings and CO2 savings are proposed, and their performance...... aiming for reducing the dynamic part of the energy consumption of the network may increase the fixed part of the energy consumption meanwhile. In the second half of the thesis, the conflict between energy efficiency and Quality of Service (QoS) is addressed by introducing a novel software defined......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...

  3. Some fractal properties of the percolating backbone in two dimensions

    International Nuclear Information System (INIS)

    Laidlaw, D.; MacKay, G.; Jan, N.

    1987-01-01

    A new algorithm is presented, based on elements of artificial intelligence theory, to determine the fractal properties of the backbone of the incipient infinite cluster. It is found that fractal dimensionality of the backbone is d/sub f//sup BB/ = 1.61 +/- 0.01, the chemical dimensionality is d/sub t/ = 1.40 +/- 0.01, and the fractal dimension of the minimum path d/sub min/ = 1.15 +/- 0.02 for the two-dimensional triangular lattice

  4. Data Acquisition Backbone Core DABC

    International Nuclear Information System (INIS)

    Adamczewski, J; Essel, H G; Kurz, N; Linev, S

    2008-01-01

    For the new experiments at FAIR new concepts of data acquisition systems have to be developed like the distribution of self-triggered, time stamped data streams over high performance networks for event building. The Data Acquisition Backbone Core (DABC) is a software package currently under development for FAIR detector tests, readout components test, and data flow investigations. All kinds of data channels (front-end systems) are connected by program plug-ins into functional components of DABC like data input, combiner, scheduler, event builder, analysis and storage components. After detailed simulations real tests of event building over a switched network (InfiniBand clusters with up to 110 nodes) have been performed. With the DABC software more than 900 MByte/s input and output per node can be achieved meeting the most demanding requirements. The software is ready for the implementation of various test beds needed for the final design of data acquisition systems at FAIR. The development of key components is supported by the FutureDAQ project of the European Union (FP6 I3HP JRA1)

  5. Overcoming barriers to membrane protein structure determination.

    Science.gov (United States)

    Bill, Roslyn M; Henderson, Peter J F; Iwata, So; Kunji, Edmund R S; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G; Vogel, Horst

    2011-04-01

    After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

  6. Structural determination of intact proteins using mass spectrometry

    Science.gov (United States)

    Kruppa, Gary [San Francisco, CA; Schoeniger, Joseph S [Oakland, CA; Young, Malin M [Livermore, CA

    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.

  7. Determination of insoluble avian eggshell matrix proteins

    Czech Academy of Sciences Publication Activity Database

    Mikšík, Ivan; Sedláková, Pavla; Lacinová, Kateřina; Pataridis, Statis; Eckhardt, Adam

    2010-01-01

    Roč. 397, č. 1 (2010), s. 205-214 ISSN 1618-2642 R&D Projects: GA MŠk(CZ) 1M0510; GA ČR(CZ) GA203/09/0675; GA ČR(CZ) GA203/08/1428 Institutional research plan: CEZ:AV0Z50110509 Keywords : eggshell proteins * insoluble proteins * matrix proteins Subject RIV: CE - Biochemistry Impact factor: 3.841, year: 2010

  8. Optical burst switching based satellite backbone network

    Science.gov (United States)

    Li, Tingting; Guo, Hongxiang; Wang, Cen; Wu, Jian

    2018-02-01

    We propose a novel time slot based optical burst switching (OBS) architecture for GEO/LEO based satellite backbone network. This architecture can provide high speed data transmission rate and high switching capacity . Furthermore, we design the control plane of this optical satellite backbone network. The software defined network (SDN) and network slice (NS) technologies are introduced. Under the properly designed control mechanism, this backbone network is flexible to support various services with diverse transmission requirements. Additionally, the LEO access and handoff management in this network is also discussed.

  9. Overcoming barriers to membrane protein structure determination

    NARCIS (Netherlands)

    Bill, Roslyn M.; Henderson, Peter J. F.; Iwata, So; Kunji, Edmund R. S.; Michel, Hartmut; Neutze, Richard; Newstead, Simon; Poolman, Bert; Tate, Christopher G.; Vogel, Horst

    After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new

  10. Spectrophotometric determination of proteins associated with ...

    African Journals Online (AJOL)

    Twenty six Aeromonas isolates from fishes, poultry and humans in Zaria were quantified for total soluble proteins (enzymes) profiles in January, 2007 by spectrophotometric (Biuret) method. Isolates were grown on Brain Heart Infusion (BHI) broth, they were incubated at 370C and centrifuged at 1,000 g/dl using harous ...

  11. On the radioimmunological determination of native and heat denaturated protein

    International Nuclear Information System (INIS)

    Menzel, E.J.; Glatz, F.; Technische Univ., Vienna

    1981-01-01

    Precipitation radioimmunoassay, solid phase radioimmunoassay and passive hemagglutination were examined for their efficiency in the determination of native or denaturated soy proteins. Native as well as autoclaved soy protein could be determined quantitatively in the precipitation radioimmunoassay, using antisera directed against the native product. In the solid phase technique only the autoclaved soy protein could be detected with high sensitivity. In the passive hemagglutination reaction, no agglutination could be observed with erythrocytes coated with autoclaved soy protein. Only antisera against the denaturated (autoclaved) soy protein agglutinated these erythrocytes. (orig.) [de

  12. Determinants of immunogenic response to protein therapeutics.

    Science.gov (United States)

    Singh, Satish K; Cousens, Leslie P; Alvarez, David; Mahajan, Pramod B

    2012-09-01

    Protein therapeutics occupy a very significant position in the biopharmaceutical market. In addition to the preclinical, clinical and post marketing challenges common to other drugs, unwanted immunogenicity is known to affect efficacy and/or safety of most biotherapeutics. A standard set of immunogenicity risk factors are routinely used to inform monitoring strategies in clinical studies. A number of in-silico, in vivo and in vitro approaches have also been employed to predict immunogenicity of biotherapeutics, but with limited success. Emerging data also indicates the role of immune tolerance mechanisms and impact of several product-related factors on modulating host immune responses. Thus, a comprehensive discussion of the impact of innate and adaptive mechanisms and molecules involved in induction of host immune responses on immunogenicity of protein therapeutics is needed. A detailed understanding of these issues is essential in order to fully exploit the therapeutic potential of this class of drugs. This Roundtable Session was designed to provide a common platform for discussing basic immunobiological and pharmacological issues related to the role of biotherapeutic-associated risk factors, as well as host immune system in immunogenicity against protein therapeutics. The session included overview presentations from three speakers, followed by a panel discussion with audience participation. Copyright © 2012. Published by Elsevier Ltd.. All rights reserved.

  13. TRX-LOGOS - a graphical tool to demonstrate DNA information content dependent upon backbone dynamics in addition to base sequence.

    Science.gov (United States)

    Fortin, Connor H; Schulze, Katharina V; Babbitt, Gregory A

    2015-01-01

    for molecular evolutionary genetics analysis to visually compare the human Forkhead box/FOX protein evolution to its binding site evolution. We also compared the DNA binding signatures of human TP53 tumor suppressor determined by two different laboratory methods (SELEX and ChIP-seq). Further analysis of the entire yeast genome, center aligned at the start codon, also revealed a distinct sequence-independent 3 bp periodic pattern in information content, present only in coding region, and perhaps indicative of the non-random organization of the genetic code. TRX-LOGOS is useful in any situation in which important information content in DNA can be better visualized at the positions of phosphate linkages (i.e. dinucleotides) where the dynamic properties of the DNA backbone functions to facilitate DNA-protein interaction.

  14. Partial sequence determination of metabolically labeled radioactive proteins and peptides

    International Nuclear Information System (INIS)

    Anderson, C.W.

    1982-01-01

    The author has used the sequence analysis of radioactive proteins and peptides to approach several problems during the past few years. They, in collaboration with others, have mapped precisely several adenovirus proteins with respect to the nucleotide sequence of the adenovirus genome; identified hitherto missed proteins encoded by bacteriophage MS2 and by simian virus 40; analyzed the aminoterminal maturation of several virus proteins; determined the cleavage sites for processing of the poliovirus polyprotein; and analyzed the mechanism of frameshifting by excess normal tRNAs during cell-free protein synthesis. This chapter is designed to aid those without prior experience at protein sequence determinations. It is based primarily on the experience gained in the studies cited above, which made use of the Beckman 890 series automated protein sequencers

  15. Accurate determination of interfacial protein secondary structure by combining interfacial-sensitive amide I and amide III spectral signals.

    Science.gov (United States)

    Ye, Shuji; Li, Hongchun; Yang, Weilai; Luo, Yi

    2014-01-29

    Accurate determination of protein structures at the interface is essential to understand the nature of interfacial protein interactions, but it can only be done with a few, very limited experimental methods. Here, we demonstrate for the first time that sum frequency generation vibrational spectroscopy can unambiguously differentiate the interfacial protein secondary structures by combining surface-sensitive amide I and amide III spectral signals. This combination offers a powerful tool to directly distinguish random-coil (disordered) and α-helical structures in proteins. From a systematic study on the interactions between several antimicrobial peptides (including LKα14, mastoparan X, cecropin P1, melittin, and pardaxin) and lipid bilayers, it is found that the spectral profiles of the random-coil and α-helical structures are well separated in the amide III spectra, appearing below and above 1260 cm(-1), respectively. For the peptides with a straight backbone chain, the strength ratio for the peaks of the random-coil and α-helical structures shows a distinct linear relationship with the fraction of the disordered structure deduced from independent NMR experiments reported in the literature. It is revealed that increasing the fraction of negatively charged lipids can induce a conformational change of pardaxin from random-coil to α-helical structures. This experimental protocol can be employed for determining the interfacial protein secondary structures and dynamics in situ and in real time without extraneous labels.

  16. Green Network Planning Model for Optical Backbones

    DEFF Research Database (Denmark)

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

    2010-01-01

    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......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...

  17. Determination of phospholipid transfer proteins in rat tissues by immunoassays

    International Nuclear Information System (INIS)

    Teerlink, T.

    1983-01-01

    Several quantitative immunoassays have been developed for two phospholipid transfer proteins from rat liver, i.e. the phosphatidylcholine transfer protein and the non-specific lipid transfer protein. The development of a double-antibody radioimmunoassay for the phosphatidylcholine transfer protein is described. The transfer protein was labelled with iodine-125 by the mild glucose oxidase-lactoperoxidase method. Although less than one tyrosine residue per molecule of transfer protein was labelled, only 20% of the labelled transfer protein was immunoprecipitable. This value could be increased to 80% by purifying the labelled protein by affinity chromatography on a column of anti-phosphatidylcholine transfer protein-IgG coupled to Sepharose 4B. The radioimmunoassay was used to determine the levels of phosphatidylcholine transfer protein in homogenates and 105 000 xg supernatants from various rat tissues as well as several Morris hepatomas. An enzyme immunoassay for the non-specific lipid transfer protein is also described. The antiserum that was raised especially by the author was cross-reactive with the non-specific lipid transfer protein present in 105 000 xg supernatants from human, mouse and bovine liver. The non-specific lipid transfer protein lost its immunoreactivity upon labelling with iodine-125 using different labelling techniques. Therefore, a regular radioimmunoassay could not be developed. The results of these different assays were compared. (Auth.)

  18. Integrating NOE and RDC using sum-of-squares relaxation for protein structure determination.

    Science.gov (United States)

    Khoo, Y; Singer, A; Cowburn, D

    2017-07-01

    We revisit the problem of protein structure determination from geometrical restraints from NMR, using convex optimization. It is well-known that the NP-hard distance geometry problem of determining atomic positions from pairwise distance restraints can be relaxed into a convex semidefinite program (SDP). However, often the NOE distance restraints are too imprecise and sparse for accurate structure determination. Residual dipolar coupling (RDC) measurements provide additional geometric information on the angles between atom-pair directions and axes of the principal-axis-frame. The optimization problem involving RDC is highly non-convex and requires a good initialization even within the simulated annealing framework. In this paper, we model the protein backbone as an articulated structure composed of rigid units. Determining the rotation of each rigid unit gives the full protein structure. We propose solving the non-convex optimization problems using the sum-of-squares (SOS) hierarchy, a hierarchy of convex relaxations with increasing complexity and approximation power. Unlike classical global optimization approaches, SOS optimization returns a certificate of optimality if the global optimum is found. Based on the SOS method, we proposed two algorithms-RDC-SOS and RDC-NOE-SOS, that have polynomial time complexity in the number of amino-acid residues and run efficiently on a standard desktop. In many instances, the proposed methods exactly recover the solution to the original non-convex optimization problem. To the best of our knowledge this is the first time SOS relaxation is introduced to solve non-convex optimization problems in structural biology. We further introduce a statistical tool, the Cramér-Rao bound (CRB), to provide an information theoretic bound on the highest resolution one can hope to achieve when determining protein structure from noisy measurements using any unbiased estimator. Our simulation results show that when the RDC measurements are

  19. “Pinning strategy”: a novel approach for predicting the backbone ...

    Indian Academy of Sciences (India)

    Prakash

    To assess the quality of the strategy, we define two measures. The first one ...... modular framework of the protein backbone; Protein Eng. 12. 1063–1073 .... Richardson J S, Getzoff E D and Richardson D C 1978 The beta bulge: a common ...

  20. Backbone Diversity Analysis in Catalyst Design

    NARCIS (Netherlands)

    Maldonado, A.G.; Hageman, J.A.; Mastroianni, S.; Rothenberg, G.

    2009-01-01

    We present a computer-based heuristic framework for designing libraries of homogeneous catalysts. In this approach, a set of given bidentate ligand-metal complexes is disassembled into key substructures (building blocks). These include metal atoms, ligating groups, backbone groups, and residue

  1. ExScal Backbone Network Architecture

    Science.gov (United States)

    2005-01-01

    802.11 battery powered nodes was laid over the sensor network. We adopted the Stargate platform for the backbone tier to serve as the basis for...its head. XSS Hardware and Network: XSS stands for eXtreme Scaling Stargate . A stargate is a linux-based single board computer. It has a 400 MHz

  2. Versatile phosphite ligands based on silsesquioxane backbones

    NARCIS (Netherlands)

    van der Vlugt, JI; Ackerstaff, J; Dijkstra, TW; Mills, AM; Kooijman, H; Spek, AL; Meetsma, A; Abbenhuis, HCL; Vogt, D

    Silsesquioxanes are employed as ligand backbones for the synthesis of novel phosphite compounds with 3,3'-5,5'-tetrakis(tert-butyl)-2,2'-di-oxa-1,1'-biphenyl substituents. Both mono- and bidentate phosphites are prepared in good yields. Two types of silsesquioxanes are employed as starting

  3. Host Proteins Determine MRSA Biofilm Structure and Integrity

    DEFF Research Database (Denmark)

    Dreier, Cindy; Nielsen, Astrid; Jørgensen, Nis Pedersen

    Human extracellular matrix (hECM) proteins aids the initial attachment and initiation of an infection, by specific binding to bacterial cell surface proteins. However, the importance of hECM proteins in structure, integrity and antibiotic resilience of a biofilm is unknown. This study aims...... to determine how specific hECM proteins affect S. aureus USA300 JE2 biofilms. Biofilms were grown in the presence of synovial fluid from rheumatoid arteritis patients to mimic in vivo conditions, where bacteria incorporate hECM proteins into the biofilm matrix. Difference in biofilm structure, with and without...... addition of hECM to growth media, was visualized by confocal laser scanning microscopy. Two enzymatic degradation experiments were used to study biofilm matrix composition and importance of hECM proteins: enzymatic removal of specific hECM proteins from growth media, before biofilm formation, and enzymatic...

  4. Selectivity determinants of GPCR-G-protein binding

    DEFF Research Database (Denmark)

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

    2017-01-01

    of the G-protein barcode through distinct residues, like multiple keys (receptors) opening the same lock (G protein) using non-identical cuts. Considering the evolutionary history of GPCRs allows the identification of these selectivity-determining residues. These findings lay the foundation...

  5. Probabilistic Determination of Native State Ensembles of Proteins

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

  6. 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.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  8. Induced helical backbone conformations of self-organizable dendronized polymers.

    Science.gov (United States)

    Rudick, Jonathan G; Percec, Virgil

    2008-12-01

    Control of function through the primary structure of a molecule presents a significant challenge with valuable rewards for nanoscience. Dendritic building blocks encoded with information that defines their three-dimensional shape (e.g., flat-tapered or conical) and how they associate with each other are referred to as self-assembling dendrons. Self-organizable dendronized polymers possess a flat-tapered or conical self-assembling dendritic side chain on each repeat unit of a linear polymer backbone. When appended to a covalent polymer, the self-assembling dendrons direct a folding process (i.e., intramolecular self-assembly). Alternatively, intermolecular self-assembly of dendrons mediated by noncovalent interactions between apex groups can generate a supramolecular polymer backbone. Self-organization, as we refer to it, is the spontaneous formation of periodic and quasiperiodic arrays from supramolecular elements. Covalent and supramolecular polymers jacketed with self-assembling dendrons self-organize. The arrays are most often comprised of cylindrical or spherical objects. The shape of the object is determined by the primary structure of the dendronized polymer: the structure of the self-assembling dendron and the length of the polymer backbone. It is therefore possible to predictably generate building blocks for single-molecule nanotechnologies or arrays of supramolecules for bottom-up self-assembly. We exploit the self-organization of polymers jacketed with self-assembling dendrons to elucidate how primary structure determines the adopted conformation and fold (i.e., secondary and tertiary structure), how the supramolecules associate (i.e., quaternary structure), and their resulting functions. A combination of experimental techniques is employed to interrogate the primary, secondary, tertiary, and quaternary structure of the self-organizable dendronized polymers. We refer to the process by which we interpolate between the various levels of structural

  9. 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.

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

    KAUST Repository

    Bhuyan, M.S.; Gao, Xin

    2011-01-01

    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.

  11. Contribution of peptide backbone to Anti-citrulline-dependent antibody reactivity

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Dam, Catharina; Olsen, Dorthe

    2015-01-01

    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...... 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...... 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....

  12. 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.

  13. Practical analysis of specificity-determining residues in protein families.

    Science.gov (United States)

    Chagoyen, Mónica; García-Martín, Juan A; Pazos, Florencio

    2016-03-01

    Determining the residues that are important for the molecular activity of a protein is a topic of broad interest in biomedicine and biotechnology. This knowledge can help understanding the protein's molecular mechanism as well as to fine-tune its natural function eventually with biotechnological or therapeutic implications. Some of the protein residues are essential for the function common to all members of a family of proteins, while others explain the particular specificities of certain subfamilies (like binding on different substrates or cofactors and distinct binding affinities). Owing to the difficulty in experimentally determining them, a number of computational methods were developed to detect these functional residues, generally known as 'specificity-determining positions' (or SDPs), from a collection of homologous protein sequences. These methods are mature enough for being routinely used by molecular biologists in directing experiments aimed at getting insight into the functional specificity of a family of proteins and eventually modifying it. In this review, we summarize some of the recent discoveries achieved through SDP computational identification in a number of relevant protein families, as well as the main approaches and software tools available to perform this type of analysis. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  14. Rapid and reliable protein structure determination via chemical shift threading.

    Science.gov (United States)

    Hafsa, Noor E; Berjanskii, Mark V; Arndt, David; Wishart, David S

    2018-01-01

    Protein structure determination using nuclear magnetic resonance (NMR) spectroscopy can be both time-consuming and labor intensive. Here we demonstrate how chemical shift threading can permit rapid, robust, and accurate protein structure determination using only chemical shift data. Threading is a relatively old bioinformatics technique that uses a combination of sequence information and predicted (or experimentally acquired) low-resolution structural data to generate high-resolution 3D protein structures. The key motivations behind using NMR chemical shifts for protein threading lie in the fact that they are easy to measure, they are available prior to 3D structure determination, and they contain vital structural information. The method we have developed uses not only sequence and chemical shift similarity but also chemical shift-derived secondary structure, shift-derived super-secondary structure, and shift-derived accessible surface area to generate a high quality protein structure regardless of the sequence similarity (or lack thereof) to a known structure already in the PDB. The method (called E-Thrifty) was found to be very fast (often chemical shift refinement, these results suggest that protein structure determination, using only NMR chemical shifts, is becoming increasingly practical and reliable. E-Thrifty is available as a web server at http://ethrifty.ca .

  15. Non-equilibrium hydrogen exchange for determination of H-bond strength and water accessibility in solid proteins.

    Science.gov (United States)

    Grohe, Kristof; Movellan, Kumar Tekwani; Vasa, Suresh Kumar; Giller, Karin; Becker, Stefan; Linser, Rasmus

    2017-05-01

    We demonstrate measurement of non-equilibrium backbone amide hydrogen-deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using 1 H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state.

  16. De novo protein structure determination using sparse NMR data

    International Nuclear Information System (INIS)

    Bowers, Peter M.; Strauss, Charlie E.M.; Baker, David

    2000-01-01

    We describe a method for generating moderate to high-resolution protein structures using limited NMR data combined with the ab initio protein structure prediction method Rosetta. Peptide fragments are selected from proteins of known structure based on sequence similarity and consistency with chemical shift and NOE data. Models are built from these fragments by minimizing an energy function that favors hydrophobic burial, strand pairing, and satisfaction of NOE constraints. Models generated using this procedure with ∼1 NOE constraint per residue are in some cases closer to the corresponding X-ray structures than the published NMR solution structures. The method requires only the sparse constraints available during initial stages of NMR structure determination, and thus holds promise for increasing the speed with which protein solution structures can be determined

  17. DNA nanotubes for NMR structure determination of membrane proteins.

    Science.gov (United States)

    Bellot, Gaëtan; McClintock, Mark A; Chou, James J; Shih, William M

    2013-04-01

    Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling-based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, toward collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes using counter ions and small DNA-binding molecules. This detergent-resistant liquid-crystal medium offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility and structural programmability. Production of sufficient nanotubes for four or five NMR experiments can be completed in 1 week by a single individual.

  18. 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.

  19. 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.

  20. Porous solid backbone impregnation for electrochemical energy conversion systems

    KAUST Repository

    Boulfrad, Samir; Jabbour, Ghassan

    2013-01-01

    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.

  1. Study of muscular skeletal apparatus’s functional state of junior sportsmen-power lifters, who have backbone verterbral abnormalities

    Directory of Open Access Journals (Sweden)

    V.R. Ilmatov

    2015-10-01

    Full Text Available Purpose: determination of abnormalities and disorders of muscular skeletal apparatuses’ status of power lifters, who have vertebral abnormalities of backbone. Material: 58 junior sportsmen participated in the research. 36 sportsmen were the main group of the research and had vertebral disorders in backbone. For posture testing visual examination was used. Backbone mobility was tested with goniometry method. Flat feet were registered with plantography method. Results: we determined posture abnormalities in sagittal and frontal planes; feet flat, limited maximal movements in thoracic and lumbar spines. It was determined that the most limited were rotational movements and backbone unbending. The next were side bents. These limitations were accompanied by pain syndrome. These observations indirectly confirmed theory of direct interaction of backbone structures with nervous structures. It is also a confirmation of vertebral abnormalities’ presence in junior sportsmen. Conclusions: it was found that in junior sportsmen - power lifters with backbone pathologies in 100% of cases symptoms are determined by local limitations of backbone mobility with pain syndrome. In 35% of cases they are accompanied by posture’s disorders and feet flat. Orientation and methodic of rehabilitation of such sportsmen have been determined.

  2. Peptoid-Peptide hybrid backbone architectures

    DEFF Research Database (Denmark)

    Olsen, Christian Adam

    2010-01-01

    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......-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...

  3. 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.

  4. 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...

  5. Identifying secondary structures in proteins using NMR chemical shift 3D correlation maps

    Science.gov (United States)

    Kumari, Amrita; Dorai, Kavita

    2013-06-01

    NMR chemical shifts are accurate indicators of molecular environment and have been extensively used as aids in protein structure determination. This work focuses on creating empirical 3D correlation maps of backbone chemical shift nuclei for use as identifiers of secondary structure elements in proteins. A correlated database of backbone nuclei chemical shifts was constructed from experimental structural data gathered from entries in the Protein Data Bank (PDB) as well as isotropic chemical shift values from the RefDB database. Rigorous statistical analysis of the maps led to the conclusion that specific correlations between triplets of backbone chemical shifts are best able to differentiate between different secondary structures such as α-helices, β-strands and turns. The method is compared with similar techniques that use NMR chemical shift information as aids in biomolecular structure determination and performs well in tests done on experimental data determined for different types of proteins, including large multi-domain proteins and membrane proteins.

  6. Structural insights into the backbone-circularized granulocyte colony-stimulating factor containing a short connector.

    Science.gov (United States)

    Miyafusa, Takamitsu; Shibuya, Risa; Honda, Shinya

    2018-06-02

    Backbone circularization is a powerful approach for enhancing the structural stability of polypeptides. Herein, we present the crystal structure of the circularized variant of the granulocyte colony-stimulating factor (G-CSF) in which the terminal helical region was circularized using a short, two-amino acid connector. The structure revealed that the N- and C-termini were indeed connected by a peptide bond. The local structure of the C-terminal region transited from an α helix to 3 10 helix with a bend close to the N-terminal region, indicating that the structural change offset the insufficient length of the connector. This is the first-ever report of a crystal structure of the backbone of a circularized protein. It will facilitate the development of backbone circularization methodology. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Fusion protein is the main determinant of metapneumovirus host tropism.

    Science.gov (United States)

    de Graaf, Miranda; Schrauwen, Eefje J A; Herfst, Sander; van Amerongen, Geert; Osterhaus, Albert D M E; Fouchier, Ron A M

    2009-06-01

    Human metapneumovirus (HMPV) and avian metapneumovirus subgroup C (AMPV-C) infect humans and birds, respectively. This study confirmed the difference in host range in turkey poults, and analysed the contribution of the individual metapneumovirus genes to host range in an in vitro cell-culture model. Mammalian Vero-118 cells supported replication of both HMPV and AMPV-C in contrast to avian quail fibroblast (QT6) cells in which only AMPV-C replicated to high titres. Inoculation of Vero-118 and QT6 cells with recombinant HMPV in which genes were exchanged with those of AMPV-C revealed that the metapneumovirus fusion (F) protein is the main determinant for host tropism. Chimeric viruses in which polymerase complex proteins were exchanged between HMPV and AMPV-C replicated less efficiently compared with HMPV in QT6 cells. Using mini-genome systems, it was shown that exchanging these polymerase proteins resulted in reduced replication and transcription efficiency in QT6 cells. Examination of infected Vero-118 and QT6 cells revealed that viruses containing the F protein of AMPV-C yielded larger syncytia compared with viruses containing the HMPV F protein. Cell-content mixing assays revealed that the F protein of AMPV-C was more fusogenic compared with the F protein of HMPV, and that the F2 region is responsible for the difference observed between AMPV-C and HMPV F-promoted fusion in QT6 and Vero-118 cells. This study provides insight into the determinants of host tropism and membrane fusion of metapneumoviruses.

  8. Nonribosomal biosynthesis of backbone-modified peptides

    Science.gov (United States)

    Niquille, David L.; Hansen, Douglas A.; Mori, Takahiro; Fercher, David; Kries, Hajo; Hilvert, Donald

    2018-03-01

    Biosynthetic modification of nonribosomal peptide backbones represents a potentially powerful strategy to modulate the structure and properties of an important class of therapeutics. Using a high-throughput assay for catalytic activity, we show here that an L-Phe-specific module of an archetypal nonribosomal peptide synthetase can be reprogrammed to accept and process the backbone-modified amino acid (S)-β-Phe with near-native specificity and efficiency. A co-crystal structure with a non-hydrolysable aminoacyl-AMP analogue reveals the origins of the 40,000-fold α/β-specificity switch, illuminating subtle but precise remodelling of the active site. When the engineered catalyst was paired with downstream module(s), (S)-β-Phe-containing peptides were produced at preparative scale in vitro (~1 mmol) and high titres in vivo (~100 mg l-1), highlighting the potential of biosynthetic pathway engineering for the construction of novel nonribosomal β-frameworks.

  9. Taking MAD to the extreme: ultrafast protein structure determination

    International Nuclear Information System (INIS)

    Walsh, M.A.; Dementieva, I.; Evans, G.; Sanishvili, R.; Joachimiak, A.

    1999-01-01

    Multiwavelength anomalous diffraction data were measured in 23 min from a 16 kDa selenomethionyl-substituted protein, producing experimental phases to 2.25 (angstrom) resolution. The data were collected on a mosaic 3 x 3 charge-coupled device using undulator radiation from the Structural Biology Center 19ID beamline at the Argonne National Laboratory's Advanced Photon Source. The phases were independently obtained semiautomatically by two crystallographic program suites, CCP4 and CNS. The quality and speed of this data acquisition exemplify the opportunities at third-generation synchrotron sources for high-throughput protein crystal structure determination

  10. Redox-controlled backbone dynamics of human cytochrome c revealed by 15N NMR relaxation measurements

    International Nuclear Information System (INIS)

    Sakamoto, Koichi; Kamiya, Masakatsu; Uchida, Takeshi; Kawano, Keiichi; Ishimori, Koichiro

    2010-01-01

    Research highlights: → The dynamic parameters for the backbone dynamics in Cyt c were determined. → The backbone mobility of Cyt c is highly restricted due to the covalently bound heme. → The backbone mobility of Cyt c is more restricted upon the oxidation of the heme. → The redox-dependent dynamics are shown in the backbone of Cyt c. → 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 15 N NMR relaxation experiments. 15 N T 1 and T 2 values and 1 H- 15 N NOEs of uniformly 15 N-labeled reduced and oxidized Cyt c were measured, and the generalized order parameters (S 2 ), the effective correlation time for internal motion (τ e ), the 15 N exchange broadening contributions (R ex ) for each residue, and the overall correlation time (τ 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 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 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.

  11. AUTOBA: automation of backbone assignment from HN(C)N suite of experiments.

    Science.gov (United States)

    Borkar, Aditi; Kumar, Dinesh; Hosur, Ramakrishna V

    2011-07-01

    Development of efficient strategies and automation represent important milestones of progress in rapid structure determination efforts in proteomics research. In this context, we present here an efficient algorithm named as AUTOBA (Automatic Backbone Assignment) designed to automate the assignment protocol based on HN(C)N suite of experiments. Depending upon the spectral dispersion, the user can record 2D or 3D versions of the experiments for assignment. The algorithm uses as inputs: (i) protein primary sequence and (ii) peak-lists from user defined HN(C)N suite of experiments. In the end, one gets H(N), (15)N, C(α) and C' assignments (in common BMRB format) for the individual residues along the polypeptide chain. The success of the algorithm has been demonstrated, not only with experimental spectra recorded on two small globular proteins: ubiquitin (76 aa) and M-crystallin (85 aa), but also with simulated spectra of 27 other proteins using assignment data from the BMRB.

  12. What determines the structures of native folds of proteins?

    International Nuclear Information System (INIS)

    Trovato, Antonio; Hoang, Trinh X; Banavar, Jayanth R; Maritan, Amos; Seno, Flavio

    2005-01-01

    We review a simple physical model (Hoang et al 2004 Proc. Natl Acad. Sci. USA 101 7960, Banavar et al 2004 Phys. Rev. E at press) which captures the essential physico-chemical ingredients that determine protein structure, such as the inherent anisotropy of a chain molecule, the geometrical and energetic constraints placed by hydrogen bonds, sterics, and hydrophobicity. Within this framework, marginally compact conformations resembling the native state folds of proteins emerge as competing minima in the free energy landscape. Here we demonstrate that a hydrophobic-polar (HP) sequence composed of regularly repeated patterns has as its ground state a β-helical structure remarkably similar to a known architecture in the Protein Data Bank

  13. 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

  14. Protein structure determination by exhaustive search of Protein Data Bank derived databases.

    Science.gov (United States)

    Stokes-Rees, Ian; Sliz, Piotr

    2010-12-14

    Parallel sequence and structure alignment tools have become ubiquitous and invaluable at all levels in the study of biological systems. We demonstrate the application and utility of this same parallel search paradigm to the process of protein structure determination, benefitting from the large and growing corpus of known structures. Such searches were previously computationally intractable. Through the method of Wide Search Molecular Replacement, developed here, they can be completed in a few hours with the aide of national-scale federated cyberinfrastructure. By dramatically expanding the range of models considered for structure determination, we show that small (less than 12% structural coverage) and low sequence identity (less than 20% identity) template structures can be identified through multidimensional template scoring metrics and used for structure determination. Many new macromolecular complexes can benefit significantly from such a technique due to the lack of known homologous protein folds or sequences. We demonstrate the effectiveness of the method by determining the structure of a full-length p97 homologue from Trichoplusia ni. Example cases with the MHC/T-cell receptor complex and the EmoB protein provide systematic estimates of minimum sequence identity, structure coverage, and structural similarity required for this method to succeed. We describe how this structure-search approach and other novel computationally intensive workflows are made tractable through integration with the US national computational cyberinfrastructure, allowing, for example, rapid processing of the entire Structural Classification of Proteins protein fragment database.

  15. Monoclonal protein reference change value as determined by gel-based serum protein electrophoresis.

    Science.gov (United States)

    Salamatmanesh, Mina; McCudden, Christopher R; McCurdy, Arleigh; Booth, Ronald A

    2018-01-01

    The International Myeloma Working Group recommendations for monitoring disease progression or response include quantitation of the involved monoclonal immunoglobulin. They have defined the minimum change criteria of ≧25% with an absolute change of no gel-based serum protein electrophoresis. Sixteen clinically stable MGUS patients were identified from our clinical hematology database. Individual biological variability (CVi) was determined and used to calculate a monoclonal protein reference change value (RCV). Analytical variability of the normal protein fractions (albumin, alpha-1, alpha-2, beta, total gamma) ranged from 1.3% for albumin to 5.8% for the alpha-1 globulins. CVa of low (5.6g/L) and high (32.2g/L) concentration monoclonal proteins were 3.1% and 22.2%, respectively. Individual CVi of stable patients ranged from 3.5% to 24.5% with a CVi of 12.9%. The reference change value (RCV) at a 95% probability was determined to be 36.7% (low) 39.6% (high) using our CVa and CVi. Serial monitoring of monoclonal protein concentration is important for MGUS and multiple myeloma patients. Accurate criteria for interpreting a change in monoclonal protein concentration are required for appropriate decision making. We used QC results and real-world conditions to assess imprecision of serum protein fractions including low and high monoclonal protein fractions and clinically stable MGUS patients to determine CVi and RCV. The calculated RCVs of 36.7% (low) and 39.6% (high) in this study were greater that reported previously and greater than the established criteria for relapse. Response criteria may be reassessed to increase sensitivity and specificity for detection of response. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  16. 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.

  17. Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family).

    Science.gov (United States)

    Kulinska, Anna; Godziszewska, Jolanta; Wojciechowska, Anna; Ludwiczak, Marta; Jagura-Burdzy, Grazyna

    2016-04-01

    The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequence parSRA3, forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacent mobC promoter driving expression of the mobC-nicoperon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions in Escherichia coli, since its overexpression in transnegatively interferes with mini-RA3 replication and stable maintenance of RA3. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. 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......, microstructural characterization and electrochemical testing are discussed. Data on polarization resistance, Rp, are obtained from impedance spectra recorded on quasi-symmetrical cells (YSZ backbones/YSZ/LSM-YSZ (screen printed)). The backbones are infiltrated with LSM and compared to a standard LSM-YSZ screen...

  19. Protein energy landscapes determined by five-dimensional crystallography

    International Nuclear Information System (INIS)

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

    2013-01-01

    Barriers of activation within the photocycle of a photoactive protein were extracted from comprehensive time courses of time resolved crystallographic data collected at multiple temperature settings. 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

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

    International Nuclear Information System (INIS)

    DAVID A. BOOTHMAN

    2006-01-01

    accumulation of nCLU signals cell death; and (3) sCLU is cytoprotective via blockage of IR-induced TGF-? signaling, which causes growth inhibition and cell death by apoptosis. These hypotheses will be examined in the following three Aims: Specific Aim No.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 No.2: Determine the functions of sCLU compared to nCLU using clusterin-deficient human or mouse cell lines. Specific Aim No.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

  1. 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.

  2. Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C’ scalar couplings (3hbJNC’)

    NARCIS (Netherlands)

    Bonvin, A.M.J.J.; Houben, K.; Guenneugues, M.N.L.; Kaptein, R.; Boelens, R.

    2001-01-01

    The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small alpha/beta protein

  3. 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.

  4. 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

  5. Backbone dynamics of oxidized and reduced D. vulgaris flavodoxin in solution

    International Nuclear Information System (INIS)

    Hrovat, Andrea; Bluemel, Markus; Loehr, Frank; Mayhew, Stephen G.; Rueterjans, Heinz

    1997-01-01

    Recombinant Desulfovibrio vulgaris flavodoxin was produced in Escherichia coli. A complete backbone NMR assignment for the two-electron reduced protein revealed significant changes of chemical shift values compared to the oxidized protein, in particular for the flavine mononucleotide (FMN)-binding site. A comparison of homo- and heteronuclear NOESY spectra for the two redox states led to the assumption that reduction is not accompanied by significant changes of the global fold of the protein.The backbone dynamics of both the oxidized and reduced forms of D. vulgaris flavodoxin were investigated using two-dimensional 15 N- 1 H correlation NMR spectroscopy.T 1 , T 2 and NOE data are obtained for 95% of the backbone amide groups in both redox states. These values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-;4559, 4559-;4570]. A comparison of the two redox states indicates that in the reduced species significantly more flexibility occurs in the two loop regions enclosing FMN.Also, a higher amplitude of local motion could be found for the N(3)H group of FMN bound to the reduced protein compared to the oxidized state

  6. Defining an essence of structure determining residue contacts in proteins.

    Science.gov (United States)

    Sathyapriya, R; Duarte, Jose M; Stehr, Henning; Filippis, Ioannis; Lappe, Michael

    2009-12-01

    The network of native non-covalent residue contacts determines the three-dimensional structure of a protein. However, not all contacts are of equal structural significance, and little knowledge exists about a minimal, yet sufficient, subset required to define the global features of a protein. Characterisation of this "structural essence" has remained elusive so far: no algorithmic strategy has been devised to-date that could outperform a random selection in terms of 3D reconstruction accuracy (measured as the Ca RMSD). It is not only of theoretical interest (i.e., for design of advanced statistical potentials) to identify the number and nature of essential native contacts-such a subset of spatial constraints is very useful in a number of novel experimental methods (like EPR) which rely heavily on constraint-based protein modelling. To derive accurate three-dimensional models from distance constraints, we implemented a reconstruction pipeline using distance geometry. We selected a test-set of 12 protein structures from the four major SCOP fold classes and performed our reconstruction analysis. As a reference set, series of random subsets (ranging from 10% to 90% of native contacts) are generated for each protein, and the reconstruction accuracy is computed for each subset. We have developed a rational strategy, termed "cone-peeling" that combines sequence features and network descriptors to select minimal subsets that outperform the reference sets. We present, for the first time, a rational strategy to derive a structural essence of residue contacts and provide an estimate of the size of this minimal subset. Our algorithm computes sparse subsets capable of determining the tertiary structure at approximately 4.8 A Ca RMSD with as little as 8% of the native contacts (Ca-Ca and Cb-Cb). At the same time, a randomly chosen subset of native contacts needs about twice as many contacts to reach the same level of accuracy. This "structural essence" opens new avenues in the

  7. Protein-protein interaction site predictions with minimum covariance determinant and Mahalanobis distance.

    Science.gov (United States)

    Qiu, Zhijun; Zhou, Bo; Yuan, Jiangfeng

    2017-11-21

    Protein-protein interaction site (PPIS) prediction must deal with the diversity of interaction sites that limits their prediction accuracy. Use of proteins with unknown or unidentified interactions can also lead to missing interfaces. Such data errors are often brought into the training dataset. In response to these two problems, we used the minimum covariance determinant (MCD) method to refine the training data to build a predictor with better performance, utilizing its ability of removing outliers. In order to predict test data in practice, a method based on Mahalanobis distance was devised to select proper test data as input for the predictor. With leave-one-validation and independent test, after the Mahalanobis distance screening, our method achieved higher performance according to Matthews correlation coefficient (MCC), although only a part of test data could be predicted. These results indicate that data refinement is an efficient approach to improve protein-protein interaction site prediction. By further optimizing our method, it is hopeful to develop predictors of better performance and wide range of application. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Backbone resonance assignments of the outer membrane lipoprotein FrpD from Neisseria meningitidis

    Czech Academy of Sciences Publication Activity Database

    Bumba, Ladislav; Sviridova, E.; Kutá-Smatanová, Ivana; Řezáčová, Pavlína; Veverka, Václav

    2014-01-01

    Roč. 8, č. 1 (2014), s. 53-55 ISSN 1874-2718 R&D Projects: GA ČR(CZ) GAP207/11/0717; GA MŠk(CZ) LK11205 Institutional support: RVO:61388963 ; RVO:61388971 ; RVO:67179843 Keywords : Neisseria meningitidis * FrpC * FrpD * backbone assignments * NMR * iron-regulated protein Subject RIV: CE - Biochemistry Impact factor: 0.760, year: 2014

  9. Live Zika virus chimeric vaccine candidate based on a yellow fever 17-D attenuated backbone

    OpenAIRE

    Nougairede, Antoine; Klitting, Raphaelle; Aubry, Fabien; Gilles, Magali; Touret, Franck; De Lamballerie, Xavier

    2018-01-01

    Zika virus (ZIKV) recently dispersed throughout the tropics and sub-tropics causing epidemics associated with congenital disease and neurological complications. There is currently no commercial vaccine for ZIKV. Here we describe the initial development of a chimeric virus containing the prM/E proteins of a ZIKV epidemic strain incorporated into a yellow fever 17-D attenuated backbone. Using the versatile and rapid ISA (Infectious Subgenomic Amplicons) reverse genetics method, we compared diff...

  10. Printing Proteins as Microarrays for High-Throughput Function Determination

    Science.gov (United States)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  11. Radiation safety system (RSS) backbones: Design, engineering, fabrication and installation

    International Nuclear Information System (INIS)

    Wilmarth, J.E.; Sturrock, J.C.; Gallegos, F.R.

    1998-01-01

    The Radiation Safety System (RSS) Backbones are part of an electrical/electronic/mechanical system insuring safe access and exclusion of personnel to areas at the Los Alamos Neutron Science Center (LANSCE) accelerator. The RSS Backbones control the safety fusible beam plugs which terminate transmission of accelerated ion beams in response to predefined conditions. Any beam or access fault of the backbone inputs will cause insertion of the beam plugs in the low energy beam transport. The Backbones serve the function of tying the beam plugs to the access control systems, beam spill monitoring systems and current-level limiting systems. In some ways the Backbones may be thought of as a spinal column with beam plugs at the head and nerve centers along the spinal column. The two Linac Backbone segments and experimental area segments form a continuous cable plant over 3,500 feet from beam plugs to the tip on the longest tail. The Backbones were installed in compliance with current safety standards, such as installation of the two segments in separate conduits or tray. Monitoring for ground-faults and input wiring verification was an added enhancement to the system. The system has the capability to be tested remotely

  12. A use of Ramachandran potentials in protein solution structure determinations

    International Nuclear Information System (INIS)

    Bertini, Ivano; Cavallaro, Gabriele; Luchinat, Claudio; Poli, Irene

    2003-01-01

    A strategy is developed to use database-derived φ-ψ constraints during simulated annealing procedures for protein solution structure determination in order to improve the Ramachandran plot statistics, while maintaining the agreement with the experimental constraints as the sole criterion for the selection of the family. The procedure, fully automated, consists of two consecutive simulated annealing runs. In the first run, the database-derived φ-ψ constraints are enforced for all aminoacids (but prolines and glycines). A family of structures is then selected on the ground of the lowest violations of the experimental constraints only, and the φ-ψ values for each residue are examined. In the second and final run, the database-derived φ-ψ constraints are enforced only for those residues which in the first run have ended in one and the same favored φ-ψ region. For residues which are either spread over different favored regions or concentrated in disallowed regions, the constraints are not enforced. The final family is then selected, after the second run, again only based on the agreement with the experimental constraints. This automated approach was implemented in DYANA and was tested on as many as 12 proteins, including some containing paramagnetic metals, whose structures had been previously solved in our laboratory. The quality of the structures, and of Ramachandran plot statistics in particular, was notably improved while preserving the agreement with the experimental constraints

  13. 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.

  14. 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....

  15. 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...

  16. Dynamic power control for wireless backbone mesh networks: a survey

    CSIR Research Space (South Africa)

    Olwal, TO

    2010-01-01

    Full Text Available points of failures, and robust against RF interference, obstacles or power outage. This is because WMRs forming wireless backbone mesh networks (WBMNs) are built on advanced physical technologies. Such nodes perform both accessing and forwarding...

  17. 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...

  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...... as the initial tag clustering result and then assign the rest tags into the corresponding clusters based on the similarity. Experimental results on three real world datasets namely MedWorm, MovieLens and Dmoz demonstrate the effectiveness and the superiority of the proposed method against the traditional...... Agglomerative Clustering on tagging data, which possess the inherent drawbacks, such as the sensitivity of initialization. In this paper, we instead make use of the approximate backbone of tag clustering results to find out better tag clusters. In particular, we propose an APProximate backbonE-based Clustering...

  19. 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...

  20. 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.

  1. Fluorometric determination of proteins using the yttrium(III)-sodium lauryl sulfate-rutin-protein system

    International Nuclear Information System (INIS)

    Liu Shufang; Yang Jinghe; Wu Xia; Wang Fei; Wang Feng; Jia Zhen

    2006-01-01

    It is found that rutin can react with yttrium(III) (Y 3+ ), and emits fluorescence of rutin. The intensity is greatly enhanced by proteins in the presence of sodium lauryl sulfate (SLS). Based on this, a new fluorimetric method of determination of proteins is developed. Under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of proteins in the range of 5.0x10 -9 -1.0x10 -5 g/mL for bovine serum albumin (BSA), 3.0x10 -8 -1.0x10 -5 g/mL for human serum albumin (HSA) and 1.0x10 -7 -2.0x10 -5 g/mL for egg albumin (EA). Their detection limits (S/N=3) are 1.6x10 -9 , 9.8x10 -9 and 2.1x10 -8 g/mL, respectively. The interaction mechanism is also studied

  2. Fluorometric determination of proteins using the yttrium(III)-sodium lauryl sulfate-rutin-protein system

    Energy Technology Data Exchange (ETDEWEB)

    Liu Shufang [Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Yang Jinghe [Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)]. E-mail: yjh@sdu.edu.cn; Wu Xia [Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Fei [Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Feng [Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Jia Zhen [Key Laboratory of Colloid and Interface Chemistry (Shandong University), Ministry of Education, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2006-04-15

    It is found that rutin can react with yttrium(III) (Y{sup 3+}), and emits fluorescence of rutin. The intensity is greatly enhanced by proteins in the presence of sodium lauryl sulfate (SLS). Based on this, a new fluorimetric method of determination of proteins is developed. Under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of proteins in the range of 5.0x10{sup -9}-1.0x10{sup -5}g/mL for bovine serum albumin (BSA), 3.0x10{sup -8}-1.0x10{sup -5}g/mL for human serum albumin (HSA) and 1.0x10{sup -7}-2.0x10{sup -5}g/mL for egg albumin (EA). Their detection limits (S/N=3) are 1.6x10{sup -9}, 9.8x10{sup -9} and 2.1x10{sup -8}g/mL, respectively. The interaction mechanism is also studied.

  3. 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.

  4. Solid state radiation chemistry of the DNA backbone

    International Nuclear Information System (INIS)

    Bernhard, W.A.

    1989-09-01

    The long term goal of this program is to determine the fundamental rules needed to predict the type and yield of damage produced in DNA due to direct effects of ionizing radiation. The focus is on damage to the sugar-phosphate backbone, damage that would lead to strand breaks. Model systems have been chosen that permit various aspects of this problem to be investigated. The emphasis will be on single crystals of monosaccharides, nucleosides, and nucleotides but will also include some powder work on polynucleotides. In these model systems, free radical products and reactions are observed by electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) techniques. The information thus gained is used in constructing rules that predict what primary free radicals are formed in single crystals of model compounds and the reactions stemming from the primary radicals. The formulation of a set of rules that work in model systems will represent a major advance toward formulating a set of rules that predict the direct damage in DNA itself. In a broader context this program is part of the effort to understand and predict the effects of exposure to ionizing radiation received at low dose rates over long periods of time. Assessment of low dose effects requires a basic understanding of the action of radiation at the molecular level. By contributing to that basic understanding, this program will help solve the problems of risk assessment under low dose conditions. 5 refs., 3 figs

  5. 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

  6. Comparison of three methods for determination of protein ...

    African Journals Online (AJOL)

    However, a six fold greater amount of protein was obtained when FastPrep was applied to lyse LAB cells. Our results also indicate that, this fast and easy extraction method allows more spot-abundant polyacrylamide gels. More clear and consistent strips were detected by SDS-PAGE when proteins were extracted by ...

  7. Standardization for cortisol determination in human blood by competitive protein-binding

    International Nuclear Information System (INIS)

    Okada, H.

    1978-01-01

    Standardization for determination of cortisol from human plasma (17-hydroxycorticosteroids) using competitive protein-binding method is presented. Activated carbon coated with dextrans is used for separation of the hormone-protein complexe and hormone labelled free [pt

  8. Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry

    NARCIS (Netherlands)

    Wang, Guanbo; de Jong, Rob N; van den Bremer, Ewald T J; Parren, Paul W H I; Heck, Albert J R

    2017-01-01

    The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

  9. Backbone Brackets and Arginine Tweezers delineate Class I and Class II aminoacyl tRNA synthetases

    Science.gov (United States)

    Haupt, V. Joachim; Schroeder, Michael; Labudde, Dirk

    2018-01-01

    The origin of the machinery that realizes protein biosynthesis in all organisms is still unclear. One key component of this machinery are aminoacyl tRNA synthetases (aaRS), which ligate tRNAs to amino acids while consuming ATP. Sequence analyses revealed that these enzymes can be divided into two complementary classes. Both classes differ significantly on a sequence and structural level, feature different reaction mechanisms, and occur in diverse oligomerization states. The one unifying aspect of both classes is their function of binding ATP. We identified Backbone Brackets and Arginine Tweezers as most compact ATP binding motifs characteristic for each Class. Geometric analysis shows a structural rearrangement of the Backbone Brackets upon ATP binding, indicating a general mechanism of all Class I structures. Regarding the origin of aaRS, the Rodin-Ohno hypothesis states that the peculiar nature of the two aaRS classes is the result of their primordial forms, called Protozymes, being encoded on opposite strands of the same gene. Backbone Brackets and Arginine Tweezers were traced back to the proposed Protozymes and their more efficient successors, the Urzymes. Both structural motifs can be observed as pairs of residues in contemporary structures and it seems that the time of their addition, indicated by their placement in the ancient aaRS, coincides with the evolutionary trace of Proto- and Urzymes. PMID:29659563

  10. ADAR RNA editing below the backbone.

    Science.gov (United States)

    Keegan, Liam; Khan, Anzer; Vukic, Dragana; O'Connell, Mary

    2017-09-01

    ADAR RNA editing enzymes ( a denosine d e a minases acting on R NA) that convert adenosine bases to inosines were first identified biochemically 30 years ago. Since then, studies on ADARs in genetic model organisms, and evolutionary comparisons between them, continue to reveal a surprising range of pleiotropic biological effects of ADARs. This review focuses on Drosophila melanogaster , which has a single Adar gene encoding a homolog of vertebrate ADAR2 that site-specifically edits hundreds of transcripts to change individual codons in ion channel subunits and membrane and cytoskeletal proteins. Drosophila ADAR is involved in the control of neuronal excitability and neurodegeneration and, intriguingly, in the control of neuronal plasticity and sleep. Drosophila ADAR also interacts strongly with RNA interference, a key antiviral defense mechanism in invertebrates. Recent crystal structures of human ADAR2 deaminase domain-RNA complexes help to interpret available information on Drosophila ADAR isoforms and on the evolution of ADARs from tRNA deaminase ADAT proteins. ADAR RNA editing is a paradigm for the now rapidly expanding range of RNA modifications in mRNAs and ncRNAs. Even with recent progress, much remains to be understood about these groundbreaking ADAR RNA modification systems. © 2017 Keegan et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  11. 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.

  12. 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

  13. Determination of protein carbonyls in plasma, cell extracts, tissue homogenates, isolated proteins

    DEFF Research Database (Denmark)

    Weber, Daniela; Davies, Michael J.; Grune, Tilman

    2015-01-01

    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...

  14. Protein Glycation in Diabetes as Determined by Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Annunziata Lapolla

    2013-01-01

    Full Text Available Diabetes is a common endocrine disorder characterized by hyperglycemia leading to nonenzymatic glycation of proteins, responsible for chronic complications. The development of mass spectrometric techniques able to give highly specific and reliable results in proteome field is of wide interest for physicians, giving them new tools to monitor the disease progression and the possible complications related to diabetes, as well as the effectiveness of therapeutic treatments. This paper reports and discusses some of the data pertaining protein glycation in diabetic subjects obtained by matrix-assisted laser desorption ionization (MALDI mass spectrometry (MS. The preliminary studies carried out by in vitro protein glycation experiments show clear differences in molecular weight of glycated and unglycated proteins. Then, the attention was focused on plasma proteins human serum albumin (HSA and immunoglobulin G (IgG. Enzymatic degradation products of in vitro glycated HSA were studied in order to simulate the in vivo enzymatic digestion of glycated species by the immunological system leading to the highly reactive advanced glycation end-products (AGEs peptides. Further studies led to the evaluation of glycated Apo A-I and glycated haemoglobin levels. A different MALDI approach was employed for the identification of markers of disease in urine samples of healthy, diabetic, nephropathic, and diabetic-nephropathic subjects.

  15. Total Protein Content Determination of Microalgal Biomass by Elemental Nitrogen Analysis and a Dedicated Nitrogen-to-Protein Conversion Factor

    Energy Technology Data Exchange (ETDEWEB)

    Laurens, Lieve M [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Olstad-Thompson, Jessica L [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Templeton, David W [National Renewable Energy Laboratory (NREL), Golden, CO (United States)

    2018-04-02

    Accurately determining protein content is important in the valorization of algal biomass in food, feed, and fuel markets, where these values are used for component balance calculations. Conversion of elemental nitrogen to protein is a well-accepted and widely practiced method, but depends on developing an applicable nitrogen-to-protein conversion factor. The methodology reported here covers the quantitative assessment of the total nitrogen content of algal biomass and a description of the methodology that underpins the accurate de novo calculation of a dedicated nitrogen-to-protein conversion factor.

  16. Performance of Flow-Aware Networking in LTE backbone

    DEFF Research Database (Denmark)

    Sniady, Aleksander; Soler, José

    2012-01-01

    technologies, such as Long Term Evolution (LTE). This paper proposes usage of a modified Flow Aware Networking (FAN) technique for enhancing Quality of Service (QoS) in the all-IP transport networks underlying LTE backbone. The results obtained with OPNET Modeler show that FAN, in spite of being relatively...

  17. 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.

  18. Internet Backbone in the Democratic Republic of Congo : Feasibility ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Internet Backbone in the Democratic Republic of Congo : Feasibility Study and Advocacy. During 7-10 February 2005, representatives of five francophone African countries (Cameroon, Morocco, Niger, Sénégal, and the Democratic Republic of Congo - DRC) met to consider ways and means of galvanizing the appropriation ...

  19. Optical alignment control of polyimide molecules containing azobenzene in the backbone structure

    International Nuclear Information System (INIS)

    Sakamoto, Kenji; Usami, Kiyoaki; Sasaki, Toru; Kanayama, Takashi; Ushioda, Sukekatsu

    2004-01-01

    Using polarized infrared absorption spectroscopy, we have determined the orientation of the polyimide backbone structure in photo-alignment films for liquid crystals (LC). The polyimide used in this study contains azobenzene in the backbone structure. Photo-alignment treatment was performed on the corresponding polyamic acid film, using a light source of wavelength 340-500 nm. The polyamic acid film (∼16 nm thick) was first irradiated at normal incidence with linearly polarized light (LP-light) of 156 J/cm 2 , and then oblique angle irradiation of unpolarized light (UP-light) was performed in the plane of incidence perpendicular to the polarization direction of the LP-light. The UP-light exposure was varied up to 882 J/cm 2 . We found that the average inclination angle of the polyimide backbone structure, measured from the surface plane, increases almost linearly with UP-light exposure. On the other hand, the in-plane anisotropy induced by the first irradiation with LP-light decreases with the increase of UP-light exposure

  20. 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 highly...... homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co...... the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme....

  1. Direct observation of backbone planarization via side-chain alignment in single bulky-substituted polythiophenes

    Science.gov (United States)

    Raithel, Dominic; Simine, Lena; Pickel, Sebastian; Schötz, Konstantin; Panzer, Fabian; Baderschneider, Sebastian; Schiefer, Daniel; Lohwasser, Ruth; Köhler, Jürgen; Thelakkat, Mukundan; Sommer, Michael; Köhler, Anna; Rossky, Peter J.; Hildner, Richard

    2018-03-01

    The backbone conformation of conjugated polymers affects, to a large extent, their optical and electronic properties. The usually flexible substituents provide solubility and influence the packing behavior of conjugated polymers in films or in bad solvents. However, the role of the side chains in determining and potentially controlling the backbone conformation, and thus the optical and electronic properties on the single polymer level, is currently under debate. Here, we investigate directly the impact of the side chains by studying the bulky-substituted poly(3-(2,5-dioctylphenyl)thiophene) (PDOPT) and the common poly(3-hexylthiophene) (P3HT), both with a defined molecular weight and high regioregularity, using low-temperature single-chain photoluminescence (PL) spectroscopy and quantum-classical simulations. Surprisingly, the optical transition energy of PDOPT is significantly (˜2,000 cm‑1 or 0.25 eV) red-shifted relative to P3HT despite a higher static and dynamic disorder in the former. We ascribe this red shift to a side-chain induced backbone planarization in PDOPT, supported by temperature-dependent ensemble PL spectroscopy. Our atomistic simulations reveal that the bulkier 2,5-dioctylphenyl side chains of PDOPT adopt a clear secondary helical structural motif and thus protect conjugation, i.e., enforce backbone planarity, whereas, for P3HT, this is not the case. These different degrees of planarity in both thiophenes do not result in different conjugation lengths, which we found to be similar. It is rather the stronger electronic coupling between the repeating units in the more planar PDOPT which gives rise to the observed spectral red shift as well as to a reduced calculated electron‑hole polarization.

  2. Quantitative determination of proteins, lipids and ascorbic acid in ...

    African Journals Online (AJOL)

    The protein content of the legumes and fruits ranged from 4.10 to 9.60 % with the highest value in snot apple, followed by governor's plum, ground nuts and cow peas. Ground nuts were found to be the richest source of lipids (mean = 45.97%) while lipids were low in all the other legume and fruit samples (0.83 to 1.63 %).

  3. Determination of protein turnover parameters in athletes using 15N tracers

    International Nuclear Information System (INIS)

    Krumbiegel, P.; Kuehne, K.; Faust, H.; Bornhak, H.; Junghans, P.; Zerbes, H.

    1985-01-01

    In 2 adolescent female athletes engaged in technical-acrobatic sports the influence of a high protein diet on the protein turnover rate under training conditions was investigated by means of 15 N-glycine. Protein synthesis was significantly increased, whereas the utilization of nutritive nitrogen was decreased as expected. The 15 N tracer technique is well suited to determine the protein requirements under special training conditions

  4. 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.

  5. Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi; Darwish, Tamim A.; Pedersen, Martin Cramer

    2018-01-01

    A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron sca...... solution structure determination of membrane proteins by SANS and subsequent data analysis available to non-specialists. This article is protected by copyright. All rights reserved....

  6. A PQL (protein quantity loci) analysis of mature pea seed proteins identifies loci determining seed protein composition.

    Science.gov (United States)

    Bourgeois, Michael; Jacquin, Françoise; Cassecuelle, Florence; Savois, Vincent; Belghazi, Maya; Aubert, Grégoire; Quillien, Laurence; Huart, Myriam; Marget, Pascal; Burstin, Judith

    2011-05-01

    Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Accurate protein structure modeling using sparse NMR data and homologous structure information.

    Science.gov (United States)

    Thompson, James M; Sgourakis, Nikolaos G; Liu, Gaohua; Rossi, Paolo; Tang, Yuefeng; Mills, Jeffrey L; Szyperski, Thomas; Montelione, Gaetano T; Baker, David

    2012-06-19

    While information from homologous structures plays a central role in X-ray structure determination by molecular replacement, such information is rarely used in NMR structure determination because it can be incorrect, both locally and globally, when evolutionary relationships are inferred incorrectly or there has been considerable evolutionary structural divergence. Here we describe a method that allows robust modeling of protein structures of up to 225 residues by combining (1)H(N), (13)C, and (15)N backbone and (13)Cβ chemical shift data, distance restraints derived from homologous structures, and a physically realistic all-atom energy function. Accurate models are distinguished from inaccurate models generated using incorrect sequence alignments by requiring that (i) the all-atom energies of models generated using the restraints are lower than models generated in unrestrained calculations and (ii) the low-energy structures converge to within 2.0 Å backbone rmsd over 75% of the protein. Benchmark calculations on known structures and blind targets show that the method can accurately model protein structures, even with very remote homology information, to a backbone rmsd of 1.2-1.9 Å relative to the conventional determined NMR ensembles and of 0.9-1.6 Å relative to X-ray structures for well-defined regions of the protein structures. This approach facilitates the accurate modeling of protein structures using backbone chemical shift data without need for side-chain resonance assignments and extensive analysis of NOESY cross-peak assignments.

  9. Determining and comparing protein function in Bacterial genome sequences

    DEFF Research Database (Denmark)

    Vesth, Tammi Camilla

    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......In November 2013, there was around 21.000 different prokaryotic genomes sequenced and publicly available, and the number is growing daily with another 20.000 or more genomes expected to be sequenced and deposited by the end of 2014. An important part of the analysis of this data is the functional...... 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...

  10. 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.

  11. 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

  12. 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

  13. Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

    Science.gov (United States)

    Radoux, Chris J; Olsson, Tjelvar S G; Pitt, Will R; Groom, Colin R; Blundell, Tom L

    2016-05-12

    Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project.

  14. Determination and Quantification of Molecular Interactions in Protein Films: A Review

    Directory of Open Access Journals (Sweden)

    Felicia Hammann

    2014-12-01

    Full Text Available Protein based films are nowadays also prepared with the aim of replacing expensive, crude oil-based polymers as environmentally friendly and renewable alternatives. The protein structure determines the ability of protein chains to form intra- and intermolecular bonds, whereas the degree of cross-linking depends on the amino acid composition and molecular weight of the protein, besides the conditions used in film preparation and processing. The functionality varies significantly depending on the type of protein and affects the resulting film quality and properties. This paper reviews the methods used in examination of molecular interactions in protein films and discusses how these intermolecular interactions can be quantified. The qualitative determination methods can be distinguished by structural analysis of solutions (electrophoretic analysis, size exclusion chromatography and analysis of solid films (spectroscopy techniques, X-ray scattering methods. To quantify molecular interactions involved, two methods were found to be the most suitable: protein film swelling and solubility. The importance of non-covalent and covalent interactions in protein films can be investigated using different solvents. The research was focused on whey protein, whereas soy protein and wheat gluten were included as further examples of proteins.

  15. Determination and Quantification of Molecular Interactions in Protein Films: A Review.

    Science.gov (United States)

    Hammann, Felicia; Schmid, Markus

    2014-12-10

    Protein based films are nowadays also prepared with the aim of replacing expensive, crude oil-based polymers as environmentally friendly and renewable alternatives. The protein structure determines the ability of protein chains to form intra- and intermolecular bonds, whereas the degree of cross-linking depends on the amino acid composition and molecular weight of the protein, besides the conditions used in film preparation and processing. The functionality varies significantly depending on the type of protein and affects the resulting film quality and properties. This paper reviews the methods used in examination of molecular interactions in protein films and discusses how these intermolecular interactions can be quantified. The qualitative determination methods can be distinguished by structural analysis of solutions (electrophoretic analysis, size exclusion chromatography) and analysis of solid films (spectroscopy techniques, X-ray scattering methods). To quantify molecular interactions involved, two methods were found to be the most suitable: protein film swelling and solubility. The importance of non-covalent and covalent interactions in protein films can be investigated using different solvents. The research was focused on whey protein, whereas soy protein and wheat gluten were included as further examples of proteins.

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

    International Nuclear Information System (INIS)

    Mathe, Christelle; Devineau, Stephanie; Aude, Jean-Christophe; Lagniel, Gilles; Chedin, Stephane; Legros, Veronique; Mathon, Marie-Helene; Renault, Jean-Philippe; Pin, Serge; Boulard, Yves; Labarre, Jean

    2013-01-01

    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 nano-technology. 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 p-p 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. (authors)

  17. 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.

  18. UET: a database of evolutionarily-predicted functional determinants of protein sequences that cluster as functional sites in protein structures.

    Science.gov (United States)

    Lua, Rhonald C; Wilson, Stephen J; Konecki, Daniel M; Wilkins, Angela D; Venner, Eric; Morgan, Daniel H; Lichtarge, Olivier

    2016-01-04

    The structure and function of proteins underlie most aspects of biology and their mutational perturbations often cause disease. To identify the molecular determinants of function as well as targets for drugs, it is central to characterize the important residues and how they cluster to form functional sites. The Evolutionary Trace (ET) achieves this by ranking the functional and structural importance of the protein sequence positions. ET uses evolutionary distances to estimate functional distances and correlates genotype variations with those in the fitness phenotype. Thus, ET ranks are worse for sequence positions that vary among evolutionarily closer homologs but better for positions that vary mostly among distant homologs. This approach identifies functional determinants, predicts function, guides the mutational redesign of functional and allosteric specificity, and interprets the action of coding sequence variations in proteins, people and populations. Now, the UET database offers pre-computed ET analyses for the protein structure databank, and on-the-fly analysis of any protein sequence. A web interface retrieves ET rankings of sequence positions and maps results to a structure to identify functionally important regions. This UET database integrates several ways of viewing the results on the protein sequence or structure and can be found at http://mammoth.bcm.tmc.edu/uet/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Protein and DNA sequence determinants of thermophilic adaptation.

    Directory of Open Access Journals (Sweden)

    Konstantin B Zeldovich

    2007-01-01

    Full Text Available There have been considerable attempts in the past to relate phenotypic trait--habitat temperature of organisms--to their genotypes, most importantly compositions of their genomes and proteomes. However, despite accumulation of anecdotal evidence, an exact and conclusive relationship between the former and the latter has been elusive. We present an exhaustive study of the relationship between amino acid composition of proteomes, nucleotide composition of DNA, and optimal growth temperature (OGT of prokaryotes. Based on 204 complete proteomes of archaea and bacteria spanning the temperature range from -10 degrees C to 110 degrees C, we performed an exhaustive enumeration of all possible sets of amino acids and found a set of amino acids whose total fraction in a proteome is correlated, to a remarkable extent, with the OGT. The universal set is Ile, Val, Tyr, Trp, Arg, Glu, Leu (IVYWREL, and the correlation coefficient is as high as 0.93. We also found that the G + C content in 204 complete genomes does not exhibit a significant correlation with OGT (R = -0.10. On the other hand, the fraction of A + G in coding DNA is correlated with temperature, to a considerable extent, due to codon patterns of IVYWREL amino acids. Further, we found strong and independent correlation between OGT and the frequency with which pairs of A and G nucleotides appear as nearest neighbors in genome sequences. This adaptation is achieved via codon bias. These findings present a direct link between principles of proteins structure and stability and evolutionary mechanisms of thermophylic adaptation. On the nucleotide level, the analysis provides an example of how nature utilizes codon bias for evolutionary adaptation to extreme conditions. Together these results provide a complete picture of how compositions of proteomes and genomes in prokaryotes adjust to the extreme conditions of the environment.

  20. Increased precision for analysis of protein-ligand dissociation constants determined from chemical shift titrations

    Energy Technology Data Exchange (ETDEWEB)

    Markin, Craig J.; Spyracopoulos, Leo, E-mail: leo.spyracopoulos@ualberta.ca [University of Alberta, Department of Biochemistry (Canada)

    2012-06-15

    NMR is ideally suited for the analysis of protein-protein and protein ligand interactions with dissociation constants ranging from {approx}2 {mu}M to {approx}1 mM, and with kinetics in the fast exchange regime on the NMR timescale. For the determination of dissociation constants (K{sub D}) of 1:1 protein-protein or protein-ligand interactions using NMR, the protein and ligand concentrations must necessarily be similar in magnitude to the K{sub D}, and nonlinear least squares analysis of chemical shift changes as a function of ligand concentration is employed to determine estimates for the parameters K{sub D} and the maximum chemical shift change ({Delta}{delta}{sub max}). During a typical NMR titration, the initial protein concentration, [P{sub 0}], is held nearly constant. For this condition, to determine the most accurate parameters for K{sub D} and {Delta}{delta}{sub max} from nonlinear least squares analyses requires initial protein concentrations that are {approx}0.5 Multiplication-Sign K{sub D}, and a maximum concentration for the ligand, or titrant, of {approx}10 Multiplication-Sign [P{sub 0}]. From a practical standpoint, these requirements are often difficult to achieve. Using Monte Carlo simulations, we demonstrate that co-variation of the ligand and protein concentrations during a titration leads to an increase in the precision of the fitted K{sub D} and {Delta}{delta}{sub max} values when [P{sub 0}] > K{sub D}. Importantly, judicious choice of protein and ligand concentrations for a given NMR titration, combined with nonlinear least squares analyses using two independent variables (ligand and protein concentrations) and two parameters (K{sub D} and {Delta}{delta}{sub max}) is a straightforward approach to increasing the accuracy of measured dissociation constants for 1:1 protein-ligand interactions.

  1. Computational design, construction, and characterization of a set of specificity determining residues in protein-protein interactions.

    Science.gov (United States)

    Nagao, Chioko; Izako, Nozomi; Soga, Shinji; Khan, Samia Haseeb; Kawabata, Shigeki; Shirai, Hiroki; Mizuguchi, Kenji

    2012-10-01

    Proteins interact with different partners to perform different functions and it is important to elucidate the determinants of partner specificity in protein complex formation. Although methods for detecting specificity determining positions have been developed previously, direct experimental evidence for these amino acid residues is scarce, and the lack of information has prevented further computational studies. In this article, we constructed a dataset that is likely to exhibit specificity in protein complex formation, based on available crystal structures and several intuitive ideas about interaction profiles and functional subclasses. We then defined a "structure-based specificity determining position (sbSDP)" as a set of equivalent residues in a protein family showing a large variation in their interaction energy with different partners. We investigated sequence and structural features of sbSDPs and demonstrated that their amino acid propensities significantly differed from those of other interacting residues and that the importance of many of these residues for determining specificity had been verified experimentally. Copyright © 2012 Wiley Periodicals, Inc.

  2. On the localisation of antigenic determinants in a Bence Jones protein

    NARCIS (Netherlands)

    Eyk, H.G. van; Myszkowska, K.

    1967-01-01

    1. 1. The presence of a low molecular weight protein (1.2 S), having antigenic determinants in common with the homologous Bence Jones protein (3.4 S), has been observed in the urine of a patient with multiple myeloma. Its amino acid composition and α-NH2-terminal amino acid residue make it likely

  3. 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 mo...

  4. [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.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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.

  6. Method for determining the concentration of adsorbed protein and cell biomass in cellulose fermentations

    Energy Technology Data Exchange (ETDEWEB)

    Moreira, A R; Phillips, J A; Humphrey, A E

    1978-09-01

    The method presented is based on the determination of the total Lowry protein of the solids and the total Kjeldahl nitrogen of the solids. Experimental data proving the validity of the method are reported. (JSR)

  7. Tyr51: Key Determinant of the Low Thermostability of the Colwellia psychrerythraea Cold-Shock Protein.

    Science.gov (United States)

    Lee, Yeongjoon; Kwak, Chulhee; Jeong, Ki-Woong; Durai, Prasannavenkatesh; Ryu, Kyoung-Seok; Kim, Eun-Hee; Cheong, Chaejoon; Ahn, Hee-Chul; Kim, Hak Jun; Kim, Yangmee

    2018-05-18

    Cold-shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of psychrophile Colwellia psychrerythraea 34H, ( Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs, including a five β-stranded barrel, with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10 -7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of the Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed for the formation of a K3-E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 and 642 s -1 ), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.

  8. Gut luminal endogenous protein: implications for the determination of ileal amino acid digestibility in humans.

    Science.gov (United States)

    Moughan, Paul J; Rutherfurd, Shane M

    2012-08-01

    The true ileal digestibility assay provides the most informative measure of digestibility to assess bioavailability of amino acids in foods for humans. To determine 'true' estimates of ileal amino acid digestibility, requires that endogenous amino acids present in digesta at the terminal ileum be quantified. The amounts of endogenous amino acids in ileal digesta can be determined after feeding an animal or human a protein-free diet (traditional approach) or by various methods after giving a protein-containing diet. When the protein-free method has been applied with adult human subjects an overall mean value (three separate studies) for endogenous ileal nitrogen flow of 800 mg N/d has been reported. This value is considerably lower than a comparable value obtained after feeding protein of 1852 mg N/d (mean of four separate studies), and thus endogenous ileal N and amino acids should be measured under conditions of protein alimentation. There is some confusion concerning the terminology used to define digestibility, with the term "true" digestibility having different adopted meanings. Here, true amino acid digestibility is defined as apparent amino acid digestibility corrected for the basal amino acid losses determined after giving either a protein-free or a protein-containing diet. Basal losses should be determined at a defined dry-matter and protein intake. The protein-free diet approach to determining endogenous amino acids is considered unphysiological and basal losses refer to ileal endogenous amino acid flows associated with digesta dry-matter flow, and not including "specific" effects of dietary factors such as non starch polysaccharides and anti nutritional factors. Arguments are advanced that the enzyme hydrolysed protein/ultra filtration method may be suitable for routine application with a cannulated pig model, to obtain physiologically-valid basal estimates of ileal endogenous amino acids to allow calculation of true ileal amino acid digestibility in the

  9. 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.

  10. 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.

  11. Diagnostic value of determination of amount of urinary excretion of proteins for early diabetic nephropathy

    International Nuclear Information System (INIS)

    Li Zhuocheng; Chen Jianxiong; Yan Dewen

    2007-01-01

    Objective: To investigate the value of detection of changes of the amount of usinary excretion of albumin, β 2 -m, Tamm- Horsfall protein and α 1 -m for diagnosis of early diabetic nephropathy. Methods: The amounts of 24h urinary excretion of albumin, β 2 -m, Tamm-Horsfall protein and α 1 -m were determined with RIA in 78 patients with diabetes mellitus and 40 controls. Results: The amounts of 24h urinary excretion of albumin, β 2 -m, α 1 -m in patients with diabetes mellitus were significantly higher than those in controls (P<0.01 ), while the amount of Tamm-Horsfall protein was significantly lower (P<0.01). Among the diabetic patients, the changes of the amount of protein excretion were more pronounced in those with advanced impairment of renal function. Conclusion: Determination of amount of urinary excretion of proteins was helpful for diagnosis and assessment of early diabetic nephropathy. (authors)

  12. 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...

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

    Science.gov (United States)

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

    2011-04-01

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

  14. Determining the sub-cellular localization of proteins within Caenorhabditis elegans body wall muscle.

    Science.gov (United States)

    Meissner, Barbara; Rogalski, Teresa; 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.

  15. Comparative In silico Study of Sex-Determining Region Y (SRY) Protein Sequences Involved in Sex-Determining.

    Science.gov (United States)

    Vakili Azghandi, Masoume; Nasiri, Mohammadreza; Shamsa, Ali; Jalali, Mohsen; Shariati, Mohammad Mahdi

    2016-04-01

    The SRY gene (SRY) provides instructions for making a transcription factor called the sex-determining region Y protein. The sex-determining region Y protein causes a fetus to develop as a male. In this study, SRY of 15 spices included of human, chimpanzee, dog, pig, rat, cattle, buffalo, goat, sheep, horse, zebra, frog, urial, dolphin and killer whale were used for determine of bioinformatic differences. Nucleotide sequences of SRY were retrieved from the NCBI databank. Bioinformatic analysis of SRY is done by CLC Main Workbench version 5.5 and ClustalW (http:/www.ebi.ac.uk/clustalw/) and MEGA6 softwares. The multiple sequence alignment results indicated that SRY protein sequences from Orcinus orca (killer whale) and Tursiopsaduncus (dolphin) have least genetic distance of 0.33 in these 15 species and are 99.67% identical at the amino acid level. Homosapiens and Pantroglodytes (chimpanzee) have the next lowest genetic distance of 1.35 and are 98.65% identical at the amino acid level. These findings indicate that the SRY proteins are conserved in the 15 species, and their evolutionary relationships are similar.

  16. Comparative In silico Study of Sex-Determining Region Y (SRY Protein Sequences Involved in Sex-Determining

    Directory of Open Access Journals (Sweden)

    Masoume Vakili Azghandi

    2016-05-01

    Full Text Available Background: The SRY gene (SRY provides instructions for making a transcription factor called the sex-determining region Y protein. The sex-determining region Y protein causes a fetus to develop as a male. In this study, SRY of 15 spices included of human, chimpanzee, dog, pig, rat, cattle, buffalo, goat, sheep, horse, zebra, frog, urial, dolphin and killer whale were used for determine of bioinformatic differences. Methods: Nucleotide sequences of SRY were retrieved from the NCBI databank. Bioinformatic analysis of SRY is done by CLC Main Workbench version 5.5 and ClustalW (http:/www.ebi.ac.uk/clustalw/ and MEGA6 softwares. Results: The multiple sequence alignment results indicated that SRY protein sequences from Orcinus orca (killer whale and Tursiopsaduncus (dolphin have least genetic distance of 0.33 in these 15 species and are 99.67% identical at the amino acid level. Homosapiens and Pantroglodytes (chimpanzee have the next lowest genetic distance of 1.35 and are 98.65% identical at the amino acid level. Conclusion: These findings indicate that the SRY proteins are conserved in the 15 species, and their evolutionary relationships are similar.

  17. Determination of chromium combined with DNA, RNA and protein in chromium-rich brewer's yeast

    International Nuclear Information System (INIS)

    Ding Wenjun; Qian Qinfang; Hou Xiaolin; Feng Weiyue; Chai Zhifang

    2000-01-01

    The contents of chromium in the DNA, RNA and protein fractions separated from chromium-rich and normal brewer's yeast were determined with the neutron activation analysis in order to study the combination of Cr with DNA, RNA and protein in chromium-rich brewer's yeast. The results showed that the extracting rats and concentrations of DNA, RNA and protein had no significant difference in two types of yeast, but the chromium contents of DNA, RNA and protein in the chromium-rich yeast were significantly higher than those in the normal. In addition, the content of chromium in DNA was much higher than that in RNA and protein, which indicated that the inorganic chromium compounds entered into the yeast cell, during the yeast cultivation in the culture medium containing chromium were converted into organic chromium compounds combined with DNA, RNA and protein

  18. 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

  19. Non-Uniform Sampling and J-UNIO Automation for Efficient Protein NMR Structure Determination.

    Science.gov (United States)

    Didenko, Tatiana; Proudfoot, Andrew; Dutta, Samit Kumar; Serrano, Pedro; Wüthrich, Kurt

    2015-08-24

    High-resolution structure determination of small proteins in solution is one of the big assets of NMR spectroscopy in structural biology. Improvements in the efficiency of NMR structure determination by advances in NMR experiments and automation of data handling therefore attracts continued interest. Here, non-uniform sampling (NUS) of 3D heteronuclear-resolved [(1)H,(1)H]-NOESY data yielded two- to three-fold savings of instrument time for structure determinations of soluble proteins. With the 152-residue protein NP_372339.1 from Staphylococcus aureus and the 71-residue protein NP_346341.1 from Streptococcus pneumonia we show that high-quality structures can be obtained with NUS NMR data, which are equally well amenable to robust automated analysis as the corresponding uniformly sampled data. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    International Nuclear Information System (INIS)

    Canales-Mayordomo, Angeles; Fayos, Rosa; Angulo, Jesus; Ojeda, Rafael; Martin-Pastor, Manuel; Nieto, Pedro M.; Martin-Lomas, Manuel; Lozano, Rosa; Gimenez-Gallego, Guillermo; Jimenez-Barbero, Jesus

    2006-01-01

    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)

  1. 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)

  2. Studies on voltammetric determination of cadmium in samples containing native and digested proteins

    Energy Technology Data Exchange (ETDEWEB)

    Drozd, Marcin; Pietrzak, Mariusz, E-mail: mariusz@ch.pw.edu.pl; Malinowska, Elżbieta

    2014-03-01

    Highlights: • Proteins exhibit diverse impact on the DPASV cadmium signals. • Proteins subjected to HNO{sub 3} introduce less interference, than the native ones. • Optimal amount of SDS depends on the kind of protein. • Presence of thiolated coating agents of QDs do not influence the analysis. - Abstract: This work focuses on determination of cadmium ions using anodic stripping voltammetry (ASV) on thin film mercury electrode in conditions corresponding to those obtained after digestion of cadmium-based quantum dots and their conjugates. It presents the impact of selected proteins, including potential receptors and surface blocking agents on the voltammetric determination of cadmium. Experiments regarding elimination of interferences related to proteins presence using sodium dodecyl sulfate (SDS) are also shown. Effect of SDS on selected analytical parameters and simplicity of analyses carried out was investigated in the framework of current studies. The significant differences of influence among tested proteins on ASV cadmium determination, as well as the variability in SDS effectiveness as the antifouling agent were observed and explained. This work is especially important for those, who design new bioassays and biosensors with a use of quantum dots as electrochemical labels, as it shows what problems may arise from presence of native and digested proteins in tested samples.

  3. 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

  4. Blind Testing of Routine, Fully Automated Determination of Protein Structures from NMR Data

    NARCIS (Netherlands)

    Rosato, A.; Aramini, J.M.; van der Schot, G.; de Vries, S.J.|info:eu-repo/dai/nl/304837717; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238

    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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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

  10. NMR backbone resonance assignments of the prodomain variants of BDNF in the urea denatured state.

    Science.gov (United States)

    Wang, Jing; Bains, Henrietta; Anastasia, Agustin; Bracken, Clay

    2018-04-01

    Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins which plays a central role in neuronal survival, growth, plasticity and memory. A single Val66Met variant has been identified in the prodomain of human BDNF that is associated with anxiety, depression and memory disorders. The structural differences within the full-length prodomain Val66 and Met66 isoforms could shed light on the mechanism of action of the Met66 and its impact on the development of neuropsychiatric-associated disorders. In the present study, we report the backbone 1 H, 13 C, and 15 N NMR assignments of both full-length Val66 and Met66 prodomains in the presence of 2 M urea. These conditions were utilized to suppress residual structure and aid subsequent native state structural investigations aimed at mapping and identifying variant-dependent conformational differences under native-state conditions.

  11. Determination of protein-bound-radioiodine level as an index for determining the form of thyrotoxicosis

    International Nuclear Information System (INIS)

    Peshev, N.; Mladenov, B.; Guginska, E.

    1977-01-01

    The diagnostic capabilities of the test in hyperthyroidism and its possibilities in determining the form and stage of the disease were studied on a rather ample case material of 243 patients. The major diagnostic value of the method in differentiating between euthyroid function and hyperthyroidism is pointed out. The great sensitivity of the test will allow detection of even the slightest disturbances of iodine metabolism. (author)

  12. 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.

  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. Cell-free protein synthesis for structure determination by X-ray crystallography.

    Science.gov (United States)

    Watanabe, Miki; Miyazono, Ken-ichi; Tanokura, Masaru; Sawasaki, Tatsuya; Endo, Yaeta; Kobayashi, Ichizo

    2010-01-01

    Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.

  15. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  17. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    Science.gov (United States)

    Xie, Jianming [San Diego, CA; Wang, Lei [San Diego, CA; Wu, Ning [Boston, MA; Schultz, Peter G [La Jolla, CA

    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.

  18. Rapid Determination of Protein Solubility and Stability Conditions for NMR Studies Using Incomplete Factorial Design

    International Nuclear Information System (INIS)

    Ducat, Thierry; Declerck, Nathalie; Gostan, Thierry; Kochoyan, Michel; Demene, Helene

    2006-01-01

    Sample preparation constitutes a crucial and limiting step in structural studies of proteins by NMR. The determination of the solubility and stability (SAS) conditions of biomolecules at millimolar concentrations stays today empirical and hence time- and material-consuming. Only few studies have been recently done in this field and they have highlighted the interest of using crystallogenesis tools to optimise sample conditions. In this study, we have adapted a method based on incomplete factorial design and making use of crystallisation plates to quantify the influence of physico-chemical parameters such as buffer pH and salts on protein SAS. A description of the experimental set up and an evaluation of the method are given by case studies on two functional domains from the bacterial regulatory protein LicT as well as two other proteins. Using this method, we could rapidly determine optimised conditions for extracting soluble proteins from bacterial cells and for preparing purified protein samples sufficiently concentrated and stable for NMR characterisation. The drastic reduction in the time and number of experiments required for searching protein SAS conditions makes this method particularly well-adapted for a systematic investigation on a large range of physico-chemical parameters

  19. The AUDANA algorithm for automated protein 3D structure determination from NMR NOE data

    International Nuclear Information System (INIS)

    Lee, Woonghee; Petit, Chad M.; Cornilescu, Gabriel; Stark, Jaime L.; Markley, John L.

    2016-01-01

    We introduce AUDANA (Automated Database-Assisted NOE Assignment), an algorithm for determining three-dimensional structures of proteins from NMR data that automates the assignment of 3D-NOE spectra, generates distance constraints, and conducts iterative high temperature molecular dynamics and simulated annealing. The protein sequence, chemical shift assignments, and NOE spectra are the only required inputs. Distance constraints generated automatically from ambiguously assigned NOE peaks are validated during the structure calculation against information from an enlarged version of the freely available PACSY database that incorporates information on protein structures deposited in the Protein Data Bank (PDB). This approach yields robust sets of distance constraints and 3D structures. We evaluated the performance of AUDANA with input data for 14 proteins ranging in size from 6 to 25 kDa that had 27–98 % sequence identity to proteins in the database. In all cases, the automatically calculated 3D structures passed stringent validation tests. Structures were determined with and without database support. In 9/14 cases, database support improved the agreement with manually determined structures in the PDB and in 11/14 cases, database support lowered the r.m.s.d. of the family of 20 structural models.

  20. The AUDANA algorithm for automated protein 3D structure determination from NMR NOE data

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Woonghee, E-mail: whlee@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison and Biochemistry Department (United States); Petit, Chad M. [University of Alabama at Birmingham, Department of Biochemistry and Molecular Genetics (United States); Cornilescu, Gabriel; Stark, Jaime L.; Markley, John L., E-mail: markley@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison and Biochemistry Department (United States)

    2016-06-15

    We introduce AUDANA (Automated Database-Assisted NOE Assignment), an algorithm for determining three-dimensional structures of proteins from NMR data that automates the assignment of 3D-NOE spectra, generates distance constraints, and conducts iterative high temperature molecular dynamics and simulated annealing. The protein sequence, chemical shift assignments, and NOE spectra are the only required inputs. Distance constraints generated automatically from ambiguously assigned NOE peaks are validated during the structure calculation against information from an enlarged version of the freely available PACSY database that incorporates information on protein structures deposited in the Protein Data Bank (PDB). This approach yields robust sets of distance constraints and 3D structures. We evaluated the performance of AUDANA with input data for 14 proteins ranging in size from 6 to 25 kDa that had 27–98 % sequence identity to proteins in the database. In all cases, the automatically calculated 3D structures passed stringent validation tests. Structures were determined with and without database support. In 9/14 cases, database support improved the agreement with manually determined structures in the PDB and in 11/14 cases, database support lowered the r.m.s.d. of the family of 20 structural models.

  1. Determining protein complex connectivity using a probabilistic deletion network derived from quantitative proteomics.

    Science.gov (United States)

    Sardiu, Mihaela E; Gilmore, Joshua M; Carrozza, Michael J; Li, Bing; Workman, Jerry L; Florens, Laurence; Washburn, Michael P

    2009-10-06

    Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.

  2. Determining protein complex connectivity using a probabilistic deletion network derived from quantitative proteomics.

    Directory of Open Access Journals (Sweden)

    Mihaela E Sardiu

    2009-10-01

    Full Text Available Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.

  3. STUDIES OF METHOD FOR DETERMINING THE PROTEIN CONCENTRATION OF "MALEIN PPD" BY THE KJELDAHL METHOD

    Directory of Open Access Journals (Sweden)

    Ciuca, V

    2017-06-01

    Full Text Available Glanders is a contagious and fatal disease of horses, donkeys, and mules, caused by infection with the bacterium Burkholderia mallei. The pathogen causes nodules and ulcerations in the upper respiratory tract and lungs. Glanders is transmissible to humans by direct contact with diseased animals or with infected or contaminated material. In the untreated acute disease, the mortality rate can reach 95% within 3 weeks Malein PPD - the diagnostic product contain max 2mg/ml Burkholderia mallei. The amount of protein in the biological product "Malein PPD" is measured as nitrogen from protein molecule, applying the Kjeldahl (method determination of nitrogen by sulphuric acid digestion. The validation study aims to demonstrate the determination of the protein of the Malein PPD, by sulphuric acid digestion, it is an appropriate analytical method, reproducible and meets the quality requirements of diagnostic reagents. The paper establishes the performance characteristics of the method considered and identify the factors that influence these characteristics. The method for determining the concentration of protein, by the Kjeldahl method is considered valid if the results obtained for each validation parameter are within the admissibility criteria.The validation procedure includes details on protocol working to determine the protein of the Malein PPD, validation criteria, experimental results, mathematical calculations.

  4. Novel determination of protein, fat, and lactose of milk by liquid scintillation counter

    International Nuclear Information System (INIS)

    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

  5. Determination of Six Transmembrane Protein of Prostate 2 Gene Expression and Intracellular Localization in Prostate Cancer

    Directory of Open Access Journals (Sweden)

    Bora İrer

    2017-12-01

    Full Text Available Objective: In this study, we aimed to determine the relationship between the RNA and protein expression profile of six transmembrane protein of prostate 2 (STAMP2 gene and androgen and the intracellular localization of STAMP2. Materials and Methods: RNA and protein were obtained from androgen treated lymph node carcinoma of the prostate (LNCaP cells, untreated LNCaP cells, DU145 cells with no androgen receptor, and STAMP2 transfected COS-7 cells. The expression profile of STAMP2 gene and the effect of androgenes on the expression was shown in RNA and protein levels by using Northern and Western blotting methods. In addition, intracellular localization of the naturally synthesized STAMP2 protein and the transfected STAMP2 protein in COS-7 cells after androgen administration in both LNCaP cells was determined by immunofluorescence microscopy. Results: We found that the RNA and protein expression of STAMP2 gene in LNCaP cells are regulated by androgenes, the power of expression is increased with the duration of androgen treatment and there is no STAMP2 expression in DU145 cells which has no androgen receptor. As a result of the immunofluorescence microscopy study we observed that STAMP2 protein was localized at golgi complex and cell membrane. Conclusion: In conclusion, we have demonstrated that STAMP2 may play an important role in the pathogenesis of the prostate cancer and in the androgen-dependent androgen-independent staging of prostate cancer. In addition, STAMP2 protein, which is localized in the intracellular golgi complex and cell membrane, may be a new target molecule for prostate cancer diagnosis and treatment.

  6. Determination of Protein by Fluorescence Enhancement of Curcumin in Lanthanum-Curcumin-Sodium Dodecyl Benzene Sulfonate-Protein System

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng [Zaozhuang University, People' s Republic of China; Huang, Wei [Zaozhuang University, People' s Republic of China; Zhang, Yunfeng [Zaozhuang University, People' s Republic of China; Wang, Mingyin [Zaozhuang University, People' s Republic of China; Sun, Lina [Zaozhuang University, People' s Republic of China; Tang, Bo [Shandong University, Jinan, China; Wang, Wei [ORNL

    2011-01-01

    We found that the fluorescence intensity of the lanthanum (La(3+))-curcumin (CU) complex can be highly enhanced by proteins in the presence of sodium dodecyl benzene sulphonate (SDBS). Based on this finding, a new fluorimetric method for the determination of protein was developed. Under optimized conditions, the enhanced intensities of fluorescence are quantitatively in proportion to the concentrations of proteins in the range 0.0080-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1) for human serum albumin (HSA) with excitation of 425 nm, and 0.00020-20.0 g mL(-1) for bovine serum albumin (BSA) and 0.00080-20.0 g mL(-1)for human serum albumin (HSA) with excitation of 280 nm, while corresponding qualitative detection limits (S/N 3) are as low as 5.368, 0.573, 0.049, 0.562 g mL(-1), respectively. Study on reaction mechanism reveals that proteins can bind with La(3+), CU and SDBS through self-assembling function with electrostatic attraction, hydrogen bonding, hydrophobic interaction and van der Waals forces, etc. The proteins form a supermolecular association with multilayer structure, in which La(3+)-CU is clamped between BSA and SDBS. The unique high fluorescence enhancement of CU is resulted through synergic effects of favorable hydrophobic microenvironment provided by BSA and SDBS, and efficient intermolecular energy transfer among BSA, SDBS and CU. In energy transfer process, La(3+) plays a crucial role because it not only shortens the distance between SDBS and CU, but also acts as a "bridge" for transferring the energy from BSA to CU.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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...

  12. A New Method for Determining Structure Ensemble: Application to a RNA Binding Di-Domain Protein.

    Science.gov (United States)

    Liu, Wei; Zhang, Jingfeng; Fan, Jing-Song; Tria, Giancarlo; Grüber, Gerhard; Yang, Daiwen

    2016-05-10

    Structure ensemble determination is the basis of understanding the structure-function relationship of a multidomain protein with weak domain-domain interactions. Paramagnetic relaxation enhancement has been proven a powerful tool in the study of structure ensembles, but there exist a number of challenges such as spin-label flexibility, domain dynamics, and overfitting. Here we propose a new (to our knowledge) method to describe structure ensembles using a minimal number of conformers. In this method, individual domains are considered rigid; the position of each spin-label conformer and the structure of each protein conformer are defined by three and six orthogonal parameters, respectively. First, the spin-label ensemble is determined by optimizing the positions and populations of spin-label conformers against intradomain paramagnetic relaxation enhancements with a genetic algorithm. Subsequently, the protein structure ensemble is optimized using a more efficient genetic algorithm-based approach and an overfitting indicator, both of which were established in this work. The method was validated using a reference ensemble with a set of conformers whose populations and structures are known. This method was also applied to study the structure ensemble of the tandem di-domain of a poly (U) binding protein. The determined ensemble was supported by small-angle x-ray scattering and nuclear magnetic resonance relaxation data. The ensemble obtained suggests an induced fit mechanism for recognition of target RNA by the protein. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  13. 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.

  14. Backbone dynamics of the human CC-chemokine eotaxin

    Energy Technology Data Exchange (ETDEWEB)

    Ye Jiqing; Mayer, Kristen L.; Stone, Martin J. [Indiana University, Department of Chemistry (United States)

    1999-10-15

    Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. {sup 15}N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human eotaxin. {sup 15}N longitudinal (R{sub 1}) and transverse (R{sub 2}) auto relaxation rates, heteronuclear {sup 1}H-{sup 15}N steady-state NOEs, and transverse cross-relaxation rates ({eta}{sub xy}) were obtained at 30 deg. C for all resolved backbone secondary amide groups using {sup 1} H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time ({tau}{sub m}) is 5.09{+-}0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D{sub parallel}/D{sub perpendicular}) is 0.81{+-}0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1-19), the C-terminus (residues 68-73) and the loop connecting the first two {beta}-strands (residues 30-37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond-millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization.

  15. Microvolume protein concentration determination using the NanoDrop 2000c spectrophotometer.

    Science.gov (United States)

    Desjardins, Philippe; Hansen, Joel B; Allen, Michael

    2009-11-04

    Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 microL. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

  16. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination.

    Science.gov (United States)

    Field, Anjalie; Field, Jeffrey

    2010-08-01

    In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Bradford and Ninhydrin assays, two common dye-based assays for protein, as well as by ammonia release, the most common assay used in the food industry. Neither compound was detected in the Ninhydrin and Bradford assays at concentrations of >100 μg/ml. The ammonia assay detected melamine but was inconclusive with respect to cyanuric acid. To develop an accurate test for food that would not detect either chemical as a protein, assays were run on cat food and reconstituted milk powder. The Bradford assay readily measured the protein content of each food, and importantly, the addition of melamine or cyanuric acid to reconstituted milk did not affect the readings. The protein concentrations obtained for reconstituted milk powder were as expected, but those for the cat food were 10 to 30-fold lower, due to its low solubility. We conclude that dye-binding assays can be employed to detect protein in food without interference from melamine and cyanuric acid, thus reducing the incentive to use them as additives.

  17. Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis.

    Science.gov (United States)

    Riond, Barbara; Wenger-Riggenbach, Bettina; Hofmann-Lehmann, Regina; Lutz, Hans

    2009-03-01

    Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and alpha-, beta-, and gamma-globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm-blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi-automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within-run and within-assay precision. Data from warm-blooded and draught horses were compared using the Mann-Whitney U test. Within-run and within-assay CVs were draught horses and so combined reference intervals (2.5-97.5%) were calculated for total protein (51.0-72.0 g/L), albumin (29.6-38.5 g/L), alpha(1)-globulin (1.9-3.1 g/L), alpha(2)-globulin (5.3-8.7 g/L), beta(1)-globulin (2.8-7.3g/L), beta(2)-globulin (2.2-6.0 g/L), and gamma-globulin (5.8-12.7 g/L) concentrations, and albumin/globulin ratio (0.93-1.65). Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.

  18. 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.

  19. Determination of selenium in BCR single cell protein via destructive neutron activation analysis

    International Nuclear Information System (INIS)

    Goeij, J.J.M. de; Zegers, C.

    1978-10-01

    The amount of selenium in single cell protein (SCP), a product of BP Research Centre at Sunbury-at-Thames, England, was determined by neutron activation analysis. The SCP-samples were irradiated in the reactor of the Interuniversity Reactor Institute at Delft, in a neutron flux of 1.0 x 10 13 n/cm 2 s for 24 hours. After chemical destruction of the samples the amount of selenium was determined by measuring the γ-peaks of selenium-75

  20. Precise determination of protein extinction coefficients under native and denaturing conditions using SV-AUC.

    Science.gov (United States)

    Hoffmann, Andreas; Grassl, Kerstin; Gommert, Janine; Schlesak, Christian; Bepperling, Alexander

    2018-04-17

    The accurate determination of protein concentration is an important though non-trivial task during the development of a biopharmaceutical. The fundamental prerequisite for this is the availability of an accurate extinction coefficient. Common approaches for the determination of an extinction coefficient for a given protein are either based on the theoretical prediction utilizing the amino acid sequence or the photometric determination combined with a measurement of absolute protein concentration. Here, we report on an improved SV-AUC based method utilizing an analytical ultracentrifuge equipped with absorbance and Rayleigh interference optics. Global fitting of datasets helped to overcome some of the obstacles encountered with the traditional method employing synthetic boundary cells. Careful calculation of dn/dc values taking glycosylation and solvent composition into account allowed the determination of the extinction coefficients of monoclonal antibodies and an Fc-fusion protein under native as well as under denaturing conditions. An intra-assay precision of 0.9% and an accuracy of 1.8% compared to the theoretical value was achieved for monoclonal antibodies. Due to the large number of data points of a single dataset, no meaningful difference between the ProteomeLab XL-I and the new Optima AUC platform could be observed. Thus, the AUC-based approach offers a precise, convenient and versatile alternative to conventional methods like total amino acid analysis (AAA).

  1. Nuclear techniques for the determination of protein content in plant material

    International Nuclear Information System (INIS)

    Niemann, E.G.

    1980-01-01

    Elemental analysis for nitrogen has gained in importance over the last decade, as protein improvement and protein control in food and feed has come to be recognized as one of the most promising ways of overcoming deficiencies in food production and distribution. The need for fast and reliable screening methods has stimulated the improvement and automation of classic chemical methods for protein and nitrogen determination and, on the other hand, the development and adaptation of physical and nuclear analysis procedures. After about ten years of work this process has come to a stage where a critical evaluation of the existing methods seems necessary and justified. The present review describes and compares nuclear techniques for nitrogen determination in plant material. These include activation analysis techniques, based on various nuclear reactions, initiated by fast and thermal neutrons, energetic photons, protons, deuterons and α-particles. Other nuclear methods have been applied for nitrogen or protein determination, like ESCA, PIXE, NMR, NQR and Moessbauer spectroscopy, some of which possess good potential as screening methods. Depending on the needs, such as sample size, analysis rate and postulated accuracy, different nuclear techniques may be selected today for nitrogen screening. Some of the techniques discussed have additional potential for carbon or oxygen determination, for measuring depth or lateral N distribution, or for the recognition of the type of chemical N binding. Though most if not all techniques need further development for routine application, they are able to compete with chemical techniques in cost, rate and accuracy. (author)

  2. A novel strategy for NMR resonance assignment and protein structure determination

    International Nuclear Information System (INIS)

    Lemak, Alexander; Gutmanas, Aleksandras; Chitayat, Seth; Karra, Murthy; Farès, Christophe; Sunnerhagen, Maria; Arrowsmith, Cheryl H.

    2011-01-01

    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.

  3. Fluorometric determination of proteins using the terbium (III)-2-thenoyltrifluoroacetone-sodium dodecyl benzene sulfonate-protein system

    Energy Technology Data Exchange (ETDEWEB)

    Jia Zhen [Key Laboratory of Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Department of Chemistry, Dezhou University, Dezhou 253023 (China); Yang Jinghe [Key Laboratory of Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)]. E-mail: yjh@sdu.edu.cn; Wu Xia [Key Laboratory of Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Fei [Key Laboratory of Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Guo Changying [Key Laboratory of Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Liu Shufang [Key Laboratory of Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2006-12-15

    It is found that in hexamethylene tetramine (HMTA)-HCl buffer of pH=8.00, proteins can enhance the fluorescence of terbium (III) (Tb{sup 3+})-2-thenoyltrifluoroacetone (TTA)-sodium dodecyl benzene sulfonate (SDBS) system. Based on this, a sensitive method for the determination of proteins is proposed. The experiments indicate that under the optimum conditions, the enhanced fluorescence intensity is in proportion to the concentration of proteins in the range of 4.0x10{sup -9}-7.5x10{sup -6}g/mL for bovine serum albumin (BSA), 5.0x10{sup -9}-1.5x10{sup -5}g/mL for human serum albumin (HSA), 1.0x10{sup -8}-7.5x10{sup -6}g/mL for egg albumin (EA). Their detection limits (S/N=3) are 0.5, 0.8 and 2.0ng/mL, respectively. The interaction mechanism is also studied.

  4. 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

  5. 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

    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

  6. A sensitive enzyme-linked immunosorbent assay for the determination of fish protein in processed foods.

    Science.gov (United States)

    Shibahara, Yusuke; Uesaka, Yoshihiko; Wang, Jun; Yamada, Shoichi; Shiomi, Kazuo

    2013-01-15

    Fish is one of the most common causes of food allergy and its major allergen is parvalbumin, a 12 kDa muscular protein. In this study, a sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of fish protein in processed foods was developed using a polyclonal antibody raised against Pacific mackerel parvalbumin. The developed sandwich ELISA showed 22.6-99.0% reactivity (based on the reactivity to Pacific mackerel parvalbumin) to parvalbumins from various species of fish. The limits of detection and quantitation were estimated to be 0.23 and 0.70 μg protein per g of food, respectively. When the sandwich ELISA was subjected to inter-laboratory validation, spiked fish protein was recovered from five model processed foods in the range of 69.4-84.8% and the repeatability and reproducibility relative standard deviations were satisfactorily low (≤ 10.5%). Thus, the sandwich ELISA was judged to be a useful tool to determine fish protein in processed foods. Copyright © 2012 Elsevier Ltd. All rights reserved.

  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. Feasibility and market potential of protein determination of wheat using californium-252

    International Nuclear Information System (INIS)

    Roberts, T.C. Jr.; Eckhoff, N.D.; Clack, R.W.; Roberts, T.C. Sr.

    1976-01-01

    To evaluate the feasibility of protein determination by capture gamma-ray analysis using californium-252 neutrons, an in-situ protein analysis system for use by grain handlers has been examined. Three 227 kilogram (approximately) lots of wheat were used to determine the amount of nitrogen present. Protein analyses by the Kjeldahl method were obtained from samples taken before and after the capture gamma-ray analyses. The 5.267-MeV gamma-ray was selected for use in this study as a compromise between efficiency and interference from other elements. The associated counting equipment was a multichannel analyzer with pulse shaping electronic and analysis computing equipment. A linear regression program was used to compare the regions of interest to the Kjeldahl protein averages. The counts composing each peak were summed and normalized using the total count of the hydrogen peak. The normalized nitrogen percentages indicate a significant correlation between the spectral regions and the Kjeldahl analyses. To a first approximation, the value of wheat is the wheat protein. At the present time, protein testing of wheat is destructive, cumbersome, and time-consuming as compared to the potential for capture gamma-ray analysis testing. Assuming that such a protein analysis unit can analyze 42 tonne of wheat per hour, over 120 units would be needed to monitor one-half the U.S. annual wheat production. A 0.5% improvement in processor realizations and grain throughput value of $167.00 per tonne will result in a projected savings of $150,000 per year per unit

  9. 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.

  10. 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.

  11. Protein Adsorption Patterns and Analysis on IV Nanoemulsions-The Key Factor Determining the Organ Distribution.

    Science.gov (United States)

    Keck, Cornelia M; Jansch, Mirko; Müller, Rainer H

    2012-12-21

    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.

  12. Integrated Structural Biology for α-Helical Membrane Protein Structure Determination.

    Science.gov (United States)

    Xia, Yan; Fischer, Axel W; Teixeira, Pedro; Weiner, Brian; Meiler, Jens

    2018-04-03

    While great progress has been made, only 10% of the nearly 1,000 integral, α-helical, multi-span membrane protein families are represented by at least one experimentally determined structure in the PDB. Previously, we developed the algorithm BCL::MP-Fold, which samples the large conformational space of membrane proteins de novo by assembling predicted secondary structure elements guided by knowledge-based potentials. Here, we present a case study of rhodopsin fold determination by integrating sparse and/or low-resolution restraints from multiple experimental techniques including electron microscopy, electron paramagnetic resonance spectroscopy, and nuclear magnetic resonance spectroscopy. Simultaneous incorporation of orthogonal experimental restraints not only significantly improved the sampling accuracy but also allowed identification of the correct fold, which is demonstrated by a protein size-normalized transmembrane root-mean-square deviation as low as 1.2 Å. The protocol developed in this case study can be used for the determination of unknown membrane protein folds when limited experimental restraints are available. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Melamine and Cyanuric Acid do not interfere with Bradford and Ninhydrin assays for protein determination

    OpenAIRE

    Field, Anjalie; Field, Jeffrey

    2010-01-01

    In the fall of 2007 pet food contaminated with melamine and cyanuric acid caused kidney stones in thousands of animals. In the summer of 2008, a more serious outbreak of adulterated dairy food caused the deaths of six infants and sickened about 290,000 children in China. In all cases, melamine was likely added to inflate the apparent protein content of the foods. To determine if we could measure protein without interference from melamine and cyanuric acid we tested these compounds in the Brad...

  14. 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.

  15. 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

  16. Determining Effects of Non-synonymous SNPs on Protein-Protein Interactions using Supervised and Semi-supervised Learning

    Science.gov (United States)

    Zhao, Nan; Han, Jing Ginger; Shyu, Chi-Ren; Korkin, Dmitry

    2014-01-01

    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 rewiring of

  17. Structure determination of an integral membrane protein at room temperature from crystals in situ

    International Nuclear Information System (INIS)

    Axford, Danny; Foadi, James; Hu, Nien-Jen; Choudhury, Hassanul Ghani; Iwata, So; Beis, Konstantinos; Evans, Gwyndaf; Alguel, Yilmaz

    2015-01-01

    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

  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. 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

    into account spatial distribution of light, diffusely transmitted through a sample. The proposed method has been tested on two sample sets prepared from industrial raw milk standards, with variable fat and protein content. Partial Least-Squares (PLS) regression on the features calculated from images......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...... of monochromatically illuminated milk samples resulted in models with high prediction performance when analysed the sets separately (best models with cross-validated R2=0.974 for protein and R2=0.973 for fat content). However when analysed the sets jointly the obtained results were significantly worse (best models...

  20. Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane.

    Science.gov (United States)

    Elazar, Assaf; Weinstein, Jonathan; Biran, Ido; Fridman, Yearit; Bibi, Eitan; Fleishman, Sarel Jacob

    2016-01-29

    Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and self-association are strongly coupled in receptor homodimers.

  1. 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

    , using the partial least square regression (PLSR) method to construct a calibration model. The linear concentration range of adsorbed BSA is from 0 to 1.75 mg/mL by using 10 mm path length cuvettes. The influence of the sedimentation in suspension, different buffers, and different aluminum hydroxide......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...

  2. Resonance light scattering technique for the determination of proteins with polymethacrylic acid (PMAA)

    Science.gov (United States)

    Chen, Yanhua; Gao, Dejiang; Tian, Yuan; Ai, Peng; Zhang, Hanqi; Yu, Aimin

    2007-07-01

    As a resonance light scattering (RLS) probe, the polyelectrolyte polymethacrylic acid (PMAA) was applied in this assay. The bovine serum albumin (BSA) and human serum albumin (HSA) were determined by the electrostatic interaction of PMAA and proteins. At pH 3.8 Na 2HPO 4-citric acid buffer solution, the RLS intensities of PMAA-BSA (HSA) system were greatly enhanced. The characteristic peaks were appeared at the wavelength 320, 546 and 594 nm. The optimization conditions of the reaction were also examined and selected. Under the selected conditions, the RLS intensities were proportional to the protein concentrations in the range of (0.0200-2.00) × 10 -6 mol/L for BSA and (0.0200-2.40) × 10 -6 mol/L for HSA. The influences of some foreign substances were also examined. The synthetic samples containing proteins and some real samples were analyzed and the results obtained were satisfactory.

  3. 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...

  4. 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

  5. Determination of ABA-binding proteins contents in subcellular fractions isolated from cotton seedlings using radioimmunoanalysis

    International Nuclear Information System (INIS)

    Tursunkhodjayeva, F.M.

    2004-01-01

    Full text: Knowledge of plants' hormone receptor sites is essential to understanding of the principles of phytohormone action in cells and tissues. The hormone abscisic acid (ABA) takes part in many important physiological processes of plants, including water balance and resistance to salt stress. The detection of salt tolerance in the early stages of ontogenesis is desirable for effective cultivation of cotton. Usually such characteristics are determined visually after genetic analysis of hybrids over several generations. This classic method of genetics requires a long time to grow several generations of cotton plants. In this connection we study ABA-binding protein contents in subcellular fractions isolated from seedlings of several kinds of cotton with different tolerance to salt stress. The contents of ABA-binding protein in nuclei and chloroplasts fractions isolated from cotton seedlings were determined using radioimmunoanalysis. The subcellular fractions were prepared by ultracentrifugation in 0,25 - 2,2 M sucrose gradient. ABA-binding protein was isolated from cotton seedlings by affinity chromatography. The antibodies against ABA-binding protein of cotton were developed in rabbits according standard protocols. Than the antibodies were labelled by radioisotope J 125 according Greenwood et al. It was shown, that the nuclei and chloroplasts fractions isolated from cotton with high tolerance to salt stress contain ABA-binding protein up to 1,5-1,8 times more, than the same fractions from cotton with low tolerance to salt stress. So, the ABA-binding protein contents in cotton seedlings may be considered as a marker for screening of cotton kinds, which may potentially have high tolerance to salt stress

  6. 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.

  7. Data Acquisition Backbone Core DABC release v1.0

    International Nuclear Information System (INIS)

    Adamczewski-Musch, J; Kurz, N; Linev, S; Essel, H G

    2010-01-01

    The Data Acquisition Backbone Core (DABC) is a general purpose software framework designed for the implementation of a wide-range of data acquisition systems - from various small detector test beds to high performance systems. DABC consists of a compact data-flow kernel and a number of plug-ins for various functional components like data inputs, device drivers, user functional modules and applications. DABC provides configurable components for implementing event building over fast networks like InfiniBand or Gigabit Ethernet. A generic Java GUI provides the dynamic control and visualization of control parameters and commands, provided by DIM servers. A first set of application plug-ins has been implemented to use DABC as event builder for the front-end components of the GSI standard DAQ system MBS (Multi Branch System). Another application covers the connection to DAQ readout chains from detector front-end boards (N-XYTER) linked to read-out controller boards (ROC) over UDP into DABC for event building, archiving and data serving. This was applied for data taking in the September 2008 test beamtime for the CBM experiment at GSI. DABC version 1.0 is released and available from the website.

  8. 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.

  9. 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

  10. Unusual estrogen-binding liver protein: additional data on the structural determinants of androgenic ligands

    International Nuclear Information System (INIS)

    Smirnov, A.N.; Shchelkunova, T.A.; Rozen, V.B.

    1986-01-01

    The relative competitive activity of a number of androstane derivatives was determined according to the 50% displacement of [ 3 H] estradiol from complexes with an unusual estrogen-binding protein (UEBP) of the liver of male rats. It was shown that: (1) the bulk of the energy of the bond of the steroid to protein is due to hydrophobic interactions; (2) the real ability to form specific complexes with the UEBP at androgen concentrations close to the physiological is determined by the 17β-hydroxyl and is enhanced by the 3α- or 2α-hydroxy group; (3) the 3- and 17-keto groups weaken the interaction of androgens with the UEBP; (4) cis-coupling of the A and B rings in the molecule of androgens does not prevent the binding of the steroids to protein. These data substantially refine the concepts of the mechanisms of the interaction of androgens with the UEBP and may promote an elucidation of the physiological function of this protein

  11. 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

    2012-07-01

    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/.

  12. Structural and biochemical characterization of the cell fate determining nucleotidyltransferase fold protein MAB21L1.

    Science.gov (United States)

    de Oliveira Mann, Carina C; Kiefersauer, Reiner; Witte, Gregor; Hopfner, Karl-Peter

    2016-06-08

    The exceptionally conserved metazoan MAB21 proteins are implicated in cell fate decisions and share considerable sequence homology with the cyclic GMP-AMP synthase. cGAS is the major innate immune sensor for cytosolic DNA and produces the second messenger 2'-5', 3'-5' cyclic GMP-AMP. Little is known about the structure and biochemical function of other proteins of the cGAS-MAB21 subfamily, such as MAB21L1, MAB21L2 and MAB21L3. We have determined the crystal structure of human full-length MAB21L1. Our analysis reveals high structural conservation between MAB21L1 and cGAS but also uncovers important differences. Although monomeric in solution, MAB21L1 forms a highly symmetric double-pentameric oligomer in the crystal, raising the possibility that oligomerization could be a feature of MAB21L1. In the crystal, MAB21L1 is in an inactive conformation requiring a conformational change - similar to cGAS - to develop any nucleotidyltransferase activity. Co-crystallization with NTP identified a putative ligand binding site of MAB21 proteins that corresponds to the DNA binding site of cGAS. Finally, we offer a structure-based explanation for the effects of MAB21L2 mutations in patients with eye malformations. The underlying residues participate in fold-stabilizing interaction networks and mutations destabilize the protein. In summary, we provide a first structural framework for MAB21 proteins.

  13. [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 < 1.90% and inter-day < 4.39%), and high recoveries (97.41%-99.91%). In addition, non-protein nitrogen (NPN) such as melamine added into the soya-bean milk had weak influence on MRBT results.

  14. The genotype-environment interaction variance in rice-seed protein determination

    International Nuclear Information System (INIS)

    Ismachin, M.

    1976-01-01

    Many environmental factors influence the protein content of cereal seed. This fact procured difficulties in breeding for protein. Yield is another example on which so many environmental factors are of influence. The length of time required by the plant to reach maturity, is also affected by the environmental factors; even though its effect is not too decisive. In this investigation the genotypic variance and the genotype-environment interaction variance which contribute to the total variance or phenotypic variance was analysed, with purpose to give an idea to the breeder how selection should be made. It was found that genotype-environment interaction variance is larger than the genotypic variance in contribution to total variance of protein-seed determination or yield. In the analysis of the time required to reach maturity it was found that genotypic variance is larger than the genotype-environment interaction variance. It is therefore clear, why selection for time required to reach maturity is much easier than selection for protein or yield. Selected protein in one location may be different from that to other locations. (author)

  15. 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.

  16. Proteins labelling with 125I and experimental determination of their specific activity

    International Nuclear Information System (INIS)

    Caro, R.A.; Ciscato, V.A.; Giacomini, S.M.V. de; Quiroga, S.; Radicella, R.

    1975-11-01

    A standardization of the labelling technique of proteins with 125 I and the control of the obtained products, principally their specific activities was performed, in order to utilize them correctly in radioimmunoassays. The quantities of chloramine-T and sodium metabisulphite were lowered, with regard to the original method, to 3.6 and 9.6 μg respectively. Under these conditions, optimal yields and radioiodinated proteins with good immunological activities were obtained. It was found that the specific activity calculated, as usual, from the yield obtained by electrophoresis, is higher than the real value. For these reasons the yields and the corresponding specific activities were determined from ascending chromatographies performed with 70 per cent methanol as solvent, during two hours in darkness. The radioimmunoassay displacement curves obtained with proteins labelled which the proposed method and the specific activities of which were calculated from their radiochromatographic patterns, were reproducible and gave a percentage of bound radioiodinated protein in the absence of cold protein of 50 +- 4. (author) [es

  17. On the accuracy of protein determination in large biological samples by prompt gamma neutron activation analysis

    Energy Technology Data Exchange (ETDEWEB)

    Kasviki, K. [Institute of Nuclear Technology and Radiation Protection, NCSR ' Demokritos' , Aghia Paraskevi, Attikis 15310 (Greece); Medical Physics Laboratory, Medical School, University of Ioannina, Ioannina 45110 (Greece); Stamatelatos, I.E. [Institute of Nuclear Technology and Radiation Protection, NCSR ' Demokritos' , Aghia Paraskevi, Attikis 15310 (Greece)], E-mail: ion@ipta.demokritos.gr; Yannakopoulou, E. [Institute of Physical Chemistry, NCSR ' Demokritos' , Aghia Paraskevi, Attikis 15310 (Greece); Papadopoulou, P. [Institute of Technology of Agricultural Products, NAGREF, Lycovrissi, Attikis 14123 (Greece); Kalef-Ezra, J. [Medical Physics Laboratory, Medical School, University of Ioannina, Ioannina 45110 (Greece)

    2007-10-15

    A prompt gamma neutron activation analysis (PGNAA) facility has been developed for the determination of nitrogen and thus total protein in large volume biological samples or the whole body of small animals. In the present work, the accuracy of nitrogen determination by PGNAA in phantoms of known composition as well as in four raw ground meat samples of about 1 kg mass was examined. Dumas combustion and Kjeldahl techniques were also used for the assessment of nitrogen concentration in the meat samples. No statistically significant differences were found between the concentrations assessed by the three techniques. The results of this work demonstrate the applicability of PGNAA for the assessment of total protein in biological samples of 0.25-1.5 kg mass, such as a meat sample or the body of small animal even in vivo with an equivalent radiation dose of about 40 mSv.

  18. On the accuracy of protein determination in large biological samples by prompt gamma neutron activation analysis

    International Nuclear Information System (INIS)

    Kasviki, K.; Stamatelatos, I.E.; Yannakopoulou, E.; Papadopoulou, P.; Kalef-Ezra, J.

    2007-01-01

    A prompt gamma neutron activation analysis (PGNAA) facility has been developed for the determination of nitrogen and thus total protein in large volume biological samples or the whole body of small animals. In the present work, the accuracy of nitrogen determination by PGNAA in phantoms of known composition as well as in four raw ground meat samples of about 1 kg mass was examined. Dumas combustion and Kjeldahl techniques were also used for the assessment of nitrogen concentration in the meat samples. No statistically significant differences were found between the concentrations assessed by the three techniques. The results of this work demonstrate the applicability of PGNAA for the assessment of total protein in biological samples of 0.25-1.5 kg mass, such as a meat sample or the body of small animal even in vivo with an equivalent radiation dose of about 40 mSv

  19. The Polerovirus Minor Capsid Protein Determines Vector Specificity and Intestinal Tropism in the Aphid

    Science.gov (United States)

    Brault, Véronique; Périgon, Sophie; Reinbold, Catherine; Erdinger, Monique; Scheidecker, Danièle; Herrbach, Etienne; Richards, Ken; Ziegler-Graff, Véronique

    2005-01-01

    Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD. PMID:16014930

  20. Determination of the nature of Gnetum africanum Welw. leaf proteins by spectrophotometry

    International Nuclear Information System (INIS)

    Lodi, O.; Mbemba, F.; Yandju, M.C.

    2011-01-01

    The plant biological material of our study consisted of Gnetum africanum leaves (wild vegetable) collected from five different countryside sites, namely Kananga, Kikwit, Mai-Ndombe, Mbandaka and Kisangani. Determining the nature of Gnetum africanum leaf proteins has shown they would be classified in the group of albumins as they absorb at U.V. wavelengths between 270 and 278 nm, like the absorption of the egg white and yellow albumin as that of a legume (Psophocarpus scandens) albumins.

  1. Algorithm for selection of optimized EPR distance restraints for de novo protein structure determination

    Science.gov (United States)

    Kazmier, Kelli; Alexander, Nathan S.; Meiler, Jens; Mchaourab, Hassane S.

    2010-01-01

    A hybrid protein structure determination approach combining sparse Electron Paramagnetic Resonance (EPR) distance restraints and Rosetta de novo protein folding has been previously demonstrated to yield high quality models (Alexander et al., 2008). However, widespread application of this methodology to proteins of unknown structures is hindered by the lack of a general strategy to place spin label pairs in the primary sequence. In this work, we report the development of an algorithm that optimally selects spin labeling positions for the purpose of distance measurements by EPR. For the α-helical subdomain of T4 lysozyme (T4L), simulated restraints that maximize sequence separation between the two spin labels while simultaneously ensuring pairwise connectivity of secondary structure elements yielded vastly improved models by Rosetta folding. 50% of all these models have the correct fold compared to only 21% and 8% correctly folded models when randomly placed restraints or no restraints are used, respectively. Moreover, the improvements in model quality require a limited number of optimized restraints, the number of which is determined by the pairwise connectivities of T4L α-helices. The predicted improvement in Rosetta model quality was verified by experimental determination of distances between spin labels pairs selected by the algorithm. Overall, our results reinforce the rationale for the combined use of sparse EPR distance restraints and de novo folding. By alleviating the experimental bottleneck associated with restraint selection, this algorithm sets the stage for extending computational structure determination to larger, traditionally elusive protein topologies of critical structural and biochemical importance. PMID:21074624

  2. A two-step recognition of signal sequences determines the translocation efficiency of proteins.

    OpenAIRE

    Belin, D; Bost, S; Vassalli, J D; Strub, K

    1996-01-01

    The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated...

  3. 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.

  4. Gradient-free determination of isoelectric points of proteins on chip.

    Science.gov (United States)

    Łapińska, Urszula; Saar, Kadi L; Yates, Emma V; Herling, Therese W; Müller, Thomas; Challa, Pavan K; Dobson, Christopher M; Knowles, Tuomas P J

    2017-08-30

    The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behaviour. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. Here, we introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. To demonstrate the general approachability of this platform, we have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes.

  5. Accuracy and precision of protein-ligand interaction kinetics determined from chemical shift titrations

    Energy Technology Data Exchange (ETDEWEB)

    Markin, Craig J.; Spyracopoulos, Leo, E-mail: leo.spyracopoulos@ualberta.ca [University of Alberta, Department of Biochemistry (Canada)

    2012-12-15

    NMR-monitored chemical shift titrations for the study of weak protein-ligand interactions represent a rich source of information regarding thermodynamic parameters such as dissociation constants (K{sub D}) in the micro- to millimolar range, populations for the free and ligand-bound states, and the kinetics of interconversion between states, which are typically within the fast exchange regime on the NMR timescale. We recently developed two chemical shift titration methods wherein co-variation of the total protein and ligand concentrations gives increased precision for the K{sub D} value of a 1:1 protein-ligand interaction (Markin and Spyracopoulos in J Biomol NMR 53: 125-138, 2012). In this study, we demonstrate that classical line shape analysis applied to a single set of {sup 1}H-{sup 15}N 2D HSQC NMR spectra acquired using precise protein-ligand chemical shift titration methods we developed, produces accurate and precise kinetic parameters such as the off-rate (k{sub off}). For experimentally determined kinetics in the fast exchange regime on the NMR timescale, k{sub off} {approx} 3,000 s{sup -1} in this work, the accuracy of classical line shape analysis was determined to be better than 5 % by conducting quantum mechanical NMR simulations of the chemical shift titration methods with the magnetic resonance toolkit GAMMA. Using Monte Carlo simulations, the experimental precision for k{sub off} from line shape analysis of NMR spectra was determined to be 13 %, in agreement with the theoretical precision of 12 % from line shape analysis of the GAMMA simulations in the presence of noise and protein concentration errors. In addition, GAMMA simulations were employed to demonstrate that line shape analysis has the potential to provide reasonably accurate and precise k{sub off} values over a wide range, from 100 to 15,000 s{sup -1}. The validity of line shape analysis for k{sub off} values approaching intermediate exchange ({approx}100 s{sup -1}), may be facilitated by

  6. Accuracy and precision of protein-ligand interaction kinetics determined from chemical shift titrations.

    Science.gov (United States)

    Markin, Craig J; Spyracopoulos, Leo

    2012-12-01

    NMR-monitored chemical shift titrations for the study of weak protein-ligand interactions represent a rich source of information regarding thermodynamic parameters such as dissociation constants (K ( D )) in the micro- to millimolar range, populations for the free and ligand-bound states, and the kinetics of interconversion between states, which are typically within the fast exchange regime on the NMR timescale. We recently developed two chemical shift titration methods wherein co-variation of the total protein and ligand concentrations gives increased precision for the K ( D ) value of a 1:1 protein-ligand interaction (Markin and Spyracopoulos in J Biomol NMR 53: 125-138, 2012). In this study, we demonstrate that classical line shape analysis applied to a single set of (1)H-(15)N 2D HSQC NMR spectra acquired using precise protein-ligand chemical shift titration methods we developed, produces accurate and precise kinetic parameters such as the off-rate (k ( off )). For experimentally determined kinetics in the fast exchange regime on the NMR timescale, k ( off ) ~ 3,000 s(-1) in this work, the accuracy of classical line shape analysis was determined to be better than 5 % by conducting quantum mechanical NMR simulations of the chemical shift titration methods with the magnetic resonance toolkit GAMMA. Using Monte Carlo simulations, the experimental precision for k ( off ) from line shape analysis of NMR spectra was determined to be 13 %, in agreement with the theoretical precision of 12 % from line shape analysis of the GAMMA simulations in the presence of noise and protein concentration errors. In addition, GAMMA simulations were employed to demonstrate that line shape analysis has the potential to provide reasonably accurate and precise k ( off ) values over a wide range, from 100 to 15,000 s(-1). The validity of line shape analysis for k ( off ) values approaching intermediate exchange (~100 s(-1)), may be facilitated by more accurate K ( D ) measurements

  7. Determination of gas phase protein ion densities via ion mobility analysis with charge reduction.

    Science.gov (United States)

    Maisser, Anne; Premnath, Vinay; Ghosh, Abhimanyu; Nguyen, Tuan Anh; Attoui, Michel; Hogan, Christopher J

    2011-12-28

    We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C, lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM aqueous ammonium acetate) and denaturing (1 : 49.5 : 49.5, formic acid : methanol : water) ESI. Charge reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal concentrations of positive and negative ions. Ions produced by the source collide with and reduce the charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions. Density inference is made via determination of the mobility diameter, and correspondingly the spherical equivalent protein volume. Through this approach it is found that for both non-denaturing and denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of 0.97 g cm(-3) and 0.86 g cm(-3), respectively. Ions from non-denaturing ESI are found to be slightly more compact than predicted from the protein crystal structures, suggesting that low charge state protein ions in the gas phase are slightly denser than their solution conformations. While a slight difference is detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are found to be much more dense than those examined previously by drift tube mobility analysis, in which charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to non-compact ions in the gas-phase, and suggests that for gas phase

  8. Determination of protein content in some foodstaffs using 14 MeV NAA

    International Nuclear Information System (INIS)

    Abdel Samei, M.B.; Elshafie, M.A.; Hanna, M.; Csikai, J.; Juhasz, M.

    1986-01-01

    The protein content of meals of various seeds (rice, lupine, sorghum, pumpkin, linseed, water melon, tomato) collected from Egypt and the USA was determined by 14 MeV neutron activation analysis via sup(14)N(n,2n)sup(13)N reaction. Non-conventional sources like tea and coffee residues are also investigated. The concentrations of trace elements determined by the x-ray fluorescence technique show definite differences even for a given type of seed produced in different geographical regions. (author)

  9. Breaking symmetry in the structure determination of (large) symmetric protein dimers

    Energy Technology Data Exchange (ETDEWEB)

    Gaponenko, Vadim; Altieri, Amanda S.; Li, Jess; Byrd, R. Andrew [National Cancer Institute, Structural Biophysics Laboratory (United States)], E-mail: rabyrd@ncifcrf.gov

    2002-10-15

    We demonstrate a novel methodology to disrupt the symmetry in the NMR spectra of homodimers. A paramagnetic probe is introduced sub-stoichiometrically to create an asymmetric system with the paramagnetic probe residing on only one monomer within the dimer. This creates sufficient magnetic anisotropy for resolution of symmetry-related overlapped resonances and, consequently, detection of pseudocontact shifts and residual dipolar couplings specific to each monomeric component. These pseudocontact shifts can be readily incorporated into existing structure refinement calculations and enable determination of monomer orientation within the dimeric protein. This methodology can be widely used for solution structure determination of symmetric dimers.

  10. 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.

  11. Relative Abundance of Integral Plasma Membrane Proteins in Arabidopsis Leaf and Root Tissue Determined by Metabolic Labeling and Mass Spectrometry

    Science.gov (United States)

    Bernfur, Katja; Larsson, Olaf; Larsson, Christer; Gustavsson, Niklas

    2013-01-01

    Metabolic labeling of proteins with a stable isotope (15N) in intact Arabidopsis plants was used for accurate determination by mass spectrometry of differences in protein abundance between plasma membranes isolated from leaves and roots. In total, 703 proteins were identified, of which 188 were predicted to be integral membrane proteins. Major classes were transporters, receptors, proteins involved in membrane trafficking and cell wall-related proteins. Forty-one of the integral proteins, including nine of the 13 isoforms of the PIP (plasma membrane intrinsic protein) aquaporin subfamily, could be identified by peptides unique to these proteins, which made it possible to determine their relative abundance in leaf and root tissue. In addition, peptides shared between isoforms gave information on the proportions of these isoforms. A comparison between our data for protein levels and corresponding data for mRNA levels in the widely used database Genevestigator showed an agreement for only about two thirds of the proteins. By contrast, localization data available in the literature for 21 of the 41 proteins show a much better agreement with our data, in particular data based on immunostaining of proteins and GUS-staining of promoter activity. Thus, although mRNA levels may provide a useful approximation for protein levels, detection and quantification of isoform-specific peptides by proteomics should generate the most reliable data for the proteome. PMID:23990937

  12. Synonymous codon bias and functional constraint on GC3-related DNA backbone dynamics in the prokaryotic nucleoid.

    Science.gov (United States)

    Babbitt, Gregory A; Alawad, Mohammed A; Schulze, Katharina V; Hudson, André O

    2014-01-01

    While mRNA stability has been demonstrated to control rates of translation, generating both global and local synonymous codon biases in many unicellular organisms, this explanation cannot adequately explain why codon bias strongly tracks neighboring intergene GC content; suggesting that structural dynamics of DNA might also influence codon choice. Because minor groove width is highly governed by 3-base periodicity in GC, the existence of triplet-based codons might imply a functional role for the optimization of local DNA molecular dynamics via GC content at synonymous sites (≈GC3). We confirm a strong association between GC3-related intrinsic DNA flexibility and codon bias across 24 different prokaryotic multiple whole-genome alignments. We develop a novel test of natural selection targeting synonymous sites and demonstrate that GC3-related DNA backbone dynamics have been subject to moderate selective pressure, perhaps contributing to our observation that many genes possess extreme DNA backbone dynamics for their given protein space. This dual function of codons may impose universal functional constraints affecting the evolution of synonymous and non-synonymous sites. We propose that synonymous sites may have evolved as an 'accessory' during an early expansion of a primordial genetic code, allowing for multiplexed protein coding and structural dynamic information within the same molecular context. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Rapid protein fold determination using secondary chemical shifts and cross-hydrogen bond 15N-13C' scalar couplings (3hbJNC')

    Energy Technology Data Exchange (ETDEWEB)

    Bonvin, Alexandre M.J.J.; Houben, Klaartje; Guenneugues, Marc; Kaptein, Robert; Boelens, Rolf [Utrecht University, Bijvoet Center for Biomolecular Research, NMR Spectroscopy (Netherlands)

    2001-11-15

    The possibility of generating protein folds at the stage of backbone assignment using structural restraints derived from experimentally measured cross-hydrogen bond scalar couplings and secondary chemical shift information is investigated using as a test case the small {alpha}/{beta} protein chymotrypsin inhibitor 2. Dihedral angle restraints for the {phi} and {psi} angles of 32 out of 64 residues could be obtained from secondary chemical shift analysis with the TALOS program (Corneliscu et al., 1999a). This information was supplemented by 18 hydrogen-bond restraints derived from experimentally measured cross-hydrogen bond {sup 3hb}J{sub NC'} coupling constants. These experimental data were sufficient to generate structures that are as close as 1.0 A backbone rmsd from the crystal structure. The fold is, however, not uniquely defined and several solutions are generated that cannot be distinguished on the basis of violations or energetic considerations. Correct folds could be identified by combining clustering methods with knowledge-based potentials derived from structural databases.

  14. Amide or Amine: Determining the Origin of the 3300 cm−1 NH Mode in Protein SFG Spectra Using 15N Isotope Labels

    Science.gov (United States)

    Weidner, Tobias; Breen, Nicholas F.; Drobny, Gary P.; Castner, David G.

    2009-01-01

    Sum frequency generation (SFG) vibrational spectroscopy has been employed in biomaterials research and protein adsorption studies with growing success in recent years. A number of studies focusing on understanding SFG spectra of proteins and peptides at different interfaces have laid the foundation for future, more complex studies. In many cases a strong NH mode near 3300 cm−1 is observed in the SFG spectra, but the relationship of this mode to the peptide structure is uncertain since it has been assigned to either a backbone amide mode or a side chain related amine resonance. A thorough understanding of the SFG spectra of these first model systems is an important first step for future experiments. To clarify the origin of the NH SFG mode we studied 15N isotopically labeled 14-amino acid amphiphilic model peptides composed of lysine (K) and leucine (L) in an α-helical secondary structure (LKα14) that were adsorbed onto charged surfaces in situ at the solid-liquid interface. 15N substitution at the terminal amine group of the lysine side chains resulted in a red-shift of the NH mode of 9 cm−1 on SiO2 and 13 cm−1 on CaF2. This clearly shows the 3300 cm−1 NH feature is associated with side chain NH stretches and not with backbone amide modes. PMID:19873996

  15. Amide or amine: determining the origin of the 3300 cm(-1) NH mode in protein SFG spectra using 15N isotope labels.

    Science.gov (United States)

    Weidner, Tobias; Breen, Nicholas F; Drobny, Gary P; Castner, David G

    2009-11-26

    Sum frequency generation (SFG) vibrational spectroscopy has been employed in biomaterials research and protein adsorption studies with growing success in recent years. A number of studies focusing on understanding SFG spectra of proteins and peptides at different interfaces have laid the foundation for future, more complex studies. In many cases, a strong NH mode near 3300 cm(-1) is observed in the SFG spectra, but the relationship of this mode to the peptide structure is uncertain, since it has been assigned to either a backbone amide mode or a side chain related amine resonance. A thorough understanding of the SFG spectra of these first model systems is an important first step for future experiments. To clarify the origin of the NH SFG mode, we studied (15)N isotopically labeled 14-amino acid amphiphilic model peptides composed of lysine (K) and leucine (L) in an alpha-helical secondary structure (LKalpha14) that were adsorbed onto charged surfaces in situ at the solid-liquid interface. (15)N substitution at the terminal amine group of the lysine side chains resulted in a red-shift of the NH mode of 9 cm(-1) on SiO(2) and 13 cm(-1) on CaF(2). This clearly shows the 3300 cm(-1) NH feature is associated with side chain NH stretches and not with backbone amide modes.

  16. Protein fiber linear dichroism for structure determination and kinetics in a low-volume, low-wavelength couette flow cell

    OpenAIRE

    Dafforn, Tim; Rajendra, Jacindra; Halsall, David J.; Serpell, Louise C.; Rodger, Alison

    2004-01-01

    High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the st...

  17. 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.

  18. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.

    Science.gov (United States)

    Takeda, Mitsuhiro; Ono, Akira M; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    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-(13)C/(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-(13)C/(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 (13)C, (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.

  19. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination

    International Nuclear Information System (INIS)

    Takeda, Mitsuhiro; Ono, Akira M.; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    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 (ε- and ζ-SAIL Phe) and tyrosine (ε-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 δ-SAIL Phe and δ-SAIL Tyr, which allow us to observe and assign δ- 13 C/ 1 H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the δ-, ε- or ζ- 13 C/ 1 H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the δ-, ε-, and ζ-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 13 C, 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 ζ-SAIL Phe and ε-SAIL Tyr would be practically the best choice for protein structural determinations.

  20. 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...... around Gln262 and the active site Cys215 reveals that the probability of finding a water molecule correctly positioned for catalysis is much larger in PTP1B than in PTP1B(R47V),(D48N),(M258C),(G259Q) and PTPalpha, in accordance with experiments....

  1. Determination of possible effects of mineral concentration on protein synthesis by rumen microbes in vitro

    International Nuclear Information System (INIS)

    Nikolic, J.A.; Jovanovic, M.; Andric, R.

    1976-01-01

    The aim of the present investigation was to determine the effect of different concentrations of sulphide, magnesium and zinc on protein synthesis by rumen micro-organisms in vitro. Rumen content was taken from a young bull fed a diet based on maize and dried sugar beet pulp (2/1) supplemented with urea. The rate of incorporation of 35 S from Na 2 35 SO 4 in relation to the mean specific radioactivity of the sulphide pool was used to estimate the overall rate of microbial protein synthesis. It was found that the rate of protein synthesis and the net rate of utilization of ammonia-N were not affected by differences in mean sulphide concentration from 3.6-8.0 mg/litre. The rate of reduction of sulphate appeared not to be affected by the addition of sodium sulphide to the medium. The rate and efficiency of protein synthesis by rumen micro-organisms were not significantly affected by increasing the concentration of total magnesium from 8.4-15.3 mg/100 ml. The values for soluble magnesium varied widely (1.2-7.8 mg/100 ml), and appeared to be partly dependent on the pH of the medium. Zinc concentrations varying from 5.2-12.4 mg/litre did not influence the overall rate of protein synthesis, although the efficiency tended to be higher when the concentration of zinc was greater. Concentrations of soluble zinc were low (0.3-1.15 mg/litre), and not influenced by changes in the concentration of total zinc. It was concluded that increasing the concentrations of the examined elements above the basic values did not lead consistently to an improved production of microbial protein but, on the other hand, had no obvious detrimental effect on microbial metabolic activity within the limits studied. (author)

  2. 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.

  3. Use of 13.5-MeV neutrons for protein determination in grain crops

    International Nuclear Information System (INIS)

    Barit, I.A.; Kuz'min, L.E.; Makarov, S.A.; Vozhzhov, V.F.; Pronman, I.M.

    1989-01-01

    One of the main objectives of the Food Supply Program, i.e., that of improving the quality of crop production, is bound up intimately with the intensification of work on the selection and genetics of high-protein grain and legume crops. High-protein stains cannot be isolated without the proper analytical service for mass testing of the nitrogen content in the grain, which is one of the main elements of protein. The neutron-activation method of nitrogen determination is based on the use of the 14 N(n, 2n) 13 N nuclear reaction (E th = 11.3 MeV) with an average neutron energy of ∼14.5 MeV. In this work the authors consider a new variant of the neutron-activation method of determining nitrogen in grain and legume crops. The method is based on the use of monoenergetic neutrons with an energy of ∼13.5 MeV, generated in relatively thin titanium-tritium targets by a mass-separated deuteron beam from neutron generators operating at 150-300 kV, in order to eliminate the interference of the reaction 39 K(n, 2n) 38 K (E thr = 13.4 MeV). The present method has been used to determine the protein content (mass %) in different grains and legumes at the All-Union Selection-Genetic Institute of the Lenin Academy of Agricultural Sciences. The correctness of the analysis was checked by comparison with the data of chemical analysis. The discrepancy between the results of the two methods does not exceed 3%, which is within the limits of the error of measurement of Δ and K s.r

  4. FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein

    NARCIS (Netherlands)

    Nazarov, P.V.; Koehorst, R.B.M.; Vos, W.L.; Apanasovich, V.V.; Hemminga, M.A.

    2007-01-01

    A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based

  5. 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......Hypochlorous acid (HOCl) is a potent oxidant, which is produced in vivo by activated phagocytes. This compound is an important antibacterial agent, but excessive or misplaced production has been implicated in a number of human diseases, including atherosclerosis, arthritis, and some cancers....... Proteins are major targets for this oxidant, and such reaction results in side-chain modification, backbone fragmentation, and cross-linking. Despite a wealth of qualitative data for such reactions, little absolute kinetic data is available to rationalize the in vitro and in vivo data. In this study...

  6. Determination of fat and total protein content in milk using conventional digital imaging.

    Science.gov (United States)

    Kucheryavskiy, Sergey; Melenteva, Anastasiia; Bogomolov, Andrey

    2014-04-01

    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 into account spatial distribution of light, diffusely transmitted through a sample. The proposed method has been tested on two sample sets prepared from industrial raw milk standards, with variable fat and protein content. Partial Least-Squares (PLS) regression on the features calculated from images of monochromatically illuminated milk samples resulted in models with high prediction performance when analysed the sets separately (best models with cross-validated R(2)=0.974 for protein and R(2)=0.973 for fat content). However when analysed the sets jointly with the obtained results were significantly worse (best models with cross-validated R(2)=0.890 for fat content and R(2)=0.720 for protein content). The results have been compared with previously published Vis/SW-NIR spectroscopic study of similar samples. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Active Gating, Molecular Pumping, and Turnover Determination in Biomimetic Lipidic Cubic Mesophases with Reconstituted Membrane Proteins.

    Science.gov (United States)

    Speziale, Chiara; Zabara, Alexandru Florian; Drummond, Calum John; Mezzenga, Raffaele

    2017-11-28

    Understanding the mechanisms controlling molecular transport in bioinspired materials is a central topic in many branches of nanotechnology. In this work, we show that biomolecules of fundamental importance in biological processes, such as glucose, can be transported in an active, controlled, and selective manner across macroscopic lipidic cubic mesophases, by correctly reconstituting within them their corresponding membrane protein transporters, such as Staphylococcus epidermidis (GlcP Se ). Importantly, by duly exploiting the symporter properties of GlcP Se of coupled glucose/H + transport, the diffusion of glucose can further be tuned by independent physiological stimuli, such as parallel or antiparallel pH gradients, offering an important model to study molecular exchange processes in cellular machinery. We finally show that by measuring the transport properties of the lipidic mesophases with and without the GlcP Se membrane protein reconstituted within, it becomes possible to determine its intrinsic conductance. We generalize these findings to other membrane proteins from the antiporters family, such as the bacterial ClC exchanger from Escherichia coli (EcClC), providing a robust method for evaluating the turnover rate of the membrane proteins in general.

  8. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Non-invasive high throughput approach for protein hydrophobicity determination based on surface tension.

    Science.gov (United States)

    Amrhein, Sven; Bauer, Katharina Christin; Galm, Lara; Hubbuch, Jürgen

    2015-12-01

    The surface hydrophobicity of a protein is an important factor for its interactions in solution and thus the outcome of its production process. Yet most of the methods are not able to evaluate the influence of these hydrophobic interactions under natural conditions. In the present work we have established a high resolution stalagmometric method for surface tension determination on a liquid handling station, which can cope with accuracy as well as high throughput requirements. Surface tensions could be derived with a low sample consumption (800 μL) and a high reproducibility (content. The protein influence on the solutions' surface tension was correlated to the hydrophobicity of lysozyme, human lysozyme, BSA, and α-lactalbumin. Differences in proteins' hydrophobic character depending on pH and species could be resolved. Within this work we have developed a pH dependent hydrophobicity ranking, which was found to be in good agreement with literature. For the studied pH range of 3-9 lysozyme from chicken egg white was identified to be the most hydrophilic. α-lactalbumin at pH 3 exhibited the most pronounced hydrophobic character. The stalagmometric method occurred to outclass the widely used spectrophotometric method with bromophenol blue sodium salt as it gave reasonable results without restrictions on pH and protein species. © 2015 Wiley Periodicals, Inc.

  10. 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

  11. Determination of the glass-transition temperature of proteins from a viscometric approach.

    Science.gov (United States)

    Monkos, Karol

    2015-03-01

    All fully hydrated proteins undergo a distinct change in their dynamical properties at glass-transition temperature Tg. To determine indirectly this temperature for dry albumins, the viscosity measurements of aqueous solutions of human, equine, ovine, porcine and rabbit serum albumin have been conducted at a wide range of concentrations and at temperatures ranging from 278 K to 318 K. Viscosity-temperature dependence of the solutions is discussed on the basis of the three parameters equation resulting from Avramov's model. One of the parameter in the Avramov's equation is the glass-transition temperature. For all studied albumins, Tg of a solution monotonically increases with increasing concentration. The glass-transition temperature of a solution depends both on Tg for a dissolved dry protein Tg,p and water Tg,w. To obtain Tg,p for each studied albumin the modified Gordon-Taylor equation was applied. This equation describes the dependence of Tg of a solution on concentration, and Tg,p and a parameter depending on the strength of the protein-solvent interaction are the fitting parameters. Thus determined the glass-transition temperature for the studied dry albumins is in the range (215.4-245.5)K. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. MCBT: Multi-Hop Cluster Based Stable Backbone Trees for Data Collection and Dissemination in WSNs

    Directory of Open Access Journals (Sweden)

    Tae-Jin Lee

    2009-07-01

    Full Text Available 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. Solution, solid phase and computational structures of apicidin and its backbone-reduced analogs.

    Science.gov (United States)

    Kranz, Michael; Murray, Peter John; Taylor, Stephen; Upton, Richard J; Clegg, William; Elsegood, Mark R J

    2006-06-01

    The recently isolated broad-spectrum antiparasitic apicidin (1) is one of the few naturally occurring cyclic tetrapeptides (CTP). Depending on the solvent, the backbone of 1 exhibits two gamma-turns (in CH(2)Cl(2)) or a beta-turn (in DMSO), differing solely in the rotation of the plane of one of the amide bonds. In the X-ray crystal structure, the peptidic C==Os and NHs are on opposite sides of the backbone plane, giving rise to infinite stacks of cyclotetrapeptides connected by three intermolecular hydrogen bonds between the backbones. Conformational searches (Amber force field) on a truncated model system of 1 confirm all three backbone conformations to be low-energy states. The previously synthesized analogs of 1 containing a reduced amide bond exhibit the same backbone conformation as 1 in DMSO, which is confirmed further by the X-ray crystal structure of a model system of the desoxy analogs of 1. This similarity helps in explaining why the desoxy analogs retain some of the antiprotozoal activities of apicidin. The backbone-reduction approach designed to facilitate the cyclization step of the acyclic precursors of the CTPs seems to retain the conformational preferences of the parent peptide backbone.

  14. Determination of protein content in seeds by prompt gamma-ray spectrometry

    International Nuclear Information System (INIS)

    Carbonari, A.W.; Pecequilo, B.R.S.

    1984-01-01

    The protein level in seeds can be directly calculated through the determination of the nitrogen content in grains. The authors show here that the radioactive thermal neutron capture prompt gamma-rays technique can be used to determine the nitrogen content in grains without chemical destruction, with good precision and relative rapidity, by detecting the prompt gamma rays emitted by the 14 N(n,γ) 15 N reaction product. The samples were irradiated in the tangential tube of the IEA-R1 research reactor, in Sao Paulo, and a pair spectrometer was used for the detection of the prompt gamma-rays. The nitrogen content was determined in several samples of soybean, common bean, peas and rice and the results compared with typical nitrogen content values for each grain. 33 references, 1 figure, 1 table

  15. 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.

  16. Evaluation of the protein concentration in enzymes via the determination of sulphur by TXRF

    International Nuclear Information System (INIS)

    Mertens, M.; Rittmeyer, C.; Kolbesen, B.O.

    2000-01-01

    Total reflection x-ray fluorescence spectrometry (TXRF) offers many advantages for the identification of trace elements in biological samples like enzymes, tissues or plants. Without any preliminary treatment elements may be determined with high accuracy especially transition metals like Fe, Ni, Cu, Mo and the alkaline earth metal Ca. A further aspect of the investigation of enzymes is the simple and simultaneous determination of light elements. Especially sulphur is of interest. The element sulphur exists mainly in the two amino, acids methionine and cysteine as well as in iron-sulphur clusters and may be used for an easy and simultaneous calculation of the protein concentration. Hence the quantitative determination of sulphur by TXRF allows a cross-check regarding of the conventional quantitative determination of protein concentration by, for example, the Lowry method. On the basis of three enzymes of different origins and molecular weights the presentation will show the influence of the bio-organic matrix and different buffer media on element determination by TXRF. As is already known the influence of the matrix on the detection of light elements is stronger than on transition metals. It can be discussed whether layer thickness and layer effects of the drying residues (characterization by SEM and thickness profilometer (ALPHA-step)) and / or self absorption effects as well as the excitation are of significance. The results indicate that with enzymes of low molecular weight a reliable determination of sulphur is possible whereas those with higher molecular weights gave poorer results on account of the matrix effects described. (author)

  17. Magic Angle Spinning NMR Structure Determination of Proteins from Pseudocontact Shifts

    KAUST Repository

    Li, Jianping; Pilla, Kala Bharath; Li, Qingfeng; Zhang, Zhengfeng; Su, Xuncheng; Huber, Thomas; Yang, Jun

    2013-01-01

    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.

  18. 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.

  19. Uganda's National Transmission Backbone Infrastructure Project: Technical Challenges and the Way Forward

    Science.gov (United States)

    Bulega, T.; Kyeyune, A.; Onek, P.; Sseguya, R.; Mbabazi, D.; Katwiremu, E.

    2011-10-01

    Several publications have identified technical challenges facing Uganda's National Transmission Backbone Infrastructure project. This research addresses the technical limitations of the National Transmission Backbone Infrastructure project, evaluates the goals of the project, and compares the results against the technical capability of the backbone. The findings of the study indicate a bandwidth deficit, which will be addressed by using dense wave division multiplexing repeaters, leasing bandwidth from private companies. Microwave links for redundancy, a Network Operation Center for operation and maintenance, and deployment of wireless interoperability for microwave access as a last-mile solution are also suggested.

  20. Solution structure and dynamics of melanoma inhibitory activity protein

    International Nuclear Information System (INIS)

    Lougheed, Julie C.; Domaille, Peter J.; Handel, Tracy M.

    2002-01-01

    Melanoma inhibitory activity (MIA) is a small secreted protein that is implicated in cartilage cell maintenance and melanoma metastasis. It is representative of a recently discovered family of proteins that contain a Src Homologous 3 (SH3) subdomain. While SH3 domains are normally found in intracellular proteins and mediate protein-protein interactions via recognition of polyproline helices, MIA is single-domain extracellular protein, and it probably binds to a different class of ligands.Here we report the assignments, solution structure, and dynamics of human MIA determined by heteronuclear NMR methods. The structures were calculated in a semi-automated manner without manual assignment of NOE crosspeaks, and have a backbone rmsd of 0.38 A over the ordered regions of the protein. The structure consists of an SH3-like subdomain with N- and C-terminal extensions of approximately 20 amino acids each that together form a novel fold. The rmsd between the solution structure and our recently reported crystal structure is 0.86 A over the ordered regions of the backbone, and the main differences are localized to the most dynamic regions of the protein. The similarity between the NMR and crystal structures supports the use of automated NOE assignments and ambiguous restraints to accelerate the calculation of NMR structures

  1. Determination of the X-ray structure of the snake venom protein omwaprin by total chemical synthesis and racemic protein crystallography.

    Science.gov (United States)

    Banigan, James R; Mandal, Kalyaneswar; Sawaya, Michael R; Thammavongsa, Vilasak; Hendrickx, Antoni P A; Schneewind, Olaf; Yeates, Todd O; Kent, Stephen B H

    2010-10-01

    The 50-residue snake venom protein L-omwaprin and its enantiomer D-omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L- and D-omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L-amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L- and D-omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2(1)/c and its structure was determined at atomic resolution (1.33 A) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high-resolution X-ray structures by direct methods.

  2. 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.

  3. 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.

  4. Determination of immune status in dogs against CPV-2 by recombinant protein based latex agglutination test.

    Science.gov (United States)

    Thomas, Jobin; Singh, Mithilesh; Goswami, T K; Glora, Philma; Chakravarti, Soumendu; Chander, Vishal; Upmanyu, Vikramaditya; Verma, Suman; Sharma, Chhavi; Mahendran, K

    2017-09-01

    Canine parvoviral enteritis is a highly contagious viral illness caused by canine parvovirus-2 (CPV-2) which affects puppies of mainly 6-20 weeks of age. Vaccination is pivotal in preventing and controlling CPV-2 infection. Determination of antibody status is a critical determinant for successful vaccination. The hemagglutination inhibition (HI) test is 'gold standard' test for quantification of antibodies specific to CPV-2, although the execution of this test is not feasible under field conditions. The present study was undertaken to develop a point of care testing to determine immune status prior to CPV-2 vaccination or to detect seroconversion in immunized dogs by latex agglutination test (LAT) using recombinant antigen. Truncated portion of VP2 protein (tVP2) of CPV-2 was selected on the basis of antigenic indices, overexpressed the recombinant protein in E. coli system and was subsequently used in development of LAT. A total of 59 serum samples obtained from vaccinated (n = 54) and healthy unvaccinated (n = 5) dogs were tested. The positivity was observed in 85% (46/54) of these dogs with varying agglutination pattern. The overall sensitivity and specificity of latex agglutination test in comparison to HI test was recorded as 90% and 88% respectively with an agreement value of 90% (CI = 95%). Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  5. 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.

  6. Unambiguous Determination of Intermolecular Hydrogen Bond of NMR Structure by Molecular Dynamics Refinement Using All-Atom Force Field and Implicit Solvent Model

    International Nuclear Information System (INIS)

    Jee, Jun Goo

    2010-01-01

    It has been shown that AMD refinement is very useful for defining an intermolecular hydrogen bond in NMR structure calculation. The refined structure also provides a clue for explaining the pH dependence in Ub and UIM complexes. As reported by Choi et al., serine-mediated hydrogen bonds are the third most populated hydrogen bonds found in protein-protein intermolecular interactions, after the backbone-backbone and backbone-aspartate ones. The abundance imposes the requirement of an method to determine the interface of protein-protein complexes. The precise geometry is particularly important in the complex structures between Ub and UBDs. Ub recognizes various targets with the same surface, where both hydrophobic and hydrophobic interactions are involved. Hence, the details of the hydrophilic interactions are necessary to find the common binding modes. The structure determination of a biomolecule by NMR depends heavily on the distance restraints derived by the NOE cross peaks that are observed between two protons within 6 A through space. Therefore, the existence of the NOE peaks and their correct assignments to two corresponding protons are essential for an accurate and precise structure determination. Recent developments of NOE assignment and calculation algorithms have enabled the determination of protein 3D structures without any manual interpretation, provided chemical shifts are assigned in most atoms and sufficient NOE peaks exist. Along with these advances, the necessity of determining complicated structures such as complexes is increasing

  7. Determination of Endosperm Protein Secondary Structure in Hard Wheat Breeding Lines using Synchrotron Infrared Microspectroscopy

    International Nuclear Information System (INIS)

    Bonwell, E.; Fisher, T.; Fritz, A.; Wetzel, D.

    2008-01-01

    substantial variance in this feature and its determination allows the prediction of relative quality for breadmaking to be taken into consideration for subsequent steps in the wheat breeding process. Data treatments include deconvolution, modeling of the individual resulting bands that contribute to the amide I band to enable measurement of the relative amounts of both forms. Results with specimens representing multiple crop years of hard winter wheat breeding are reported. It is evident that a range is available for the breeder to choose from, that allows including this protein molecular structural attribute in the selection process.

  8. Development of a chimeric Zika vaccine using a licensed live-attenuated flavivirus vaccine as backbone.

    Science.gov (United States)

    Li, Xiao-Feng; Dong, Hao-Long; Wang, Hong-Jiang; Huang, Xing-Yao; Qiu, Ye-Feng; Ji, Xue; Ye, Qing; Li, Chunfeng; Liu, Yang; Deng, Yong-Qiang; Jiang, Tao; Cheng, Gong; Zhang, Fu-Chun; Davidson, Andrew D; Song, Ya-Jun; Shi, Pei-Yong; Qin, Cheng-Feng

    2018-02-14

    The global spread of Zika virus (ZIKV) and its unexpected association with congenital defects necessitates the rapid development of a safe and effective vaccine. Here we report the development and characterization of a recombinant chimeric ZIKV vaccine candidate (termed ChinZIKV) that expresses the prM-E proteins of ZIKV using the licensed Japanese encephalitis live-attenuated vaccine SA14-14-2 as the genetic backbone. ChinZIKV retains its replication activity and genetic stability in vitro, while exhibiting an attenuation phenotype in multiple animal models. Remarkably, immunization of mice and rhesus macaques with a single dose of ChinZIKV elicits robust and long-lasting immune responses, and confers complete protection against ZIKV challenge. Significantly, female mice immunized with ChinZIKV are protected against placental and fetal damage upon ZIKV challenge during pregnancy. Overall, our study provides an alternative vaccine platform in response to the ZIKV emergency, and the safety, immunogenicity, and protection profiles of ChinZIKV warrant further clinical development.

  9. A systematic analysis of backbone amide assignments achieved via combinatorial selective labelling of amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Jeremy Craven, C. [University of Sheffield, Department of Biotechnology and Molecular Biology (United Kingdom); Al-Owais, Moza; Parker, Martin J. [University of Leeds, Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology (United Kingdom)], E-mail: m.j.parker@leeds.ac.uk

    2007-06-15

    With the advent of high-yield cell-free expressions systems, many researchers are exploiting selective isotope labelling of amino acids to increase the efficiency and accuracy of the NMR assignment process. We developed recently a combinatorial selective labelling (CSL) method capable of yielding large numbers of residue-type and sequence-specific backbone amide assignments, which involves comparing cross-peak intensities in {sup 1}H-{sup 15}N HSQC and 2D {sup 1}H-{sup 15}N HNCO spectra collected for five samples containing different combinations of {sup 13}C- and {sup 15}N-labelled amino acids [Parker MJ, Aulton-Jones M, Hounslow A, Craven C J (2004) J Am Chem Soc 126:5020-5021]. In this paper we develop a robust method for establishing the reliability of these assignments. We have performed a detailed statistical analysis of the CSL data collected for a model system (the B1 domain of protein G from Streptococcus), developing a scoring method which allows the confidence in assignments to be assessed, and which enables the effects of overlap on assignment fidelity to be predicted. To further test the scoring method and also to assess the performance of CSL in relation to sample quality, we have applied the method to the CSL data collected for GFP in our previous study.

  10. 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.

  11. Characterization of hydrogen bonding motifs in proteins: hydrogen elimination monitoring by ultraviolet photodissociation mass spectrometry.

    Science.gov (United States)

    Morrison, Lindsay J; Chai, Wenrui; Rosenberg, Jake A; Henkelman, Graeme; Brodbelt, Jennifer S

    2017-08-02

    Determination of structure and folding of certain classes of proteins remains intractable by conventional structural characterization strategies and has spurred the development of alternative methodologies. Mass spectrometry-based approaches have a unique capacity to differentiate protein heterogeneity due to the ability to discriminate populations, whether minor or major, featuring modifications or complexation with non-covalent ligands on the basis of m/z. Cleavage of the peptide backbone can be further utilized to obtain residue-specific structural information. Here, hydrogen elimination monitoring (HEM) upon ultraviolet photodissociation (UVPD) of proteins transferred to the gas phase via nativespray ionization is introduced as an innovative approach to deduce backbone hydrogen bonding patterns. Using well-characterized peptides and a series of proteins, prediction of the engagement of the amide carbonyl oxygen of the protein backbone in hydrogen bonding using UVPD-HEM is demonstrated to show significant agreement with the hydrogen-bonding motifs derived from molecular dynamics simulations and X-ray crystal structures.

  12. NMR in a crystallography-based high-throughput protein structure-determination environment

    International Nuclear Information System (INIS)

    Wüthrich, Kurt

    2010-01-01

    As an introduction to three papers on comparisons of corresponding crystal and NMR solution structures determined by the Joint Center for Structural Genomics (JCSG), an outline is provided of the JCSG strategy for combined use of the two techniques. A special commentary addresses the potentialities of the concept of ‘reference crystal structures’, which is introduced in the following three papers. An introduction is provided to three papers which compare corresponding protein crystal and NMR solution structures determined by the Joint Center for Structural Genomics (JCSG). Special mention is made of the JCSG strategy for combined use of the two techniques, and of potential applications of the concept of ‘reference crystal structures’, which is introduced in the following three papers

  13. Determination of trace elements in BCR single cell protein via destructive neutron activation analyses

    International Nuclear Information System (INIS)

    Tjioe, P.S.; Goeij, J.J.M. de; Nooijen, J.L.; Kroon, J.J.

    1978-10-01

    The amount of some trace elements in single cell protein (SCP), a product of BP Research Centre at Sunbury-at-Thames, England, was determined by neutron activation analysis. The SCP-samples were irradiated in the reactor of the Interuniversity Reactor Institute at Delft in a neutron flux of 1.0x10 13 n/cm 2 s for 12 hours. Samples of Bowen's Kale were used as reference material. After a decay of two or three days the samples were chemically destroyed, and the trace elements were separated. The quantity of the following elements was determined by measuring the γ-activity by means of a scintillation counter: antimony, cadmium, mercury, arsenic and selenium. The amounts of these elements in the SCP and in the reference material were tabled

  14. Self-consistent residual dipolar coupling based model-free analysis for the robust determination of nanosecond to microsecond protein dynamics

    International Nuclear Information System (INIS)

    Lakomek, Nils-Alexander; Walter, Korvin F. A.; Fares, Christophe; Lange, Oliver F.; Groot, Bert L. de; Grubmueller, Helmut; Brueschweiler, Rafael; Munk, Axel; Becker, Stefan; Meiler, Jens; Griesinger, Christian

    2008-01-01

    Residual dipolar couplings (RDCs) provide information about the dynamic average orientation of inter-nuclear vectors and amplitudes of motion up to milliseconds. They complement relaxation methods, especially on a time-scale window that we have called supra-τ c (τ c c rdc > = 0.72 ± 0.02 compared to LS 2 > = 0.778 ± 0.003 for the Lipari-Szabo order parameters, indicating that the inclusion of the supra-τ c window increases the averaged amplitude of mobility observed in the sub-τ c window by about 34%. For the β-strand spanned by residues Lys48 to Leu50, an alternating pattern of backbone NH RDC order parameter S rdc 2 (NH) = (0.59, 0.72, 0.59) was extracted. The backbone of Lys48, whose side chain is known to be involved in the poly-ubiquitylation process that leads to protein degradation, is very mobile on the supra-τ c time scale (S rdc 2 (NH) = 0.59 ± 0.03), while it is inconspicuous (S LS 2 (NH) = 0.82) on the sub-τ c as well as on μs-ms relaxation dispersion time scales. The results of this work differ from previous RDC dynamics studies of ubiquitin in the sense that the results are essentially independent of structural noise providing a much more robust assessment of dynamic effects that underlie the RDC data

  15. Spectrophotometric method for determination of bifunctional macrocyclic ligands in macrocyclic ligand-protein conjugates

    International Nuclear Information System (INIS)

    Dadachova, E.; Chappell, L.L.; Brechbiel, M.W.

    1999-01-01

    A simple spectrophotometric assay for determination of bifunctional polyazacarboxylate-macrocyclic ligands of different sizes that are conjugated to proteins has been developed for: 12-membered macrocycle DOTA (2-[4-nitrobenzyl]-1, 4, 7, 10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and analogs, the 15-membered PEPA macrocycle (2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N''',N'''' -pentaacetic acid), and the large 18-membered macrocycle HEHA (1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N''''-hexaacetic acid). The method is based on titration of the blue-colored 1:1 Pb(II)-Arsenazo III (AAIII) complex with the polyazacarboxylate macrocyclic ligand in the concentration range of 0-2.5 μM, wherein color change occurring upon transchelation of the Pb(II) from the AAIII to the polyazamacrocyclic ligand is monitored at 656 nm. The assay is performed at ambient temperature within 20 min without any interfering interaction between the protein and Pb(II)-AA(III) complex. Thus, this method also provides a ligand-to-protein ratio (L/P ratio) that reflects the effective number of ligands per protein molecule available to radiolabeling. The method is not suitable for 14-membered TETA macrocycle (2-[4-nitrobenzyl]-1, 4, 8, 11-tetraazacyclotetradecane N,N',N'',N'''-tetraacetic acid) because of low stability constant of Pb(II)-TETA complex. The method is rapid, simple and may be customized for other polyazacarboxylate macrocyclic ligands

  16. 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.

  17. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. [Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71].

    Science.gov (United States)

    Huang, Xueyong; Liu, Guohua; Hu, Xiaoning; Du, Yanhua; Li, Xingle; Xu, Yuling; Chen, Haomin; Xu, Bianli

    2014-04-01

    To clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen. VP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected. VP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay. VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

  19. Green IGP Link Weights for Energy-efficiency and Load-balancing in IP Backbone Networks

    OpenAIRE

    Francois, Frederic; Wang, Ning; Moessner, Klaus; Georgoulas, Stylianos; Xu, Ke

    2013-01-01

    The energy consumption of backbone networks has become a primary concern for network operators and regulators due to the pervasive deployment of wired backbone networks to meet the requirements of bandwidth-hungry applications. While traditional optimization of IGP link weights has been used in IP based load-balancing operations, in this paper we introduce a novel link weight setting algorithm, the Green Load-balancing Algorithm (GLA), which is able to jointly optimize both energy efficiency ...

  20. Backbone assignment of the little finger domain of a Y-family DNA polymerase.

    Science.gov (United States)

    Ma, Dejian; Fowler, Jason D; Suo, Zucai

    2011-10-01

    Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

  1. 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.

  2. Quantitative determination of in vitro immunoglobulin secretion with protein A from Staphylococcus aureus

    International Nuclear Information System (INIS)

    Manciulea, M.

    1982-01-01

    A micromethod for the quantitative determination of Ig secreted in vitro by mice lymphocytes isolated from the spleen of normal animals is described. The indicator system consists in sheep erythrocytes radiolabelled with sodium chromate ( 51 Cr) and coated with protein A of Staphylococcus aureus ( 51 Cr-labelled ES). When splenocytes were incubated in fluid phase at 37 0 C for 7/2 h with rabbit antisera to mouse Ig (IgM and IgG) and with guinea pig complement, the immune complexes formed between the secreted Ig and its specific IgG antibody are bound to protein A on the erythrocyte surface allowing the complement-mediated lysis of 51 Cr-labelled ES. The degree of haemolysis produced in this experimental system, which reflects the amount of in vitro secreted Ig, was quantitatively measured by radioactive determination of 51 Cr release. In combination with the ES plaque assay the method also gives information as immunoglobulin secretion per plaque forming cell. (Auth.)

  3. 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

  4. Chemical characteristics and antithrombotic effect of chondroitin sulfates from sturgeon skull and sturgeon backbone.

    Science.gov (United States)

    Gui, Meng; Song, Juyi; Zhang, Lu; Wang, Shun; Wu, Ruiyun; Ma, Changwei; Li, Pinglan

    2015-06-05

    Chondroitin sulfates (CSs) were extracted from sturgeon skull and backbone, and their chemical composition, anticoagulant, anti-platelet and thrombolysis activities were evaluated. The average molecular weights of CS from sturgeon skull and backbone were 38.5kDa and 49.2kDa, respectively. Disaccharide analysis indicated that the sturgeon backbone CS was primarily composed of disaccharide monosulfated in position four of the GalNAc (37.8%) and disaccharide monosulfated in position six of the GalNAc (59.6%) while sturgeon skull CS was primarily composed of nonsulfated disaccharide (74.2%). Sturgeon backbone CS showed stronger antithrombotic effect than sturgeon skull CS. Sturgeon backbone CS could significantly prolong activated partial thromboplastin time (APTT) and thrombin time (TT), inhibited ADP-induced platelet aggregation and dissolved platelet plasma clots in vitro. The results suggested that sturgeon backbone CS can be explored as a functional food with antithrombotic function. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Improved protein surface comparison and application to low-resolution protein structure data

    Directory of Open Access Journals (Sweden)

    Kihara Daisuke

    2010-12-01

    Full Text Available Abstract Background Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM, which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs. The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. Results The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Conclusions Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy.

  6. Improved protein surface comparison and application to low-resolution protein structure data.

    Science.gov (United States)

    Sael, Lee; Kihara, Daisuke

    2010-12-14

    Recent advancements of experimental techniques for determining protein tertiary structures raise significant challenges for protein bioinformatics. With the number of known structures of unknown function expanding at a rapid pace, an urgent task is to provide reliable clues to their biological function on a large scale. Conventional approaches for structure comparison are not suitable for a real-time database search due to their slow speed. Moreover, a new challenge has arisen from recent techniques such as electron microscopy (EM), which provide low-resolution structure data. Previously, we have introduced a method for protein surface shape representation using the 3D Zernike descriptors (3DZDs). The 3DZD enables fast structure database searches, taking advantage of its rotation invariance and compact representation. The search results of protein surface represented with the 3DZD has showngood agreement with the existing structure classifications, but some discrepancies were also observed. The three new surface representations of backbone atoms, originally devised all-atom-surface representation, and the combination of all-atom surface with the backbone representation are examined. All representations are encoded with the 3DZD. Also, we have investigated the applicability of the 3DZD for searching protein EM density maps of varying resolutions. The surface representations are evaluated on structure retrieval using two existing classifications, SCOP and the CE-based classification. Overall, the 3DZDs representing backbone atoms show better retrieval performance than the original all-atom surface representation. The performance further improved when the two representations are combined. Moreover, we observed that the 3DZD is also powerful in comparing low-resolution structures obtained by electron microscopy.

  7. 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. © 2015 Published by Elsevier Ltd.

  8. 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

  9. Convenient method for resolving degeneracies due to symmetry of the magnetic susceptibility tensor and its application to pseudo contact shift-based protein-protein complex structure determination

    Energy Technology Data Exchange (ETDEWEB)

    Kobashigawa, Yoshihiro; Saio, Tomohide [Hokkaido University, Department of Structural Biology, Faculty of Advanced Life Science (Japan); Ushio, Masahiro [Hokkaido University, Graduate School of Life Science (Japan); Sekiguchi, Mitsuhiro [Astellas Pharma Inc., Analysis and Pharmacokinetics Research Labs, Department of Drug Discovery (Japan); Yokochi, Masashi; Ogura, Kenji; Inagaki, Fuyuhiko, E-mail: finagaki@pharm.hokudai.ac.jp [Hokkaido University, Department of Structural Biology, Faculty of Advanced Life Science (Japan)

    2012-05-15

    Pseudo contact shifts (PCSs) induced by paramagnetic lanthanide ions fixed in a protein frame provide long-range distance and angular information, and are valuable for the structure determination of protein-protein and protein-ligand complexes. We have been developing a lanthanide-binding peptide tag (hereafter LBT) anchored at two points via a peptide bond and a disulfide bond to the target proteins. However, the magnetic susceptibility tensor displays symmetry, which can cause multiple degenerated solutions in a structure calculation based solely on PCSs. Here we show a convenient method for resolving this degeneracy by changing the spacer length between the LBT and target protein. We applied this approach to PCS-based rigid body docking between the FKBP12-rapamycin complex and the mTOR FRB domain, and demonstrated that degeneracy could be resolved using the PCS restraints obtained from two-point anchored LBT with two different spacer lengths. The present strategy will markedly increase the usefulness of two-point anchored LBT for protein complex structure determination.

  10. 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.

  11. Quantitative Determination of Serum Proteins in Persons Occupationally Exposed to Radar

    International Nuclear Information System (INIS)

    Kasuba, V.; Garaj-Vrhovac, V.

    1998-01-01

    Radio-wave communications are used extensively in the modern society. We are all subject to radio frequency radiation (RFR) created by a radio, television, wireless telephony, emergency communications, and radar. The interest in the health effects of RFR has been motivated the rapid growth in wireless communications. Recently, many investigations are headed to the influence of nonionizing electromagnetic radiation to people. The interpretation of nonionizing radiation effects depends on apsorption characteristics of biological material and on thermoregulative system of exposed persons. This article includes serum protein analysis of 14 people who work in a radar zone. All the examinees are men aged 39 in average. The average exposure period in the radar zone was 16 years (twelve hours each second day). Human serum proteins in all samples were detected by electrophoresis on cellulose acetate membranes (Cellogel 500, Chemeton, Italy) in sodium-Veronal-Veronal-TRIS buffer pH 8.6. The duration of electrophoresis was two hours at 130-140 V at 11 cm bridge. Electroferograms were stained with 0.1 % Ponceau,s (w/v) (Chemetron, Italy) in 5% trichloracetic acid (v/v). Quantitative determination, i.e. densitometry of five serum protein fractions (albumin, α 1 - , α 2 - , β 1 - and γ -globulin) was carried out by photometry at multipolar densitometer. The results show gradual decrease of albumin 58.0 - 44.0 g/l versus control (64.05 g/l), except in three examinees where the values were almost equal to the normal value. One can also observe the changes in γ -globulin levels. All examinees except one showed increase in g -globulin levels (19.0 - 30.0 g/l vs. normal 15.0 g/l). The effect of nonionizing electromagnetic and microwave radiation on those who work in related field certainly needs much more investigation. (author)

  12. Ab initio structure determination and refinement of a scorpion protein toxin.

    Science.gov (United States)

    Smith, G D; Blessing, R H; Ealick, S E; Fontecilla-Camps, J C; Hauptman, H A; Housset, D; Langs, D A; Miller, R

    1997-09-01

    The structure of toxin II from the scorpion Androctonus australis Hector has been determined ab initio by direct methods using SnB at 0.96 A resolution. For the purpose of this structure redetermination, undertaken as a test of the minimal function and the SnB program, the identity and sequence of the protein was withheld from part of the research team. A single solution obtained from 1 619 random atom trials was clearly revealed by the bimodal distribution of the final value of the minimal function associated with each individual trial. Five peptide fragments were identified from a conservative analysis of the initial E-map, and following several refinement cycles with X-PLOR, a model was built of the complete structure. At the end of the X-PLOR refinement, the sequence was compared with the published sequence and 57 of the 64 residues had been correctly identified. Two errors in sequence resulted from side chains with similar size while the rest of the errors were a result of severe disorder or high thermal motion in the side chains. Given the amino-acid sequence, it is estimated that the initial E-map could have produced a model containing 99% of all main-chain and 81% of side-chain atoms. The structure refinement was completed with PROFFT, including the contributions of protein H atoms, and converged at a residual of 0.158 for 30 609 data with F >or= 2sigma(F) in the resolution range 8.0-0.964 A. The final model consisted of 518 non-H protein atoms (36 disordered), 407 H atoms, and 129 water molecules (43 with occupancies less than unity). This total of 647 non-H atoms represents the largest light-atom structure solved to date.

  13. Detection of the sulfhydryl groups in proteins with slow hydrogen exchange rates and determination of their proton/deuteron fractionation factors using the deuterium-induced effects on the 13C(beta) NMR signals.

    Science.gov (United States)

    Takeda, Mitsuhiro; Jee, JunGoo; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-05-05

    A method for identifying cysteine (Cys) residues with sulfhydryl (SH) groups exhibiting slow hydrogen exchange rates has been developed for proteins in aqueous media. The method utilizes the isotope shifts of the C(beta) chemical shifts induced by the deuteration of the SH groups. The 18.2 kDa E. coli peptidyl prolyl cis-trans isomerase b (EPPIb), which was selectively labeled with [3-(13)C;3,3-(2)H(2)]Cys, showed much narrower line widths for the (13)C(beta) NMR signals, as compared to those of the proteins labeled with either [3-(13)C]Cys or (3R)-[3-(13)C;3-(2)H]Cys. The (13)C(beta) signals of the two Cys residues of EPPIb, i.e. Cys-31 and Cys-121, labeled with [3-(13)C;3,3-(2)H(2)]Cys, split into four signals in H(2)O/D(2)O (1:1) at 40 degrees C and pH 7.5, indicating that the exchange rates of the side-chain SH's and the backbone amides are too slow to average the chemical shift differences of the (13)C(beta) signals, due to the two- and three-bond isotope shifts. By virtue of the well-separated signals, the proton/deuteron fractional factors for both the SH and amide groups of the two Cys residues in EPPIb could be directly determined, as approximately 0.4-0.5 for [SD]/[SH] and 0.9-1.0 for [ND]/[NH], by the relative intensities of the NMR signals for the isotopomers. The proton NOE's of the two slowly exchanging SH's were clearly identified in the NOESY spectra and were useful for the determining the local structure of EPPIb around the Cys residues.

  14. 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.

  15. Role of monomer sequence and backbone chemistry in polypeptoid copolymers for marine antifouling coatings

    Science.gov (United States)

    Patterson, Anastasia; Wenning, Brandon; Rizis, Georgios; Calabrese, David; Finlay, John; Franco, Sofia; Clare, Anthony; Kramer, Edward; Ober, Christopher; Segalman, Rachel

    The design rules elucidated in this work suggest that antifouling coatings bearing pendant peptoid side chains perform better overall in marine fouling tests than those with peptide side chains, with extremely low attachment of N. incerta and high removal of U. linza. This difference in performance is likely due to the lack of a hydrogen bond donor in the peptoid backbone. Furthermore, we show that the bulk polymer material of these hierarchical coatings (based on PEO or PDMS) plays a key role in determining both surface presentation and fouling release performance. We demonstrate these trends utilizing a modular coating based on a triblock copolymer consisting of polystyrene and a vinyl-containing midblock, to which sequence-defined pendant oligomers (peptides or peptoids with sequences of oligo-PEO and fluoroalkyl groups) are attached via thiol-ene ``click'' chemistry. Surface presentation was analyzed with X-ray photoelectron spectroscopy and captive bubble water contact angle, and antifouling performance was evaluated with attachment and removal bioassays of the marine macroalga U. linza and diatom N. incerta. NSF GRFP and ONR PECASE.

  16. Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

    Science.gov (United States)

    Geary, Bethany; Magee, Kieran; Cash, Phillip; Young, Iain S; Whitfield, Phillip D; Doherty, Mary K

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Correlation as a Determinant of Configurational Entropy in Supramolecular and Protein Systems

    Science.gov (United States)

    2015-01-01

    For biomolecules in solution, changes in configurational entropy are thought to contribute substantially to the free energies of processes like binding and conformational change. In principle, the configurational entropy can be strongly affected by pairwise and higher-order correlations among conformational degrees of freedom. However, the literature offers mixed perspectives regarding the contributions that changes in correlations make to changes in configurational entropy for such processes. Here we take advantage of powerful techniques for simulation and entropy analysis to carry out rigorous in silico studies of correlation in binding and conformational changes. In particular, we apply information-theoretic expansions of the configurational entropy to well-sampled molecular dynamics simulations of a model host–guest system and the protein bovine pancreatic trypsin inhibitor. The results bear on the interpretation of NMR data, as they indicate that changes in correlation are important determinants of entropy changes for biologically relevant processes and that changes in correlation may either balance or reinforce changes in first-order entropy. The results also highlight the importance of main-chain torsions as contributors to changes in protein configurational entropy. As simulation techniques grow in power, the mathematical techniques used here will offer new opportunities to answer challenging questions about complex molecular systems. PMID:24702693

  18. A two-step recognition of signal sequences determines the translocation efficiency of proteins.

    Science.gov (United States)

    Belin, D; Bost, S; Vassalli, J D; Strub, K

    1996-02-01

    The cytosolic and secreted, N-glycosylated, forms of plasminogen activator inhibitor-2 (PAI-2) are generated by facultative translocation. To study the molecular events that result in the bi-topological distribution of proteins, we determined in vitro the capacities of several signal sequences to bind the signal recognition particle (SRP) during targeting, and to promote vectorial transport of murine PAI-2 (mPAI-2). Interestingly, the six signal sequences we compared (mPAI-2 and three mutated derivatives thereof, ovalbumin and preprolactin) were found to have the differential activities in the two events. For example, the mPAI-2 signal sequence first binds SRP with moderate efficiency and secondly promotes the vectorial transport of only a fraction of the SRP-bound nascent chains. Our results provide evidence that the translocation efficiency of proteins can be controlled by the recognition of their signal sequences at two steps: during SRP-mediated targeting and during formation of a committed translocation complex. This second recognition may occur at several time points during the insertion/translocation step. In conclusion, signal sequences have a more complex structure than previously anticipated, allowing for multiple and independent interactions with the translocation machinery.

  19. Determination of amino acid and protein peroxides by the xylenol orange-Fe(III) complex

    International Nuclear Information System (INIS)

    Collins, J.; Craig, G.; Gebicki, J.

    1996-01-01

    Oxidative stress imposed on living organisms is believed to lead to the depletion of their antioxidant defences, followed by chemical changes in the cell constituents. These may ultimately develop into pathological conditions such as cancer or cardiovascular disease. An assay of peroxides which could be applied to tissues or simple tissue extracts would prove extremely useful in the studies of the phenomenon of oxidative stress. With this purpose, the authors have tested the ability of two peroxide assay techniques to measure the formation of amino acid and protein peroxides in aqueous solutions irradiated with gamma rays, using a modification of the method based on the oxidation of Fe(II)) by peroxides and complexing of the Fe(III) produced by xylenol orange. The molar extinction coefficients of the peroxides tested were determined by comparison with the well-tested iodometric assay. This work was extended to the detection of all organic peroxides in human blood plasma or serum subjected to oxidative stress, where the iodometric assay proved difficult to apply and unreliable because of the binding of iodine to the blood components. Preliminary results suggest that exposure of serum to gamma radiation leads to immediate peroxidation of the proteins, with a delay before generation of lipid peroxides

  20. Clinical significance of serum sex hormones protein and lipid determination in patients with ulcerative colitis

    International Nuclear Information System (INIS)

    Song Qingzhang; Zhang Min

    2010-01-01

    Objective: To investigate the relationships between changes of serum sex hormones levels and protein-lipid metabolism in patients with ulcerative colitis. Methods: Serum levels of estradiol (E 2 ) pregnenedione (P), prolactin(PRL), luteinizing hormone (LH), follicle-stimulating hormone (FSH) (with CLIA), sree testos (T, with RIA) and total-protein (TP), albumin (Alb), globulin (G), albumin/globulinratio (A/G) total-cholesterd (TC), high density lipoprotein cholesterols (LDL-C) (with biochemistry were determined in 72 patients) with ulcerative colitis and 72 controls. Results: The serum levels of T, LH, FSH, TP, Alb, A/G, TC, LDL-C in patients with ulcerative colitis were significantly lower than those in controls (P 2 , PRL in patients with ulcerative colitis were significantly higher than those in controls (P 2 were negatively correlated with TP, A/G and TC (P 2 levels in the female sex (P>0.05) as well as between LH, FSH and T levels in the male sex (P>0.05). Conclusion: The abnormal serum levels of sex hormone might contribute to the development of hypoproteinaemia and lowered lipid levels in patients with ulcerative colitis. Treatment with correction of serum sex hormones levels might be beneficial to the patients. (authors)

  1. Determination of Clara cell protein urinary elimination as a marker of tubular dysfunction.

    Science.gov (United States)

    Martín-Granado, Ascensión; Vázquez-Moncholí, Carmen; Luis-Yanes, María Isabel; López-Méndez, Marisela; García-Nieto, Víctor

    2009-04-01

    Clara cell 16-kDa protein (CC16) is a protein expressed primarily by the bronchial cells. It is rapidly eliminated by glomerular filtration, reabsorbed almost entirely, and catabolized in proximal tubule cells. To date, normal values for urinary CC16 in healthy children have not been determined. We have studied 63 pediatric patients (mean age 8.17 +/- 3.91 years) and 31 healthy children (control group; mean age 8.83 +/- 3.65 years). In the control group, the CC16/creatinine ratio was 1.22 +/- 1.52 microg/g. In 16 out of 31 control children, the value of the ratio was zero. Fourteen patients (22.2%) showed a high CC16/creatinine ratio; in contrast, among these same patients, the ratio N-acetyl-beta-D: -glucosaminidase (NAG)/creatinine was elevated in seven cases (11.1%) and the ratio beta2-microglobulin/creatinine was elevated in seven cases (11.1%). The three parameters were in agreement in 51 patients (80.9%). Among the patients, the CC16/creatinine ratio was correlated with both the beta2-microglobulin/creatinina ratio (r = 0.76, P < 0.001) and the NAG/creatinine ratio (r = 0.6, P < 0.001). Our findings indicate that CC16 is a good marker of proximal tubular function in childhood. The highest observed values were in children with proximal tubulopathies, in children with chronic renal failure, and in those treated with cyclosporine.

  2. 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.

  3. 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

    2014-11-04

    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.

  4. The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

    Directory of Open Access Journals (Sweden)

    Chenjing Shang

    Full Text Available Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

  5. Backbone and stereospecific (13)C methyl Ile (δ1), Leu and Val side-chain chemical shift assignments of Crc.

    Science.gov (United States)

    Sharma, Rakhi; Sahu, Bhubanananda; Ray, Malay K; Deshmukh, Mandar V

    2015-04-01

    Carbon catabolite repression (CCR) allows bacteria to selectively assimilate a preferred compound among a mixture of several potential carbon sources, thus boosting growth and economizing the cost of adaptability to variable nutrients in the environment. The RNA-binding catabolite repression control (Crc) protein acts as a global post-transcriptional regulator of CCR in Pseudomonas species. Crc triggers repression by inhibiting the expression of genes involved in transport and catabolism of non-preferred substrates, thus indirectly favoring assimilation of preferred one. We report here a nearly complete backbone and stereospecific (13)C methyl side-chain chemical shift assignments of Ile (δ1), Leu and Val of Crc (~ 31 kDa) from Pseudomonas syringae Lz4W.

  6. An efficient randomized algorithm for contact-based NMR backbone resonance assignment.

    Science.gov (United States)

    Kamisetty, Hetunandan; Bailey-Kellogg, Chris; Pandurangan, Gopal

    2006-01-15

    Backbone resonance assignment is a critical bottleneck in studies of protein structure, dynamics and interactions by nuclear magnetic resonance (NMR) spectroscopy. A minimalist approach to assignment, which we call 'contact-based', seeks to dramatically reduce experimental time and expense by replacing the standard suite of through-bond experiments with the through-space (nuclear Overhauser enhancement spectroscopy, NOESY) experiment. In the contact-based approach, spectral data are represented in a graph with vertices for putative residues (of unknown relation to the primary sequence) and edges for hypothesized NOESY interactions, such that observed spectral peaks could be explained if the residues were 'close enough'. Due to experimental ambiguity, several incorrect edges can be hypothesized for each spectral peak. An assignment is derived by identifying consistent patterns of edges (e.g. for alpha-helices and beta-sheets) within a graph and by mapping the vertices to the primary sequence. The key algorithmic challenge is to be able to uncover these patterns even when they are obscured by significant noise. This paper develops, analyzes and applies a novel algorithm for the identification of polytopes representing consistent patterns of edges in a corrupted NOESY graph. Our randomized algorithm aggregates simplices into polytopes and fixes inconsistencies with simple local modifications, called rotations, that maintain most of the structure already uncovered. In characterizing the effects of experimental noise, we employ an NMR-specific random graph model in proving that our algorithm gives optimal performance in expected polynomial time, even when the input graph is significantly corrupted. We confirm this analysis in simulation studies with graphs corrupted by up to 500% noise. Finally, we demonstrate the practical application of the algorithm on several experimental beta-sheet datasets. Our approach is able to eliminate a large majority of noise edges and to

  7. 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. © 2017 American Society of Plant Biologists. All Rights Reserved.

  8. 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.

  9. Zero in on Key Open Problems in Automated NMR Protein Structure Determination

    KAUST Repository

    Abbas, Ahmed

    2015-11-12

    Nuclear magnetic resonance (NMR) is one of the main approaches for protein struc- ture determination. The biggest advantage of this approach is that it can determine the three-dimensional structure of the protein in the solution phase. Thus, the natural dynamics of the protein can be studied. However, NMR protein structure determina- tion is an expertise intensive and time-consuming process. If the structure determi- nation process can be accelerated or even automated by computational methods, that will significantly advance the structural biology field. Our goal in this dissertation is to propose highly efficient and error tolerant methods that can work well on real and noisy data sets of NMR. Our first contribution in this dissertation is the development of a novel peak pick- ing method (WaVPeak). First, WaVPeak denoises the NMR spectra using wavelet smoothing. A brute force method is then used to identify all the candidate peaks. Af- ter that, the volume of each candidate peak is estimated. Finally, the peaks are sorted according to their volumes. WaVPeak is tested on the same benchmark data set that was used to test the state-of-the-art method, PICKY. WaVPeak shows significantly better performance than PICKY in terms of recall and precision. Our second contribution is to propose an automatic method to select peaks pro- duced by peak picking methods. This automatic method is used to overcome the limitations of fixed number-based methods. Our method is based on the Benjamini- Hochberg (B-H) algorithm. The method is used with both WaVPeak and PICKY to automatically select the number of peaks to return from out of hundreds of candidate peaks. The volume (in WaVPeak) and the intensity (in PICKY) are converted into p-values. Peaks that have p-values below some certain threshold are selected. Ex- perimental results show that the new method is better than the fixed number-based method in terms of recall. To improve precision, we tried to eliminate false peaks using

  10. Determination of free and bound riboflavin in cow's milk using a novel flavin-binding protein.

    Science.gov (United States)

    Koop, Julia; Monschein, Stefanie; Pauline Macheroux, E; Knaus, Tanja; Macheroux, Peter

    2014-03-01

    A recently described putative protease from the gut bacterium Bacteroides thetaiotaomicron (termed ppBat) exhibits two tryptophan residues in the interface which enable specific binding of the isoalloxazine heterocycle of riboflavin and its two cofactor forms, FMN and FAD. Recombinant ppBat was used to capture riboflavin from bovine milk directly without any prior preparation steps. The flavin-loaded protein was then re-isolated by means of affinity chromatography to identify and quantify the captured flavins. Free riboflavin concentrations were determined to 197 and 151μg/l for milk with 3.5% and 0.5% fat content, respectively. Total riboflavin concentrations were also determined after acid-treatment of milk and were 4-5 times higher than for free riboflavin. Free FMN and FAD were not detectable and only trace amounts of FMN were found in milk following acid treatment. The method appears to be amenable to develop a direct assay for free riboflavin in milk and other foods. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Determination of protein concentration in raw milk by mid-infrared fourier transform infrared/attenuated total reflectance spectroscopy.

    Science.gov (United States)

    Etzion, Y; Linker, R; Cogan, U; Shmulevich, I

    2004-09-01

    This study investigates the potential use of attenuated total reflectance spectroscopy in the mid-infrared range for determining protein concentration in raw cow milk. The determination of protein concentration is based on the characteristic absorbance of milk proteins, which includes 2 absorbance bands in the 1500 to 1700 cm(-1) range, known as the amide I and amide II bands, and absorbance in the 1060 to 1100 cm(-1) range, which is associated with phosphate groups covalently bound to casein proteins. To minimize the influence of the strong water band (centered around 1640 cm(-1)) that overlaps with the amide I and amide II bands, an optimized automatic procedure for accurate water subtraction was applied. Following water subtraction, the spectra were analyzed by 3 methods, namely simple band integration, partial least squares (PLS) and neural networks. For the neural network models, the spectra were first decomposed by principal component analysis (PCA), and the neural network inputs were the spectra principal components scores. In addition, the concentrations of 2 constituents expected to interact with the protein (i.e., fat and lactose) were also used as inputs. These approaches were tested with 235 spectra of standardized raw milk samples, corresponding to 26 protein concentrations in the 2.47 to 3.90% (weight per volume) range. The simple integration method led to very poor results, whereas PLS resulted in prediction errors of about 0.22% protein. The neural network approach led to prediction errors of 0.20% protein when based on PCA scores only, and 0.08% protein when lactose and fat concentrations were also included in the model. These results indicate the potential usefulness of Fourier transform infrared/attenuated total reflectance spectroscopy for rapid, possibly online, determination of protein concentration in raw milk.

  12. Comparison of methods for determination of testosterone and non-protein bound testosterone in men with alcoholic liver disease

    DEFF Research Database (Denmark)

    Gluud, C; Bennett, Patrick

    1986-01-01

    The serum concentrations of testosterone and of non-protein bound testosterone were determined in 28 men with alcoholic liver disease having normal to decreased serum albumin concentrations and normal to raised SHBG concentrations. Serum testosterone concentrations determined with two...... radioimmunoassays using different purification procedures and antibody batches did not differ significantly and correlated significantly (r=0.91; p less than 0.001). The median serum concentration of non-protein bound testosterone was 0.265 nmol/l (range 0.068-0.495 nmol/l) when determined by equilibrium dialysis...... and 0.232 nmol/l (range 0.042-0.610 nmol/l) when calculated according to the law of mass action. This difference is insignificant. The concentrations of non-protein bound testosterone determined by the two methods correlated significantly (r=0.83; p less than 0.001). In the calculation of non...

  13. Mnn10 Maintains Pathogenicity in Candida albicans by Extending α-1,6-Mannose Backbone to Evade Host Dectin-1 Mediated Antifungal Immunity.

    Directory of Open Access Journals (Sweden)

    Shi Qun Zhang

    2016-05-01

    Full Text Available The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface β-(1,3-glucan, a crucial pathogen-associated molecular pattern (PAMP of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1β and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding β-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans.

  14. Small heat shock proteins are necessary for heart migration and laterality determination in zebrafish

    Science.gov (United States)

    Lahvic, Jamie L.; Ji, Yongchang; Marin, Paloma; Zuflacht, Jonah P.; Springel, Mark W.; Wosen, Jonathan E.; Davis, Leigh; Hutson, Lara D.; Amack, Jeffrey D.; Marvin, Martha J.

    2013-01-01

    Small heat shock proteins (sHsps) regulate cellular functions not only under stress, but also during normal development, when they are expressed in organ-specific patterns. Here we demonstrate that two small heat shock proteins expressed in embryonic zebrafish heart, hspb7 and hspb12, have roles in the development of left-right asymmetry. In zebrafish, laterality is determined by the motility of cilia in Kupffer’s vesicle (KV), where hspb7 is expressed; knockdown of hspb7 causes laterality defects by disrupting the motility of these cilia. In embryos with reduced hspb7, the axonemes of KV cilia have a 9+0 structure, while control embyros have a predominately 9+2 structure. Reduction of either hspb7 or hspb12 alters the expression pattern of genes that propagate the signals that establish left-right asymmetry: the nodal-related gene southpaw (spaw) in the lateral plate mesoderm, and its downstream targets pitx2, lefty1 and lefty2. Partial depletion of hspb7 causes concordant heart, brain and visceral laterality defects, indicating that loss of KV cilia motility leads causes coordinated but randomized laterality. Reducing hspb12 leads to similar alterations in the expression of downstream laterality genes, but at a lower penetrance. Simultaneous reduction of hspb7 and hspb12 randomizes heart, brain and visceral laterality, suggesting that these two genes have partially redundant functions in the establishment of left-right asymmetry. In addition, both hspb7 and hspb12 are expressed in the precardiac mesoderm and in the yolk syncytial layer, which supports the migration and fusion of mesodermal cardiac precursors. In embryos in which the reduction of hspb7 or hspb12 was limited to the yolk, migration defects predominated, suggesting that the yolk expression of these genes rather than heart expression is responsible for the migration defects. PMID:24140541

  15. Denatured state is critical in determining the properties of model proteins designed on different folds

    DEFF Research Database (Denmark)

    Amatori, Andrea; Ferkinghoff-Borg, Jesper; Tiana, Guido

    2008-01-01

    The thermodynamics of proteins designed on three common folds (SH3, chymotrypsin inhibitor 2 [CI2], and protein G) is studied with a simplified C alpha, model and compared with the thermodynamics of proteins designed on random-generated folds. The model allows to design sequences to fold within a...

  16. Protein Requirements Are Elevated in Endurance Athletes after Exercise as Determined by the Indicator Amino Acid Oxidation Method.

    Directory of Open Access Journals (Sweden)

    Hiroyuki Kato

    Full Text Available A higher protein intake has been recommended for endurance athletes compared with healthy non-exercising individuals based primarily on nitrogen balance methodology. The aim of this study was to determine the estimated average protein requirement and recommended protein intake in endurance athletes during an acute 3-d controlled training period using the indicator amino acid oxidation method. After 2-d of controlled diet (1.4 g protein/kg/d and training (10 and 5km/d, respectively, six male endurance-trained adults (28±4 y of age; Body weight, 64.5±10.0 kg; VO2peak, 60.3±6.7 ml·kg-1·min-1; means±SD performed an acute bout of endurance exercise (20 km treadmill run prior to consuming test diets providing variable amounts of protein (0.2-2.8 g·kg-1·d-1 and sufficient energy. Protein was provided as a crystalline amino acid mixture based on the composition of egg protein with [1-13C]phenylalanine provided to determine whole body phenylalanine flux, 13CO2 excretion, and phenylalanine oxidation. The estimated average protein requirement was determined as the breakpoint after biphasic linear regression analysis with a recommended protein intake defined as the upper 95% confidence interval. Phenylalanine flux (68.8±8.5 μmol·kg-1·h-1 was not affected by protein intake. 13CO2 excretion displayed a robust bi-phase linear relationship (R2 = 0.86 that resulted in an estimated average requirement and a recommended protein intake of 1.65 and 1.83 g protein·kg-1·d-1, respectively, which was similar to values based on phenylalanine oxidation (1.53 and 1.70 g·kg-1·d-1, respectively. We report a recommended protein intake that is greater than the RDA (0.8 g·kg-1·d-1 and current recommendations for endurance athletes (1.2-1.4 g·kg-1·d-1. Our results suggest that the metabolic demand for protein in endurance-trained adults on a higher volume training day is greater than their sedentary peers and current recommendations for athletes based

  17. Analysis of stationary availability factor of two-level backbone computer networks with arbitrary topology

    Science.gov (United States)

    Rahman, P. A.

    2018-05-01

    This scientific paper deals with the two-level backbone computer networks with arbitrary topology. A specialized method, offered by the author for calculation of the stationary availability factor of the two-level backbone computer networks, based on the Markov reliability models for the set of the independent repairable elements with the given failure and repair rates and the methods of the discrete mathematics, is also discussed. A specialized algorithm, offered by the author for analysis of the network connectivity, taking into account different kinds of the network equipment failures, is also observed. Finally, this paper presents an example of calculation of the stationary availability factor for the backbone computer network with the given topology.

  18. Impact of Backbone Fluorination on π-Conjugated Polymers in Organic Photovoltaic Devices: A Review

    Directory of Open Access Journals (Sweden)

    Nicolas Leclerc

    2016-01-01

    Full Text Available Solution-processed bulk heterojunction solar cells have experienced a remarkable acceleration in performances in the last two decades, reaching power conversion efficiencies above 10%. This impressive progress is the outcome of a simultaneous development of more advanced device architectures and of optimized semiconducting polymers. Several chemical approaches have been developed to fine-tune the optoelectronics and structural polymer parameters required to reach high efficiencies. Fluorination of the conjugated polymer backbone has appeared recently to be an especially promising approach for the development of efficient semiconducting polymers. As a matter of fact, most currently best-performing semiconducting polymers are using fluorine atoms in their conjugated backbone. In this review, we attempt to give an up-to-date overview of the latest results achieved on fluorinated polymers for solar cells and to highlight general polymer properties’ evolution trends related to the fluorination of their conjugated backbone.

  19. The rapid determination of fat and protein content in fresh raw milk using the laser light scattering technology

    Science.gov (United States)

    Xin, Qi; Zhi Ling, Hou; Jian Long, Tian; Zhu, Yu

    2006-08-01

    The aim was to develop a simple and rapid method for determination of fat and protein content in milk. Based on the laser light scattering theory, the ratio of the scattered light (at 90±0.05° scattering angles) intensity to the transmitted light intensity, which is called scattered-transmitted-ratio method, is adopted as the optical parameter representing the milk fat content and the protein content. In this way, the influence of the fluctuation of the power of the light source is eliminated and the accuracy of determination is improved accordingly. The system we use is real-time and can satisfy the challenging requirements of dairy farming. Results of this study indicate the feasibility of using this technology for fresh milk fat and protein analysis. The fat contents and protein contents of 50 milk samples determined by this method were consistent with the values obtained by the reference methods based on Rose-Gottlieb method and Kjeldahl determination of N method. In this paper, the operating principle of the instrument is introduced and the influence of the environmental conditions, such as the homogenization pressure and homogenization temperature, etc. on the result of the test is analyzed. Through data analysis, the concrete schemes for testing the fat using the curve fitting and testing the protein using the surface fitting technique are determined. Finally, the difference from the reference values of the test is discussed.

  20. Rediscovery and Revival of Analytical Refractometry for Protein Determination: Recombining Simplicity With Accuracy in the Digital Era.

    Science.gov (United States)

    Anderle, Heinz; Weber, Alfred

    2016-03-01

    Among "vintage" methods of protein determination, quantitative analytical refractometry has received far less attention than well-established pharmacopoeial techniques based on protein nitrogen content, such as Dumas combustion (1831) and Kjeldahl digestion (1883). Protein determination by quantitative refractometry dates back to 1903 and has been extensively investigated and characterized in the following 30 years, but has since vanished into a few niche applications that may not require the degree of accuracy and precision essential for pharmaceutical analysis. However, because high-resolution and precision digital refractometers have replaced manual instruments, reducing time and resource consumption, the method appears particularly attractive from an economic, ergonomic, and environmental viewpoint. The sample solution can be measured without dilution or other preparation procedures than the separation of the protein-free matrix by ultrafiltration, which might even be omitted for a constant matrix and excipient composition. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  1. Determination of chromium combined with DNA, RNA and proteins in chromium-rich brewer's yeast by NAA

    International Nuclear Information System (INIS)

    Ding, W.J.; Qian, Q.F.; Hou, X.L.; Feng, W.Y.; Chai, Z.F.

    2000-01-01

    The content of chromium in the DNA, RNA and protein fractions separated from chromium-rich and normal brewer's yeast was determined by neutron activation analysis (NAA). Our results show that the extracted relative amounts and concentrations of DNA, RNA and proteins have no significant difference for two types of yeast, but the chromium content in DNA, RNA and proteins fractions extracted from the chromium-rich yeast are substantially higher than those from the normal. In addition, the concentration of chromium in DNA is much higher than that in RNA and proteins. It is evident that the inorganic chromium compounds can enter the yeast cell during the yeast cultivation in the chromium-containing culture medium and are converted into organic chromium species, which are combined with DNA, RNA and proteins. (author)

  2. Determination of phase diagrams via computer simulation: methodology and applications to water, electrolytes and proteins

    International Nuclear Information System (INIS)

    Vega, C; Sanz, E; Abascal, J L F; Noya, E G

    2008-01-01

    In this review we focus on the determination of phase diagrams by computer simulation, with particular attention to the fluid-solid and solid-solid equilibria. The methodology to compute the free energy of solid phases will be discussed. In particular, the Einstein crystal and Einstein molecule methodologies are described in a comprehensive way. It is shown that both methodologies yield the same free energies and that free energies of solid phases present noticeable finite size effects. In fact, this is the case for hard spheres in the solid phase. Finite size corrections can be introduced, although in an approximate way, to correct for the dependence of the free energy on the size of the system. The computation of free energies of solid phases can be extended to molecular fluids. The procedure to compute free energies of solid phases of water (ices) will be described in detail. The free energies of ices Ih, II, III, IV, V, VI, VII, VIII, IX, XI and XII will be presented for the SPC/E and TIP4P models of water. Initial coexistence points leading to the determination of the phase diagram of water for these two models will be provided. Other methods to estimate the melting point of a solid, such as the direct fluid-solid coexistence or simulations of the free surface of the solid, will be discussed. It will be shown that the melting points of ice Ih for several water models, obtained from free energy calculations, direct coexistence simulations and free surface simulations agree within their statistical uncertainty. Phase diagram calculations can indeed help to improve potential models of molecular fluids. For instance, for water, the potential model TIP4P/2005 can be regarded as an improved version of TIP4P. Here we will review some recent work on the phase diagram of the simplest ionic model, the restricted primitive model. Although originally devised to describe ionic liquids, the model is becoming quite popular to describe the behavior of charged colloids

  3. Backbone conformation affects duplex initiation and duplex propagation in hybridisation of synthetic H-bonding oligomers.

    Science.gov (United States)

    Iadevaia, Giulia; Núñez-Villanueva, Diego; Stross, Alexander E; Hunter, Christopher A

    2018-06-06

    Synthetic oligomers equipped with complementary H-bond donor and acceptor side chains form multiply H-bonded duplexes in organic solvents. Comparison of the duplex forming properties of four families of oligomers with different backbones shows that formation of an extended duplex with three or four inter-strand H-bonds is more challenging than formation of complexes that make only two H-bonds. The stabilities of 1 : 1 complexes formed between length complementary homo-oligomers equipped with either phosphine oxide or phenol recognition modules were measured in toluene. When the backbone is very flexible (pentane-1,5-diyl thioether), the stability increases uniformly by an order of magnitude for each additional base-pair added to the duplex: the effective molarities for formation of the first intramolecular H-bond (duplex initiation) and subsequent intramolecular H-bonds (duplex propagation) are similar. This flexible system is compared with three more rigid backbones that are isomeric combinations of an aromatic ring and methylene groups. One of the rigid systems behaves in exactly the same way as the flexible backbone, but the other two do not. For these systems, the effective molarity for formation of the first intramolecular H-bond is the same as that found for the other two backbones, but additional H-bonds are not formed between the longer oligomers. The effective molarities are too low for duplex propagation in these systems, because the oligomer backbones cannot adopt conformations compatible with formation of an extended duplex.

  4. Protein oxidation and peroxidation

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard...... to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners...... and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals...

  5. Determination of antioxidant and chelating activity of protein hydrolysates from spirulina (Arthrospira maxima) obtained by simulated gastrointestinal digestion

    OpenAIRE

    Martínez-Palma, N.; Martínez-Ayala, A.; Dávila-Ortíz, G.

    2015-01-01

    Spirulina is a cyanobacteria that has been used as food since ancient times, for example in Mexico it was consumed by the Aztecs. Its high protein content, distribution and amino acid composition suggests the presence of important peptides encrypted within the sequences of parent proteins, that after been released by digestive process they could show an antioxidant effect. Our present study examined the above hypothesis through the determination of the antioxidant and chelating activity of tw...

  6. Polyolefin backbone substitution in binders for low temperature powder injection moulding feedstocks.

    Science.gov (United States)

    Hausnerova, Berenika; Kuritka, Ivo; Bleyan, Davit

    2014-02-27

    This paper reports the substitution of polyolefin backbone binder components with low melting temperature carnauba wax for powder injection moulding applications. The effect of various binder compositions of Al₂O₃ feedstock on thermal degradation parameters is investigated by thermogravimetric analysis. Within the experimental framework 29 original feedstock compositions were prepared and the superiority of carnauba wax over the polyethylene binder backbone was demonstrated in compositions containing polyethylene glycol as the initial opening agent and governing the proper mechanism of the degradation process. Moreover, the replacement of synthetic polymer by the natural wax contributes to an increase of environmental sustainability of modern industrial technologies.

  7. Polyolefin Backbone Substitution in Binders for Low Temperature Powder Injection Moulding Feedstocks

    Directory of Open Access Journals (Sweden)

    Berenika Hausnerova

    2014-02-01

    Full Text Available This paper reports the substitution of polyolefin backbone binder components with low melting temperature carnauba wax for powder injection moulding applications. The effect of various binder compositions of Al2O3 feedstock on thermal degradation parameters is investigated by thermogravimetric analysis. Within the experimental framework 29 original feedstock compositions were prepared and the superiority of carnauba wax over the polyethylene binder backbone was demonstrated in compositions containing polyethylene glycol as the initial opening agent and governing the proper mechanism of the degradation process. Moreover, the replacement of synthetic polymer by the natural wax contributes to an increase of environmental sustainability of modern industrial technologies.

  8. Electric field induced localization phenomena in a ladder network with superlattice configuration: Effect of backbone environment

    Energy Technology Data Exchange (ETDEWEB)

    Dutta, Paramita; Karmakar, S. N. [Condensed Matter Physics Division, Saha Institute of Nuclear Physics, Sector-I, Block-AF, Bidhannagar, Kolkata-700 064 (India); Maiti, Santanu K., E-mail: santanu.maiti@isical.ac.in [Physics and Applied Mathematics Unit, Indian Statistical Institute, 203 Barrackpore Trunk Road, Kolkata-700 108 (India)

    2014-09-15

    Electric field induced localization properties of a tight-binding ladder network in presence of backbone sites are investigated. Based on Green's function formalism we numerically calculate two-terminal transport together with density of states for different arrangements of atomic sites in the ladder and its backbone. Our results lead to a possibility of getting multiple mobility edges which essentially plays a switching action between a completely opaque to fully or partly conducting region upon the variation of system Fermi energy, and thus, support in fabricating mesoscopic or DNA-based switching devices.

  9. Five Principles of Industrialized Transformation for Successfully Building an Operational Backbone

    DEFF Research Database (Denmark)

    Winkler, Till J.; Kettunen, Petteri

    2018-01-01

    approach that is underpinned by five principles—template-based, business-driven, matrix-organized, tight supplier steering and cascaded planning. The UPM case provides important lessons for transformation leaders seeking to build, expand or develop a value-adding operational backbone.......To move into the digital age, a globally operating company needs to have in place an operational backbone, but many struggle with achieving this and the associated transformation program. Based on the experience of UPM, a Finnish forest industry company, we describe an industrialized transformation...

  10. 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

    Previous studies have demonstrated that gamma-irradiation of some free amino acids in the presence of oxygen gives high yields of side-chain hydroperoxides. It is shown in the present study that N-acetyl amino acids and peptides also give high levels of hydroperoxides on gamma-irradiation, even...

  11. Multi-signal sedimentation velocity analysis with mass conservation for determining the stoichiometry of protein complexes.

    Directory of Open Access Journals (Sweden)

    Chad A Brautigam

    Full Text Available Multi-signal sedimentation velocity analytical ultracentrifugation (MSSV is a powerful tool for the determination of the number, stoichiometry, and hydrodynamic shape of reversible protein complexes in two- and three-component systems. In this method, the evolution of sedimentation profiles of macromolecular mixtures is recorded simultaneously using multiple absorbance and refractive index signals and globally transformed into both spectrally and diffusion-deconvoluted component sedimentation coefficient distributions. For reactions with complex lifetimes comparable to the time-scale of sedimentation, MSSV reveals the number and stoichiometry of co-existing complexes. For systems with short complex lifetimes, MSSV reveals the composition of the reaction boundary of the coupled reaction/migration process, which we show here may be used to directly determine an association constant. A prerequisite for MSSV is that the interacting components are spectrally distinguishable, which may be a result, for example, of extrinsic chromophores or of different abundances of aromatic amino acids contributing to the UV absorbance. For interacting components that are spectrally poorly resolved, here we introduce a method for additional regularization of the spectral deconvolution by exploiting approximate knowledge of the total loading concentrations. While this novel mass conservation principle does not discriminate contributions to different species, it can be effectively combined with constraints in the sedimentation coefficient range of uncomplexed species. We show in theory, computer simulations, and experiment, how mass conservation MSSV as implemented in SEDPHAT can enhance or even substitute for the spectral discrimination of components. This should broaden the applicability of MSSV to the analysis of the composition of reversible macromolecular complexes.

  12. Extracellular Determinants of Anion Discrimination of the Cl−/H+ Antiporter Protein CLC-5*

    Science.gov (United States)

    De Stefano, Silvia; Pusch, Michael; Zifarelli, Giovanni

    2011-01-01

    Mammalian CLC proteins comprise both Cl− channels and Cl−/H+ antiporters that carry out fundamental physiological tasks by transporting Cl− across plasma membrane and intracellular compartments. The NO3− over Cl− preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl−/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3− over Cl− preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl−. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext. PMID:21921031

  13. Extracellular determinants of anion discrimination of the Cl-/H+ antiporter protein CLC-5.

    Science.gov (United States)

    De Stefano, Silvia; Pusch, Michael; Zifarelli, Giovanni

    2011-12-23

    Mammalian CLC proteins comprise both Cl- channels and Cl-/H+ antiporters that carry out fundamental physiological tasks by transporting Cl- across plasma membrane and intracellular compartments. The NO3- over Cl- preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl-/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3- over Cl- preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl-. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext.

  14. Determination of ortho-tyrosine in irradiated protein containing foods by HPLC

    International Nuclear Information System (INIS)

    Mischke, J.; Voehringer, M.; Helle, N.; Boegl K.W.; Schreiber, G.A.

    1993-01-01

    In order to control the processing and trading of irradiated foodstuffs several chemical and physical methods have been developed to identify irradiation induced changes. The three most promising methods are gas chromatorgraphic determination of radiation induced volatiles from the lipid content of foods, thermoluminescence measurements on minerals and e.s.r.-spectroscopic measurements on solids and food contents with a low water amount. There is a lack in detecting the irradiation in foods with a high protein content. It is based on the radiation induced hydroxylation of phenylalanine, forming small amounts of ortho- (and meta-) tyrosine. This method can be useful for foods with a low lipid content such as shrimps and pure egg-white. The results obtained on shrimps and egg-white are promising. All shrimp samples showed a good dose dependence which was similar to results reported by Chuaqui-Offermanns and McDougall obtained on frozen materials (chicken) irradiated at a slightly higher dose rate. There are not enough data about o-tyrosine-contents in different kinds of unirradiated shrimps. Therfore next step will be the analysis of a great number of various samples. With these information and by the use of an internal standard it should be possible to apply the HPLC method for routine analysis. As internal standards α-methyltyrosine or 4-hydroxyphenylglycine could be used. (orig./vhe)

  15. Protein fiber linear dichroism for structure determination and kinetics in a low-volume, low-wavelength couette flow cell.

    Science.gov (United States)

    Dafforn, Timothy R; Rajendra, Jacindra; Halsall, David J; Serpell, Louise C; Rodger, Alison

    2004-01-01

    High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the structure of such biomolecular systems. However, existing systems are not optimized for the requirements of fibrous proteins. We have designed and built a low-volume (200 microL), low-wavelength (down to 180 nm), low-pathlength (100 microm), high-alignment flow-alignment system (couette) to perform ultraviolet linear dichroism studies on the fibers formed by a range of biomolecules. The apparatus has been tested using a number of proteins for which longer wavelength linear dichroism spectra had already been measured. The new couette cell has also been used to obtain data on two medically important protein fibers, the all-beta-sheet amyloid fibers of the Alzheimer's derived protein Abeta and the long-chain assemblies of alpha1-antitrypsin polymers.

  16. Application of artificial neural networks for the determination of proteins with CPA-pI by rayleigh light scattering technique

    Energy Technology Data Exchange (ETDEWEB)

    Dong Lijun [Department of Chemistry, Lanzhou University, Lanzhou 730000 (China); Chen Xingguo [Department of Chemistry, Lanzhou University, Lanzhou 730000 (China)]. E-mail: chenxg@lzu.edu.cn; Hu Zhide [Department of Chemistry, Lanzhou University, Lanzhou 730000 (China)

    2007-05-15

    The determination of proteins with 2-(4-chloro-2-phosphonophenylazo)-7-(4-iodophenylazo) -1,8-dihydroxynaphthalene-3,6-disulfonic acid (CPA-pI) by Rayleigh light scattering (RLS) was studied in this paper. The weak RLS of CPA-pI and BSA can be enhanced greatly by the addition of Al{sup 3+} at the pH 5.6 and an enhanced RLS signal was produced at 365-385 nm. Based on the reaction of CPA-pI, Al{sup 3+} and proteins, a new quantitative determination method for proteins has been developed. The effect of three variables for the determination of proteins was optimized by means of artificial neural networks (ANNs) using extended delta-bar-delta (EDBD) algorithms with the optimal network structure of 3-5-1. This method is very sensitive (2.5-35.4 {mu}g/ml for bovine serum albumin (BSA)), rapid (<2 min), simple (one step) and tolerance of most interfering substances. Six samples of protein in human serum were determined and the maximum relative error is no more than 2% and the recovery is between 95% and 105%.

  17. Combining automated peak tracking in SAR by NMR with structure-based backbone assignment from 15N-NOESY

    KAUST Repository

    Jang, Richard; Gao, Xin; Li, Ming

    2012-01-01

    Background: Chemical shift mapping is an important technique in NMR-based drug screening for identifying the atoms of a target protein that potentially bind to a drug molecule upon the molecule's introduction in increasing concentrations. The goal is to obtain a mapping of peaks with known residue assignment from the reference spectrum of the unbound protein to peaks with unknown assignment in the target spectrum of the bound protein. Although a series of perturbed spectra help to trace a path from reference peaks to target peaks, a one-to-one mapping generally is not possible, especially for large proteins, due to errors, such as noise peaks, missing peaks, missing but then reappearing, overlapped, and new peaks not associated with any peaks in the reference. Due to these difficulties, the mapping is typically done manually or semi-automatically, which is not efficient for high-throughput drug screening.Results: We present PeakWalker, a novel peak walking algorithm for fast-exchange systems that models the errors explicitly and performs many-to-one mapping. On the proteins: hBclXL, UbcH5B, and histone H1, it achieves an average accuracy of over 95% with less than 1.5 residues predicted per target peak. Given these mappings as input, we present PeakAssigner, a novel combined structure-based backbone resonance and NOE assignment algorithm that uses just 15N-NOESY, while avoiding TOCSY experiments and 13C-labeling, to resolve the ambiguities for a one-to-one mapping. On the three proteins, it achieves an average accuracy of 94% or better.Conclusions: Our mathematical programming approach for modeling chemical shift mapping as a graph problem, while modeling the errors directly, is potentially a time- and cost-effective first step for high-throughput drug screening based on limited NMR data and homologous 3D structures. 2012 Jang et al.; licensee BioMed Central Ltd.

  18. Combining automated peak tracking in SAR by NMR with structure-based backbone assignment from 15N-NOESY

    KAUST Repository

    Jang, Richard

    2012-03-21

    Background: Chemical shift mapping is an important technique in NMR-based drug screening for identifying the atoms of a target protein that potentially bind to a drug molecule upon the molecule\\'s introduction in increasing concentrations. The goal is to obtain a mapping of peaks with known residue assignment from the reference spectrum of the unbound protein to peaks with unknown assignment in the target spectrum of the bound protein. Although a series of perturbed spectra help to trace a path from reference peaks to target peaks, a one-to-one mapping generally is not possible, especially for large proteins, due to errors, such as noise peaks, missing peaks, missing but then reappearing, overlapped, and new peaks not associated with any peaks in the reference. Due to these difficulties, the mapping is typically done manually or semi-automatically, which is not efficient for high-throughput drug screening.Results: We present PeakWalker, a novel peak walking algorithm for fast-exchange systems that models the errors explicitly and performs many-to-one mapping. On the proteins: hBclXL, UbcH5B, and histone H1, it achieves an average accuracy of over 95% with less than 1.5 residues predicted per target peak. Given these mappings as input, we present PeakAssigner, a novel combined structure-based backbone resonance and NOE assignment algorithm that uses just 15N-NOESY, while avoiding TOCSY experiments and 13C-labeling, to resolve the ambiguities for a one-to-one mapping. On the three proteins, it achieves an average accuracy of 94% or better.Conclusions: Our mathematical programming approach for modeling chemical shift mapping as a graph problem, while modeling the errors directly, is potentially a time- and cost-effective first step for high-throughput drug screening based on limited NMR data and homologous 3D structures. 2012 Jang et al.; licensee BioMed Central Ltd.

  19. Combining ambiguous chemical shift mapping with structure-based backbone and NOE assignment from 15N-NOESY

    KAUST Repository

    Jang, Richard

    2011-01-01

    Chemical shift mapping is an important technique in NMRbased drug screening for identifying the atoms of a target protein that potentially bind to a drug molecule upon the molecule\\'s introduction in increasing concentrations. The goal is to obtain a mapping of peaks with known residue assignment from the reference spectrum of the unbound protein to peaks with unknown assignment in the target spectrum of the bound protein. Although a series of perturbed spectra help to trace a path from reference peaks to target peaks, a one-to-one mapping generally is not possible, especially for large proteins, due to errors, such as noise peaks, missing peaks, missing but then reappearing, overlapped, and new peaks not associated with any peaks in the reference. Due to these difficulties, the mapping is typically done manually or semi-automatically. However, automated methods are necessary for high-throughput drug screening. We present PeakWalker, a novel peak walking algorithm for fast-exchange systems that models the errors explicitly and performs many-to-one mapping. On the proteins: hBclXL, UbcH5B, and histone H1, it achieves an average accuracy of over 95% with less than 1.5 residues predicted per target peak. Given these mappings as input, we present PeakAssigner, a novel combined structure-based backbone resonance and NOE assignment algorithm that uses just 15N-NOESY, while avoiding TOCSY experiments and 13C- labeling, to resolve the ambiguities for a one-toone mapping. On the three proteins, it achieves an average accuracy of 94% or better. Copyright © 2011 ACM.

  20. The Purine Bias of Coding Sequences is Determined by Physicochemical Constraints on Proteins.

    Science.gov (United States)

    Ponce de Leon, Miguel; de Miranda, Antonio Basilio; Alvarez-Valin, Fernando; Carels, Nicolas

    2014-01-01

    For this report, we analyzed protein secondary structures in relation to the statistics of three nucleotide codon positions. The purpose of this investigation was to find which properties of the ribosome, tRNA or protein level, could explain the purine bias (Rrr) as it is observed in coding DNA. We found that the Rrr pattern is the consequence of a regularity (the codon structure) resulting from physicochemical constraints on proteins and thermodynamic constraints on ribosomal machinery. The physicochemical constraints on proteins mainly come from the hydropathy and molecular weight (MW) of secondary structures as well as the energy cost of amino acid synthesis. These constraints appear through a network of statistical correlations, such as (i) the cost of amino acid synthesis, which is in favor of a higher level of guanine in the first codon position, (ii) the constructive contribution of hydropathy alternation in proteins, (iii) the spatial organization of secondary structure in proteins according to solvent accessibility, (iv) the spatial organization of secondary structure according to amino acid hydropathy, (v) the statistical correlation of MW with protein secondary structures and their overall hydropathy, (vi) the statistical correlation of thymine in the second codon position with hydropathy and the energy cost of amino acid synthesis, and (vii) the statistical correlation of adenine in the second codon position with amino acid complexity and the MW of secondary protein structures. Amino acid physicochemical properties and functional constraints on proteins constitute a code that is translated into a purine bias within the coding DNA via tRNAs. In that sense, the Rrr pattern within coding DNA is the effect of information transfer on nucleotide composition from protein to DNA by selection according to the codon positions. Thus, coding DNA structure and ribosomal machinery co-evolved to minimize the energy cost of protein coding given the functional

  1. Evaluation of the furosine and homoarginine methods for determining reactive lysine in rumen-undegraded protein.

    Science.gov (United States)

    Boucher, S E; Pedersen, C; Stein, H H; Schwab, C G

    2009-08-01

    Three samples of soybean meal (SBM), 3 samples of expeller SBM (SoyPlus, West Central Cooperative, Ralston, IA), 5 samples of distillers dried grains with solubles (DDGS), and 5 samples of fish meal were used to evaluate the furosine and homoarginine procedures to estimate reactive Lys in the rumen-undegraded protein fraction (RUP-Lys). One sample each of SBM, expeller SBM, and DDGS were subjected to additional heat treatment in the lab to ensure there was a wide range in reactive RUP-Lys content among the samples. Furosine is a secondary product of the initial stages of the Maillard reaction and can be used to calculate blocked Lys. Homoarginine is formed via the reaction of reactive Lys with O-methylisourea and can be used to calculate the concentration of reactive Lys. In previous experiments, each sample was ruminally incubated in situ for 16 h, and standardized RUP-Lys digestibility of the samples was determined in cecectomized roosters. All rumen-undegraded residue (RUR) samples were analyzed for furosine and Lys; however, only 9 of the 16 samples contained furosine, and only the 4 unheated DDGS samples contained appreciable amounts of furosine. Blocked RUP-Lys was calculated from the furosine and Lys concentrations of the RUR. Both the intact feed and RUR samples were evaluated using the homoarginine method. All samples were incubated with an O-methylisourea/BaOH solution for 72 h and analyzed for Lys and homoarginine concentrations. Reactive Lys concentrations of the intact feeds and RUR were calculated. Results of the experiment indicate that blocked RUP-Lys determined via the furosine method was negatively correlated with standardized RUP-Lys digestibility, and reactive RUP-Lys determined via the guanidination method was positively correlated with standardized RUP-Lys digestibility. Reactive Lys concentrations of the intact samples were also highly correlated with RUP-Lys digestibility. In conclusion, the furosine assay is useful in predicting RUP

  2. Determinants of protein-energy malnutrition in community-dwelling older adults: a systematic review of observational studies.

    Science.gov (United States)

    van der Pols-Vijlbrief, Rachel; Wijnhoven, Hanneke A H; Schaap, Laura A; Terwee, Caroline B; Visser, Marjolein

    2014-11-01

    Protein-energy malnutrition is associated with numerous poor health outcomes, including high health care costs, mortality rates and poor physical functioning in older adults. This systematic literature review aims to identify and provide an evidence based overview of potential determinants of protein-energy malnutrition in community-dwelling older adults. A systematic search was conducted in PUBMED, EMBASE, CINAHL and COCHRANE from the earliest possible date through January 2013. Observational studies that examined determinants of protein-energy malnutrition were selected and a best evidence synthesis was performed to summarize the results. In total 28 studies were included in this review from which 122 unique potential determinants were derived. Thirty-seven determinants were examined in sufficient number of studies and were included in a best evidence synthesis. The best evidence score comprised design (cross-sectional, longitudinal) and quality of the study (high, moderate) to grade the evidence level. Strong evidence for an association with protein-energy malnutrition was found for poor appetite, and moderate evidence for edentulousness, having no diabetes, hospitalization and poor self-reported health. Strong evidence for no association was found for anxiety, chewing difficulty, few friends, living alone, feeling lonely, death of spouse, high number of diseases, heart failure and coronary failure, stroke (CVA) and the use of anti-inflammatory medications. This review shows that protein-energy malnutrition is a multifactorial problem and that different domains likely play a role in the pathway of developing protein-energy malnutrition. These results provide important knowledge for the development of targeted, multifactorial interventions that aim to prevent the development of protein-energy malnutrition in community-dwelling older adults. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Enhancing the efficiency of thiomers: Utilizing a highly mucoadhesive polymer as backbone for thiolation and preactivation.

    Science.gov (United States)

    Prüfert, Felix; Bonengel, Sonja; Menzel, Claudia; Bernkop-Schnürch, Andreas

    2017-01-01

    The objective of this study was to develop a novel thiomer with enhanced mucoadhesive properties using a highly mucoadhesive polymeric backbone. Fixomer™ A-30 (poly(methacrylic acid-co-sodium acrylamidomethyl propane sulfonate)), exhibiting a mucoadhesive strength superior to that of all other polymers, was thiolated by conjugation with l-cysteine and furthermore preactivated with 2-mercaptonicotinic acid (MNA). The resulting derivatives Fix-SH and Fix-S-MNA exhibited coupling rates of 755μmol thiol groups and 304μmol MNA per gram polymer, respectively. The mucoadhesive profile was evaluated with three different methods: tensile studies, rotating cylinder and rheological synergism. In tensile studies, a total work of adhesion of above 500μJ was determined for the unmodified polymer that increased to around 750μJ after thiolation and around 1500μJ after preactivation. The adhesion time of Fix-SH on the rotating cylinder was 3.7-fold and that of Fix-S-MNA 6.8-fold longer compared to the unmodified polymer. A rheological synergism was observed for the unmodified polymer as well as the derivatives with a non-significant difference for Fix-SH but a 5.44-fold improvement for Fix-S-MNA. Fix-S-MNA showed a significantly improved swelling behavior with a water-uptake up to the 30-fold of its initial weight over >50h whereas thiolation showed only slight improvements. Derivatization had no significant influence on cell viability. According to the results, Fix-S-MNA seems to be a suitable polymer for mucoadhesive drug delivery systems. Copyright © 2016. Published by Elsevier B.V.

  4. Oxygen-independent direct deoxyribonucleic acid backbone breakage caused by rose bengal and visible light

    Energy Technology Data Exchange (ETDEWEB)

    Peak, M J; Peak, J G; Foote, C S; Krinsky, N I

    1984-01-01

    An oxygen enhancement ratio of 10 for the induction of backbone single-strand breaks (SSBs) in purified deoxyribonucleic acid (DNA) by monochromatic 365 nm UV radiation was obtained. Similarly, a dose reduction factor of 10 was observed when the DNA was irradiated in the presence of 0.1 M diazabicyclo(2.2.2)octane (DABCO). To determine whether this breakage of DNA was due to the action of a reactive oxygen species such as singlet oxygen, we used the photosensitizing dye Rose Bengal and visible light as a system for generating singlet oxygen. Treatment of the DNA with Rose Bengal and 545 nm monochromatic light enhanced the rate of induction of SSBs six times, compared with the rate we obtained when the light was used alone. Elimination of oxygen or addition of 0.1 M DABCO during the 545 nm irradiation in the presence of Rose Bengal did not alter the enhancement of SSBs in the DNA caused by Rose Bengal and 545 nm radiation. The induction of SSBs in the DNA caused by irradiation of the DNA by 545 nm light in the presence of Rose Bengal was not enhanced by the use of D/sub 2/O instead of H/sub 2/O as a solvent. The results indicate that Rose Bengal plus visible light can cause biological damage without the intermediacy of reactive oxygen species, i.e. Rose Bengal and visible light can react directly with biological material, in reactions that appear to be type I photosensitized processes, independent of singlet oxygen as an intermediate.

  5. Combining sequence-based prediction methods and circular dichroism and infrared spectroscopic data to improve protein secondary structure determinations

    Directory of Open Access Journals (Sweden)

    Lees Jonathan G

    2008-01-01

    Full Text Available Abstract Background A number of sequence-based methods exist for protein secondary structure prediction. Protein secondary structures can also be determined experimentally from circular dichroism, and infrared spectroscopic data using empirical analysis methods. It has been proposed that comparable accuracy can be obtained from sequence-based predictions as from these biophysical measurements. Here we have examined the secondary structure determination accuracies of sequence prediction methods with the empirically determined values from the spectroscopic data on datasets of proteins for which both crystal structures and spectroscopic data are available. Results In this study we show that the sequence prediction methods have accuracies nearly comparable to those of spectroscopic methods. However, we also demonstrate that combining the spectroscopic and sequences techniques produces significant overall improvements in secondary structure determinations. In addition, combining the extra information content available from synchrotron radiation circular dichroism data with sequence methods also shows improvements. Conclusion Combining sequence prediction with experimentally determined spectroscopic methods for protein secondary structure content significantly enhances the accuracy of the overall results obtained.

  6. Polyubiquitin chain assembly and organisation determine the dynamics of protein activation and degradation

    Directory of Open Access Journals (Sweden)

    Lan K. Nguyen

    2014-01-01

    Full Text Available Protein degradation via ubiquitination is a major proteolytic mechanism in cells. Once a protein is destined for degradation, it is tagged by multiple ubiquitin molecules. The synthesised polyubiquitin chains can be recognised by the 26S proteosome where proteins are degraded. These chains form through multiple ubiquitination cycles that are similar to multi-site phosphorylation cycles. As kinases and phosphatases, two opposing enzymes (E3 ligases and deubiquitinases DUBs catalyse (deubiquitination cycles. Although multi-ubiquitination cycles are fundamental mechanisms of controlling protein concentrations within a cell, their dynamics have never been explored. Here, we fill this knowledge gap. We show that under permissive physiological conditions, the formation of polyubiquitin chain of length greater than two and subsequent degradation of the ubiquitinated protein, which is balanced by protein synthesis, can display bistable, switch-like responses. Interestingly, the occurrence of bistability becomes pronounced, as the chain grows, giving rise to all-or-none regulation at the protein levels. We give predictions of protein distributions under bistable regime awaiting experimental verification. Importantly, we show for the first time that sustained oscillations can robustly arise in the process of formation of ubiquitin chain, largely due to the degradation of the target protein. This new feature is opposite to the properties of multi-site phosphorylation cycles, which are incapable of generating oscillation if the total abundance of interconverted protein forms is conserved. We derive structural and kinetic constraints for the emergence of oscillations, indicating that a competition between different substrate forms and the E3 and DUB is critical for oscillation. Our work provides the first detailed elucidation of the dynamical features brought about by different molecular setups of the polyubiquitin chain assembly process responsible for

  7. Electronic properties of the charge carriers on oligofluorene backbone

    International Nuclear Information System (INIS)

    Koizumi, Yoshiko; Seki, Shu; Saeki, Akinori; Tagawa, Seiichi

    2007-01-01

    The transient absorption of radical anions and cations of 9,9'-di-n-hexyl-oligofluorene was measured using pulse radiolysis and low-temperature γ-radiolysis techniques. The infrared absorption maxima of both radical anions and cations exhibit red-shift continuously upon elongation of the chain length. The absorption bands are blue-shifted by 0.04-0.07 eV with increasing the temperature from 80 to 106 K. The extinction coefficients were determined by scavenging technique, demonstrating an increase with the elongation of the chain length. The optimized geometry of fluorene trimers, calculated using density functional theory, shows that the oligofluorene molecules are more planar in its charged state than in its neutral state

  8. Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides

    Science.gov (United States)

    Chai, Wengang; Zhang, Yibing; Mauri, Laura; Ciampa, Maria G.; Mulloy, Barbara; Sonnino, Sandro; Feizi, Ten

    2018-05-01

    Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series.

  9. MFPred: Rapid and accurate prediction of protein-peptide recognition multispecificity using self-consistent mean field theory.

    Directory of Open Access Journals (Sweden)

    Aliza B Rubenstein

    2017-06-01

    Full Text Available Multispecificity-the ability of a single receptor protein molecule to interact with multiple substrates-is a hallmark of molecular recognition at protein-protein and protein-peptide interfaces, including enzyme-substrate complexes. The ability to perform structure-based prediction of multispecificity would aid in the identification of novel enzyme substrates, protein interaction partners, and enable design of novel enzymes targeted towards alternative substrates. The relatively slow speed of current biophysical, structure-based methods limits their use for prediction and, especially, design of multispecificity. Here, we develop a rapid, flexible-backbone self-consistent mean field theory-based technique, MFPred, for multispecificity modeling at protein-peptide interfaces. We benchmark our method by predicting experimentally determined peptide specificity profiles for a range of receptors: protease and kinase enzymes, and protein recognition modules including SH2, SH3, MHC Class I and PDZ domains. We observe robust recapitulation of known specificities for all receptor-peptide complexes, and comparison with other methods shows that MFPred results in equivalent or better prediction accuracy with a ~10-1000-fold decrease in computational expense. We find that modeling bound peptide backbone flexibility is key to the observed accuracy of the method. We used MFPred for predicting with high accuracy the impact of receptor-side mutations on experimentally determined multispecificity of a protease enzyme. Our approach should enable the design of a wide range of altered receptor proteins with programmed multispecificities.

  10. Quantum Chemical Benchmark Study on 46 RNA Backbone Families Using a Dinucleotide Unit

    Czech Academy of Sciences Publication Activity Database

    Kruse, H.; Mládek, Arnošt; Gkionis, Konstantinos; Hansen, A.; Grimme, S.; Šponer, Jiří

    2015-01-01

    Roč. 11, č. 10 (2015), s. 4972-4991 ISSN 1549-9618 R&D Projects: GA ČR(CZ) GBP305/12/G034 Institutional support: RVO:68081707 Keywords : MOLECULAR-DYNAMICS SIMULATIONS * DENSITY-FUNCTIONAL THEORY * SUGAR-PHOSPHATE BACKBONE Subject RIV: BO - Biophysics Impact factor: 5.301, year: 2015

  11. Comparing the Reliability of Regular Topologies on a Backbone Network. A Case Study

    DEFF Research Database (Denmark)

    Cecilio, Sergio Labeage; Gutierrez Lopez, Jose Manuel; Riaz, M. Tahir

    2009-01-01

    The aim of this paper is to compare the reliability of regular topologies on a backbone network. The study is focused on a large-scale fiberoptic network. Different regular topological solutions as single ring, double ring or 4-Regular grid are applied to the case study, and compared in terms...

  12. Directional virtual backbone based data aggregation scheme for Wireless Visual Sensor Networks.

    Science.gov (United States)

    Zhang, Jing; Liu, Shi-Jian; Tsai, Pei-Wei; Zou, Fu-Min; Ji, Xiao-Rong

    2018-01-01

    Data gathering is a fundamental task in Wireless Visual Sensor Networks (WVSNs). Features of directional antennas and the visual data make WVSNs more complex than the conventional Wireless Sensor Network (WSN). The virtual backbone is a technique, which is capable of constructing clusters. The version associating with the aggregation operation is also referred to as the virtual backbone tree. In most of the existing literature, the main focus is on the efficiency brought by the construction of clusters that the existing methods neglect local-balance problems in general. To fill up this gap, Directional Virtual Backbone based Data Aggregation Scheme (DVBDAS) for the WVSNs is proposed in this paper. In addition, a measurement called the energy consumption density is proposed for evaluating the adequacy of results in the cluster-based construction problems. Moreover, the directional virtual backbone construction scheme is proposed by considering the local-balanced factor. Furthermore, the associated network coding mechanism is utilized to construct DVBDAS. Finally, both the theoretical analysis of the proposed DVBDAS and the simulations are given for evaluating the performance. The experimental results prove that the proposed DVBDAS achieves higher performance in terms of both the energy preservation and the network lifetime extension than the existing methods.

  13. Treatment Results of Injuries of Thoracic and Lumbar Backbone Departments at Osteoporosis Patients

    Directory of Open Access Journals (Sweden)

    D.Y. Sumin

    2009-06-01

    Full Text Available Information relates to radiologic (computer tomography manifestations providing the visualization of thoracic and lumbar backbone department injuries at osteoporotic patients. Contemporary methods of transcutaneous and trans-pedicle vertebroplasty with bone cement allows to obtain a stable positive healing effect against such pathologies.

  14. SEVA Linkers: A Versatile and Automatable DNA Backbone Exchange Standard for Synthetic Biology

    DEFF Research Database (Denmark)

    Kim, Se Hyeuk; Cavaleiro, Mafalda; Rennig, Maja

    2016-01-01

    flexibility, and different researchers prefer and master different molecular technologies. Here, we describe a new, highly versatile and automatable standard “SEVA linkers” for vector exchange. SEVA linkers enable backbone swapping with 20 combinations of classical enzymatic restriction/ligation, Gibson...

  15. Integrative technology of massage manipulations in physical rehabilitation of students with backbone pathology

    Directory of Open Access Journals (Sweden)

    V.I. Kotelevskiy

    2016-06-01

    Full Text Available Purpose:to analyze effectiveness of massage manipulations’ integrative technology in physical rehabilitation of higher educational establishments’ students with backbone pathology. Material: in the research 195 students of 19-20 years’ age participated. All students had periodical initial neurological symptoms of functional pathology and first stage osteochondrosis in different parts of backbone. We conducted a course of 10 sessions of therapeutic massage. Results: the sense of massage integrative technology is that every specialist shall have certain optimal set of skills and knowledge in technique of manipulation sessions of massage. Integrative technology of massage manipulations consists of psycho-corrective and manipulation parts. It considers psycho-somatic, mechanical and reflex rehabilitation aspects of patho-genesis of backbone functional disorders and vertebral osteochondrosis. Conclusions: depending on pathological process or backbone functional state of every person (peculiarities of his (her psycho-somatic status or, even, his (her bents. Individual approach in choice of strategy, tactic and methodological provisioning of massage session shall be used.

  16. Influence of structures of polymer backbones on cooperative photoreorientation behavior of p-cyanoazobenzene side chains

    DEFF Research Database (Denmark)

    Han, Mina; Kidowaki, Masatoshi; Ichimura, Kunihiro

    2001-01-01

    Photoinduced orientational behavior of a polymethacrylate (CN6) and a polyester (p6a12) with p-cyanoazobenzene side chains was studied to reveal the structural effect of the liquid crystalline polymer backbones. Irradiation with linearly polarized W light resulted in the reorientation of the azob...

  17. HISTORY MAY BE THE BEST GUIDE FOR DETERMINING THE ATHLETE'S DIETARY PROTEIN NEEDS

    Directory of Open Access Journals (Sweden)

    Linda S. Lamont

    2009-03-01

    Full Text Available I was encouraged to read Dr. Longo and colleagues' Letter to the Editor entitled "The best athletes in ancient Rome were vegetarian!"(Longo et al., 2008. These writers ask that we rethink the issue of what is an optimal dietary protein content for athletes by considering the diets that sustained the ancient gladiators of Rome. Historical evidence shows that humans of ancient times performed at intense levels while consuming 78% of their diets' as plant protein (Kanz and Grosschmidt, 2007. This anthropological fact, and some recent laboratory evidence, argues against the need to increase the protein RDA for athletes from 0.8 g of protein per kilogram of body weight per day to 1.2 to 1.4 g per kilogram per day (ACSM, 2000. Our research group found that amino acids make a small contribution (2 - 3% of total to endurance energy needs (Lamont et al, 1999 and that athlete's have similar oxidation rates if corrections are made for oxygen consumption and fat-free body mass. Others report that a short-term training program of 38-days reduces amino acid use during exercise and down-regulates a critical enzyme in the oxidative pathway in order to spare this nutrient (McKenzie et al., 2000 Yet sport nutritionists and physiologists continue to recommend an increased protein RDA for this group. If one does a Google search using the words exercise and protein you would literally get millions of citations recommending the athlete to increase their protein intake. One reason for this continued recommendation, I believe, is that the laboratory procedure used to justify an increased protein RDA (field-based nitrogen balance measurements has many methodological shortcomings that are not recognized by the sports science community (Lamont, 2008. The problems with this technique are so great that its scientific fidelity has been questioned (Lamont, 2008. And as Longo and colleagues have highlighted (2008 the Institute of Medicine concluded that the evidence for

  18. The exploitation of ISFETs to determine the acid-base behaviour of proteins

    NARCIS (Netherlands)

    Olthuis, Wouter; Bergveld, Piet; Kruise, J.; Kruise, J.

    1998-01-01

    Investigation of the acid–base behaviour of proteins can result in information on both their composition and the concentration. Volumetric titration is the standard method to obtain this kind of data. In this paper four different ways of protein titration are presented, applied on membrane-covered

  19. Introduction to Biological Mass Spectroscopy: Determining Identity and Species of Origin of Two Proteins

    Science.gov (United States)

    Reimann, Curt T.; Mie, Axel; Nilsson, Carina; Cohen, Arieh

    2005-01-01

    An examination of the two proteins, namely, cytochrome c from horse and cow is conducted and it is indicated that cytochrome c is a mitochondrial protein. Mitochondria multiply by cell division and do not undergo sexual reproduction and mitochondria DNA is passed on via the mitochondria that are inherited from the female parent organism.

  20. Lactobacillus plantarum possesses the capability for wall teichoic acid backbone alditol switching

    Directory of Open Access Journals (Sweden)

    Bron Peter A

    2012-09-01

    Full Text Available Abstract Background Specific strains of Lactobacillus plantarum are marketed as health-promoting probiotics. The role and interplay of cell-wall compounds like wall- and lipo-teichoic acids (WTA and LTA in bacterial physiology and probiotic-host interactions remain obscure. L. plantarum WCFS1 harbors the genetic potential to switch WTA backbone alditol, providing an opportunity to study the impact of WTA backbone modifications in an isogenic background. Results Through genome mining and mutagenesis we constructed derivatives that synthesize alternative WTA variants. The mutants were shown to completely lack WTA, or produce WTA and LTA that lack D-Ala substitution, or ribitol-backbone WTA instead of the wild-type glycerol-containing backbone. DNA micro-array experiments established that the tarIJKL gene cluster is required for the biosynthesis of this alternative WTA backbone, and suggest ribose and arabinose are precursors thereof. Increased tarIJKL expression was not observed in any of our previously performed DNA microarray experiments, nor in qRT-PCR analyses of L. plantarum grown on various carbon sources, leaving the natural conditions leading to WTA backbone alditol switching, if any, to be identified. Human embryonic kidney NF-κB reporter cells expressing Toll like receptor (TLR-2/6 were exposed to purified WTAs and/or the TA mutants, indicating that WTA is not directly involved in TLR-2/6 signaling, but attenuates this signaling in a backbone independent manner, likely by affecting the release and exposure of immunomodulatory compounds such as LTA. Moreover, human dendritic cells did not secrete any cytokines when purified WTAs were applied, whereas they secreted drastically decreased levels of the pro-inflammatory cytokines IL-12p70 and TNF-α after stimulation with the WTA mutants as compared to the wild-type. Conclusions The study presented here correlates structural differences in WTA to their functional characteristics, thereby

  1. Determination of glutaredoxin enzyme activity and protein S-glutathionylation using fluorescent eosin-glutathione.

    Science.gov (United States)

    Coppo, Lucia; Montano, Sergio J; Padilla, Alicia C; Holmgren, Arne

    2016-04-15

    Glutaredoxins catalyze glutathione-dependent disulfide oxidoreductions, particularly reduction of glutathione (GSH)-protein mixed disulfides. Mammalian glutaredoxins are present in the cytosol/nucleus as Grx1 or in mitochondria as Grx2a. Here we describe di-eosin-glutathione disulfide (Di-E-GSSG) as a new tool to study glutaredoxin (Grx) activity. Di-E-GSSG has almost no fluorescence in its disulfide form due to self-quenching, whereas the reduced form (E-GSH) has a large fluorescence emission at 545 nm after excitation at 520 nm. Di-E-GSSG was a very poor substrate for glutathione reductase, but we discovered that the molecule was an excellent substrate for glutaredoxin in a coupled assay system with GSH, nicotinamide adenine dinucleotide phosphate (NADPH), and glutathione reductase or with lipoamide, NADH, and lipoamide dehydrogenase. In addition, Di-E-GSSG was used to glutathionylate the free SH group of bovine serum albumin (BSA), yielding eosin-glutathionylated BSA (E-GS-BSA) readily observed in ultraviolet (UV) light. E-GS-BSA also displayed a quenched fluorescence, and its Grx-catalyzed reduction could be followed by the formation of E-GSH by fluorescence emission using microtiter plates. This way of measuring Grx activity provided an ultrasensitive method that detected Grx1 and Grx2 at picomolar levels. Human Grx1 was readily quantified in 40 μl of plasma and determined to be 680 ± 208 pM in healthy controls. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Lysosomes and unfolded protein response, determinants of differential resistance of melanoma cells to vinca alkaloids.

    Science.gov (United States)

    Vincent, Laure-Anais; Attaoua, Chaker; Bellis, Michel; Rozkydalova, Lucie; Hadj-Kaddour, Kamel; Vian, Laurence; Cuq, Pierre

    2015-04-01

    On account of its strong ability to become chemoresistant after a primary response to drugs, malignant melanoma (MM) remains a therapeutic challenge. This study focuses on acquired resistance to vinca alkaloids (VAs) using VA-resistant MM cell lines (CAL1R-VCR, CAL1R-VDS, and CAL1R-VRB), established by long-term continuous exposure of parental CAL1-wt cells to vincristine (VCR), vindesine (VDS), or vinorelbine (VRB), respectively. Transcriptomic profiling using rma and rdam methods led to distinguish two cell groups: CAL1R-VCR and CAL1R-VDS, CAL1R-VRB, and CAL1-wt. mgsa of the specifically altered genes in the first group evidenced the GO terms 'lysosomal lumen' and 'vacuolar lumen' linked to underexpressed genes, and 'endoplasmic reticulum (ER) stress response' associated with overexpressed genes. A specific reduction of lysosomal enzymes, independent of acidic vacuole organelle (AVO) turnover, was observed (LTG probe) in CAL1R-VCR and CAL1R-VDS cells. It was associated with the specific lowering of cathepsin B and L, known to be involved in the lysosomal pathway of apoptosis. Confirming gene profiling, the same groups (CAL1R-VCR and CAL1R-VDS, CAL1-wt and CAL1R-VRB) could be distinguished regarding the VA-mediated changes on mean size areas and on acidic compartment volumes. These two parameters were reduced in CAL1R-VCR and CAL1R-VDS cells, suggesting a smaller AVO accumulation and thus a reduced sensitivity to lysosomal membrane permeabilization-mediated apoptosis. In addition, 'ER stress response' inhibition by tauroursodeoxycholic acid induced a higher VA sensitization of the first cell group. In conclusion, lysosomes and unfolded protein response could be key determinants of the differential resistance of MM to VAs. © 2015 Société Française de Pharmacologie et de Thérapeutique.

  3. Markers of protein oxidation

    DEFF Research Database (Denmark)

    Headlam, Henrietta A; Davies, Michael Jonathan

    2004-01-01

    Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed...... of this process depends on the extent of oxidation at C-3 compared with other sites. HO*, generated by gamma radiolysis, gave the highest total carbonyl yield, with protein-bound carbonyls predominating over released. In contrast, metal ion/H2O2 systems, gave more released than bound carbonyls, with this ratio...... modulated by EDTA. This is ascribed to metal ion-protein interactions affecting the sites of initial oxidation. Hypochlorous acid gave low concentrations of released carbonyls, but high yields of protein-bound material. The peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride...

  4. Determination of proteins and carbohydrates in the effluents from wastewater treatment bioreactors using resonance light-scattering method.

    Science.gov (United States)

    Zhang, Meng-Lin; Sheng, Guo-Ping; Yu, Han-Qing

    2008-07-01

    A simple and sensitive method was developed for the determination of low-concentration proteins and carbohydrates in the effluents from biological wastewater treatment reactors using resonance light-scattering (RLS) technique. Two ionic dyes, Congo red and Neutral red were, respectively used as an RLS probes for the determination of proteins and carbohydrates. This method is based on the interactions between biomacromolecules and dyes, which cause a substantial increase in the resonance scattering signal of dyes in the wavelength range of 200-650 nm. The characteristics of RLS spectra of the macromolecule-dye complexes, influencing factors, and optimum analytical conditions for the measurement were explored. The method was satisfactorily applied to the measurement of proteins and carbohydrates in the effluents from 10 aerobic or anaerobic bioreactors, and a high sensitivity were achieved.

  5. ESIprot: a universal tool for charge state determination and molecular weight calculation of proteins from electrospray ionization mass spectrometry data.

    Science.gov (United States)

    Winkler, Robert

    2010-02-01

    Electrospray ionization (ESI) ion trap mass spectrometers with relatively low resolution are frequently used for the analysis of natural products and peptides. Although ESI spectra of multiply charged protein molecules also can be measured on this type of devices, only average spectra are produced for the majority of naturally occurring proteins. Evaluating such ESI protein spectra would provide valuable information about the native state of investigated proteins. However, no suitable and freely available software could be found which allows the charge state determination and molecular weight calculation of single proteins from average ESI-MS data. Therefore, an algorithm based on standard deviation optimization (scatter minimization) was implemented for the analysis of protein ESI-MS data. The resulting software ESIprot was tested with ESI-MS data of six intact reference proteins between 12.4 and 66.7 kDa. In all cases, the correct charge states could be determined. The obtained absolute mass errors were in a range between -0.2 and 1.2 Da, the relative errors below 30 ppm. The possible mass accuracy allows for valid conclusions about the actual condition of proteins. Moreover, the ESIprot algorithm demonstrates an extraordinary robustness and allows spectral interpretation from as little as two peaks, given sufficient quality of the provided m/z data, without the necessity for peak intensity data. ESIprot is independent from the raw data format and the computer platform, making it a versatile tool for mass spectrometrists. The program code was released under the open-source GPLv3 license to support future developments of mass spectrometry software. Copyright 2010 John Wiley & Sons, Ltd.

  6. Rapid and accurate processing method for amide proton exchange rate measurement in proteins

    International Nuclear Information System (INIS)

    Koskela, Harri; Heikkinen, Outi; Kilpelaeinen, Ilkka; Heikkinen, Sami

    2007-01-01

    Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing

  7. Determination of the Influence of Substrate Concentration on Enzyme Selectivity Using Whey Protein Isolate and Bacillus licheniformis Protease

    NARCIS (Netherlands)

    Butré, C.I.; Sforza, S.; Gruppen, H.; Wierenga, P.A.

    2014-01-01

    Increasing substrate concentration during enzymatic protein hydrolysis results in a decrease in hydrolysis rate. To test if changes in the mechanism of hydrolysis also occur, the enzyme selectivity was determined. The selectivity is defined quantitatively as the relative rate of hydrolysis of each

  8. Comparison of protein fermentation characteristics in rumen fluid determined with the gas production technique and the nylon bag technique

    NARCIS (Netherlands)

    Cone, J.W.; Rodrigues, M.A.M.; Guedes, C.M.; Blok, M.C.

    2009-01-01

    In this study, a modified version of the gas production technique was used to determine protein fermentation characteristics in rumen fluid of 19 feedstuffs. Performing the incubations in a N-free environment, and with an excess of rapidly fermentable carbohydrates, made N the limiting factor to

  9. Experimental determination of net protein charge and A(tot) and K(a) of nonvolatile buffers in canine plasma.

    Science.gov (United States)

    Constable, Peter D; Stämpfli, Henry R

    2005-01-01

    Acid-base abnormalities frequently are present in sick dogs. The mechanism for an acid-base disturbance can be determined with the simplified strong ion approach, which requires accurate values for the total concentration of plasma nonvolatile buffers (A(tot)) and the effective dissociation constant for plasma weak acids (K(a)). The aims of this study were to experimentally determine A(tot) and K(a) values for canine plasma. Plasma was harvested from 10 healthy dogs; the concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, L-lactate) and nonvolatile buffer ions (total protein, albumin, phosphate) were determined; and the plasma was tonometered with CO2 at 37 degrees C. Strong ion difference (SID) was calculated from the measured strong ion concentrations, and nonlinear regression was used to estimate values for A(tot) and K(a), which were validated with data from an in vitro and in vivo study. Mean (+/- SD) values for canine plasma were A(tot) = (17.4 +/- 8.6) mM (equivalent to 0.273 mmol/g of total protein or 0.469 mmol/g of albumin); K(a) = (0.17 +/- 0.11) x 10(-7); pK(a) = 7.77. The calculated SID for normal canine plasma (pH = 7.40; P(CO2) = 37 mm Hg; [total protein] = 64 g/L) was 27 mEq/L. The net protein charge for normal canine plasma was 0.25 mEq/g of total protein or 0.42 mEq/g of albumin. Application of the experimentally determined values for A(tot), K(a), and net protein charge should improve understanding of the mechanism for complex acid-base disturbances in dogs.

  10. Methodical investigation of the protein metabolism and of the bioenergetics of protein retention in growing animals. 1. Determination of parameters of growth and protein retention of chickens after long-term labelling with /sup 15/NH/sub 4/ acetate

    Energy Technology Data Exchange (ETDEWEB)

    Schiemann, R.; Bock, H.D.; Keller, J.; Hoffmann, L.; Krawielitzki, K.; Klein, M. (Akademie der Landwirtschaftswissenschaften der DDR, Dummerstorf-Rostock. Forschungszentrum fuer Tierproduktion)

    1983-01-01

    The influence of different protein levels in the feed (group R1 20%, R2 38% crude protein) and of different energy levels (group J1 low, J2 high energy level) on the composition of the carcass and the apparent half-life periods of the body proteins were determined in 4 groups of 15 male broiler chickens labelled with /sup 15/NH/sub 4/ acetate. In all slaughtering phases the higher protein level resulted in a higher weight of the feathers, breast and leg muscles, higher amounts of N in all parts of the body and a higher percentage of feathers, breast and leg muscles of the total carcass than the lower protein level. Between 13 and 19% of the N in the carcass contributed to the feathers, 24-31% to the breast and leg muscles and 50-63% to the rest of the carcass. The relative quotas of the sum of breast and leg muscles in the carcass were higher for the low energy level than for the high energy level. There were no remarkable differences as to the protein content of the muscles in dependence on the energy level, the quota of sarcoplasmatic proteins, however, was higher on the high level in contrast to the low energy level, that of the myofibrillar proteins was lower. The apparent half-life period of the total body protein after normal protein supply was 22 days (group R1) and 14 after high protein supply. The energy levels in groups J1 and J2 had no significant influence on the half-life period of the total body protein. In the body fractions examined the apparent half-life periods were highest in the breast muscle and lowest in the rest of the carcass. The protein stored in the feathers did not undergo decomposition. The protein fractions 'sarcoplasmatic protein' and 'myofibrillar protein' of breast and leg muscle neither differed from one another nor from the respective total muscle fractions as regards their half-life period.

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

    Science.gov (United States)

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

    2017-05-01

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

  12. Relationship between recombinant protein expression and host metabolome as determined by two-dimensional NMR spectroscopy.

    Directory of Open Access Journals (Sweden)

    Young Kee Chae

    Full Text Available Escherichia coli has been the most widely used host to produce large amounts of heterologous proteins. However, given an input plasmid DNA, E. coli may produce soluble protein, produce only inclusion bodies, or yield little or no protein at all. Many efforts have been made to surmount these problems, but most of them have involved time-consuming and labor-intensive trial-and-error. We hypothesized that different metabolomic fingerprints might be associated with different protein production outcomes. If so, then it might be possible to change the expression pattern by manipulating the metabolite environment. As a first step in testing this hypothesis, we probed a subset of the intracellular metabolites by partially labeling it with 13C-glucose. We tested 71 genes and identified 17 metabolites by employing the two-dimensional NMR spectroscopy. The statistical analysis showed that there existed the metabolite compositions favoring protein production. We hope that this work would help devise a systematic and predictive approach to the recombinant protein production.

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

    Science.gov (United States)

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

    2018-02-05

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

  14. Investigation of the utility of selective methyl protonation for determination of membrane protein structures

    International Nuclear Information System (INIS)

    Shih, Steve C. C.; Stoica, Ileana; Goto, Natalie K.

    2008-01-01

    Polytopic α-helical membrane proteins present one of the final frontiers for protein structural biology, with significant challenges causing severe under-representation in the protein structure databank. However, with the advent of hardware and methodology geared to the study of large molecular weight complexes, solution NMR is being increasingly considered as a tool for structural studies of these types of membrane proteins. One method that has the potential to facilitate these studies utilizes uniformly deuterated samples with protons reintroduced at one or two methyl groups of leucine, valine and isoleucine. In this work we demonstrate that in spite of the increased proportion of these amino acids in membrane proteins, the quality of structures that can be obtained from this strategy is similar to that obtained for all α-helical water soluble proteins. This is partly attributed to the observation that NOEs between residues within the transmembrane helix did not have an impact on structure quality. Instead the most important factors controlling structure accuracy were the strength of dihedral angle restraints imposed and the number of unique inter-helical pairs of residues constrained by NOEs. Overall these results suggest that the most accurate structures will arise from accurate identification of helical segments and utilization of inter-helical distance restraints from various sources to maximize the distribution of long-range restraints

  15. Virus-producing cells determine the host protein profiles of HIV-1 virion cores

    Science.gov (United States)

    2012-01-01

    Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of

  16. Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

    International Nuclear Information System (INIS)

    Lehto, Markku; Hynynen, Riikka; Karjalainen, Katja; Kuismanen, Esa; Hyvaerinen, Kati; Olkkonen, Vesa M.

    2005-01-01

    The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P 2 and PI(3,4,5)P 3 . A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein

  17. Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.

    Energy Technology Data Exchange (ETDEWEB)

    Haskins, William E.; Leavell, Michael D.; Lane, Pamela; Jacobsen, Richard B.; Hong, Joohee; Ayson, Marites J.; Wood, Nichole L.; Schoeniger, Joseph S.; Kruppa, Gary Hermann; Sale, Kenneth L.; Young, Malin M.; Novak, Petr

    2005-03-01

    Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.

  18. Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120

    DEFF Research Database (Denmark)

    Prihandoko, Rudi; Alvarez-Curto, Elisa; Hudson, Brian D

    2016-01-01

    of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment...... activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode...

  19. Principles and determinants of G-protein coupling by the rhodopsin-like thyrotropin receptor.

    Directory of Open Access Journals (Sweden)

    Gunnar Kleinau

    Full Text Available In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR and different subtypes of G-proteins. The thyrotropin receptor (TSHR binds G-proteins promiscuously and activates both Gs (cAMP and Gq (IP. Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop (ICL 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits G alpha as well as G betagamma. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443 and helix 8 (R687 that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only. Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein

  20. Principles and determinants of G-protein coupling by the rhodopsin-like thyrotropin receptor.

    Science.gov (United States)

    Kleinau, Gunnar; Jaeschke, Holger; Worth, Catherine L; Mueller, Sandra; Gonzalez, Jorge; Paschke, Ralf; Krause, Gerd

    2010-03-18

    In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR) and different subtypes of G-proteins. The thyrotropin receptor (TSHR) binds G-proteins promiscuously and activates both Gs (cAMP) and Gq (IP). Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop (ICL) 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits G alpha as well as G betagamma. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443) and helix 8 (R687) that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only. Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein subtypes.

  1. Ner protein of phage Mu: Assignments using {sup 13}C/{sup 15}N-labeled protein

    Energy Technology Data Exchange (ETDEWEB)

    Strzelecka, T.; Gronenborn, A.M.; Clore, G.M. [National Institutes of Health, Bethesda, MD (United States)

    1994-12-01

    The Ner protein is a small (74-amino acid) DNA-binding protein that regulates a switch between the lysogenic and lytic stages of phage Mu. It inhibits expression of the C repressor gene and down-regulates its own expression. Two-dimensional NMR experiments on uniformly {sup 15}N-labeled protein provided most of the backbone and some of the sidechain proton assignments. The secondary structure determination using two-dimensional NOESY experiments showed that Ner consists of five {alpha}-helices. However, because most of the sidechain protons could not be assigned, the full structure was not determined. Using uniformly {sup 13}C/{sup 15}N-labeled Ner and a set of three-dimensional experiments, we were able to assign all of the backbone and 98% of the sidechain protons. In particular, the CBCANH and CBCA(CO)NH experiments were used to sequentially assign the C{alpha} and C{beta} resonances; the HCCH-CTOCSY and HCCH-COSY were used to assign sidechain carbon and proton resonances.

  2. Ion Binding Energies Determining Functional Transport of ClC Proteins

    Science.gov (United States)

    Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping

    2014-06-01

    The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.

  3. Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

    Science.gov (United States)

    Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

    2012-01-01

    The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

  4. Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants

    DEFF Research Database (Denmark)

    Petersen, Steen Vang; Andersen, Kell kleiner; Enghild, Jan J.

    2009-01-01

    Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and catio......Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS......) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant-robust broad-specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS-PAGE and N-terminal sequencing. We observe well-defined cleavage fragments, which suggest......, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded...

  5. 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...... fibroblasts (MEFs) and white preadipocytes by expression of simian virus 40 large T antigen results in the expression of the brown fat-specific uncoupling protein 1 (UCP-1) in the adipose state. Retinoblastoma gene-deficient (Rb-/-) MEFs and stem cells, but not the corresponding wild-type cells, differentiate...

  6. Ca-C backbone fragmentation dominates in electron detachment dissociation of gas-phase polypeptide polyanions

    DEFF Research Database (Denmark)

    Kjeldsen, Frank; Silivra, Oleg A; Ivonin, Igor A

    2005-01-01

    the dissociation of oxidized radical anions [M-nH]((n-1)-*. We demonstrate that C(alpha)-C cleavages, which are otherwise rarely observed in tandem mass spectrometry, can account for most of the backbone fragmentation, with even-electron x fragments dominating over radical a* ions. Ab initio calculations at the B3...... LYP level of theory with the 6-311+G(2 p,2 d)//6-31+G(d,p) basis set suggested a unidirectional mechanism for EDD (cleavage always N-terminal to the radical site), with a*, x formation being favored over a, x* fragmentation by 74.2 kJ mol(-1). Thus, backbone C(alpha)-C bonds N-terminal to proline...

  7. Effect of backbone structure on charge transport along isolated conjugated polymer chains

    International Nuclear Information System (INIS)

    Siebbeles, Laurens D.A.; Grozema, Ferdinand C.; Haas, Matthijs P. de; Warman, John M.

    2005-01-01

    Fast charge transport in conjugated polymers is essential for their application in opto-electronic devices. In the present paper, measurements and theoretical modeling of the mobility of excess charges along isolated chains of conjugated polymers in dilute solution are presented. Charge carriers were produced by irradiation of the polymer solution with 3-MeV electrons from a Van de Graaff accelerator. The mobilities of the charges along the polymer chains were obtained from time-resolved microwave conductivity measurements. The mobilities are strongly dependent on the chemical nature of the polymer backbone. Comparison of the experimental data with results from ab initio quantum mechanical calculations shows that the measured mobilities are strongly limited by torsional disorder, chemical defects and chain ends. Improvement of the structure of polymer backbones is therefore expected to significantly enhance the performance of these materials in 'plastic electronics'

  8. Smart-Grid Backbone Network Real-Time Delay Reduction via Integer Programming.

    Science.gov (United States)

    Pagadrai, Sasikanth; Yilmaz, Muhittin; Valluri, Pratyush

    2016-08-01

    This research investigates an optimal delay-based virtual topology design using integer linear programming (ILP), which is applied to the current backbone networks such as smart-grid real-time communication systems. A network traffic matrix is applied and the corresponding virtual topology problem is solved using the ILP formulations that include a network delay-dependent objective function and lightpath routing, wavelength assignment, wavelength continuity, flow routing, and traffic loss constraints. The proposed optimization approach provides an efficient deterministic integration of intelligent sensing and decision making, and network learning features for superior smart grid operations by adaptively responding the time-varying network traffic data as well as operational constraints to maintain optimal virtual topologies. A representative optical backbone network has been utilized to demonstrate the proposed optimization framework whose simulation results indicate that superior smart-grid network performance can be achieved using commercial networks and integer programming.

  9. Determination of structural fluctuations of proteins from structure-based calculations of residual dipolar couplings