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Sample records for native protein structure

  1. Solid state NMR strategy for characterizing native membrane protein structures.

    PubMed

    Murray, Dylan T; Das, Nabanita; Cross, Timothy A

    2013-09-17

    Unlike water soluble proteins, the structures of helical transmembrane proteins depend on a very complex environment. These proteins sit in the midst of dramatic electrical and chemical gradients and are often subject to variations in the lateral pressure profile, order parameters, dielectric constant, and other properties. Solid state NMR is a collection of tools that can characterize high resolution membrane protein structure in this environment. Indeed, prior work has shown that this complex environment significantly influences transmembrane protein structure. Therefore, it is important to characterize such structures under conditions that closely resemble its native environment. Researchers have used two approaches to gain protein structural restraints via solid state NMR spectroscopy. The more traditional approach uses magic angle sample spinning to generate isotropic chemical shifts, much like solution NMR. As with solution NMR, researchers can analyze the backbone chemical shifts to obtain torsional restraints. They can also examine nuclear spin interactions between nearby atoms to obtain distances between atomic sites. Unfortunately, for membrane proteins in lipid preparations, the spectral resolution is not adequate to obtain complete resonance assignments. Researchers have developed another approach for gaining structural restraints from membrane proteins: the use of uniformly oriented lipid bilayers, which provides a method for obtaining high resolution orientational restraints. When the bilayers are aligned with respect to the magnetic field of the NMR spectrometer, researchers can obtain orientational restraints in which atomic sites in the protein are restrained relative to the alignment axis. However, this approach does not allow researchers to determine the relative packing between helices. By combining the two approaches, we can take advantage of the information acquired from each technique to minimize the challenges and maximize the quality of the

  2. Structure of the native Sec61 protein-conducting channel

    PubMed Central

    Pfeffer, Stefan; Burbaum, Laura; Unverdorben, Pia; Pech, Markus; Chen, Yuxiang; Zimmermann, Richard; Beckmann, Roland; Förster, Friedrich

    2015-01-01

    In mammalian cells, secretory and membrane proteins are translocated across or inserted into the endoplasmic reticulum (ER) membrane by the universally conserved protein-conducting channel Sec61, which has been structurally studied in isolated, detergent-solubilized states. Here we structurally and functionally characterize native, non-solubilized ribosome-Sec61 complexes on rough ER vesicles using cryo-electron tomography and ribosome profiling. Surprisingly, the 9-Å resolution subtomogram average reveals Sec61 in a laterally open conformation, even though the channel is not in the process of inserting membrane proteins into the lipid bilayer. In contrast to recent mechanistic models for polypeptide translocation and insertion, our results indicate that the laterally open conformation of Sec61 is the only conformation present in the ribosome-bound translocon complex, independent of its functional state. Consistent with earlier functional studies, our structure suggests that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61 in a lipid environment. PMID:26411746

  3. Capturing native/native like structures with a physico-chemical metric (pcSM) in protein folding.

    PubMed

    Mishra, Avinash; Rao, Satyanarayan; Mittal, Aditya; Jayaram, B

    2013-08-01

    Specification of the three dimensional structure of a protein from its amino acid sequence, also called a "Grand Challenge" problem, has eluded a solution for over six decades. A modestly successful strategy has evolved over the last couple of decades based on development of scoring functions (e.g. mimicking free energy) that can capture native or native-like structures from an ensemble of decoys generated as plausible candidates for the native structure. A scoring function must be fast enough in discriminating the native from unfolded/misfolded structures, and requires validation on a large data set(s) to generate sufficient confidence in the score. Here we develop a scoring function called pcSM that detects true native structure in the top 5 with 93% accuracy from an ensemble of candidate structures. If we eliminate the native from ensemble of decoys then pcSM is able to capture near native structure (RMSD<=5Ǻ) in top 10 with 86% accuracy. The parameters considered in pcSM are a C-alpha Euclidean metric, secondary structural propensity, surface areas and an intramolecular energy function. pcSM has been tested on 415 systems consisting 142,698 decoys (public and CASP-largest reported hitherto in literature). The average rank for the native is 2.38, a significant improvement over that existing in literature. In-silico protein structure prediction requires robust scoring technique(s). Therefore, pcSM is easily amenable to integration into a successful protein structure prediction strategy. The tool is freely available at http://www.scfbio-iitd.res.in/software/pcsm.jsp. PMID:23665455

  4. Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures

    NASA Astrophysics Data System (ADS)

    Lammert, Heiko; Noel, Jeffrey K.; Haglund, Ellinor; Schug, Alexander; Onuchic, José N.

    2015-12-01

    The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein's functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism.

  5. Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures.

    PubMed

    Lammert, Heiko; Noel, Jeffrey K; Haglund, Ellinor; Schug, Alexander; Onuchic, José N

    2015-12-28

    The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein's functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism. PMID:26723626

  6. Constructing a folding model for protein S6 guided by native fluctuations deduced from NMR structures

    SciTech Connect

    Lammert, Heiko; Noel, Jeffrey K.; Haglund, Ellinor; Onuchic, José N.; Schug, Alexander

    2015-12-28

    The diversity in a set of protein nuclear magnetic resonance (NMR) structures provides an estimate of native state fluctuations that can be used to refine and enrich structure-based protein models (SBMs). Dynamics are an essential part of a protein’s functional native state. The dynamics in the native state are controlled by the same funneled energy landscape that guides the entire folding process. SBMs apply the principle of minimal frustration, drawn from energy landscape theory, to construct a funneled folding landscape for a given protein using only information from the native structure. On an energy landscape smoothed by evolution towards minimal frustration, geometrical constraints, imposed by the native structure, control the folding mechanism and shape the native dynamics revealed by the model. Native-state fluctuations can alternatively be estimated directly from the diversity in the set of NMR structures for a protein. Based on this information, we identify a highly flexible loop in the ribosomal protein S6 and modify the contact map in a SBM to accommodate the inferred dynamics. By taking into account the probable native state dynamics, the experimental transition state is recovered in the model, and the correct order of folding events is restored. Our study highlights how the shared energy landscape connects folding and function by showing that a better description of the native basin improves the prediction of the folding mechanism.

  7. Automated search of natively folded protein fragments for high-throughput structure determination in structural genomics.

    PubMed Central

    Kuroda, Y.; Tani, K.; Matsuo, Y.; Yokoyama, S.

    2000-01-01

    Structural genomic projects envision almost routine protein structure determinations, which are currently imaginable only for small proteins with molecular weights below 25,000 Da. For larger proteins, structural insight can be obtained by breaking them into small segments of amino acid sequences that can fold into native structures, even when isolated from the rest of the protein. Such segments are autonomously folding units (AFU) and have sizes suitable for fast structural analyses. Here, we propose to expand an intuitive procedure often employed for identifying biologically important domains to an automatic method for detecting putative folded protein fragments. The procedure is based on the recognition that large proteins can be regarded as a combination of independent domains conserved among diverse organisms. We thus have developed a program that reorganizes the output of BLAST searches and detects regions with a large number of similar sequences. To automate the detection process, it is reduced to a simple geometrical problem of recognizing rectangular shaped elevations in a graph that plots the number of similar sequences at each residue of a query sequence. We used our program to quantitatively corroborate the premise that segments with conserved sequences correspond to domains that fold into native structures. We applied our program to a test data set composed of 99 amino acid sequences containing 150 segments with structures listed in the Protein Data Bank, and thus known to fold into native structures. Overall, the fragments identified by our program have an almost 50% probability of forming a native structure, and comparable results are observed with sequences containing domain linkers classified in SCOP. Furthermore, we verified that our program identifies AFU in libraries from various organisms, and we found a significant number of AFU candidates for structural analysis, covering an estimated 5 to 20% of the genomic databases. Altogether, these

  8. Macro-to-Micro Structural Proteomics: Native Source Proteins for High-Throughput Crystallization

    PubMed Central

    Nanao, Max; Gee, Christine L.; Moskaleva, Alisa; Gradia, Scott; Iavarone, Anthony T.; Berger, James M.; May, Andrew P.; Zubieta, Chloe; Alber, Tom

    2012-01-01

    Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography. PMID:22393408

  9. Predicting Peptide Structures in Native Proteins from Physical Simulations of Fragments

    PubMed Central

    Voelz, Vincent A.; Shell, M. Scott; Dill, Ken A.

    2009-01-01

    It has long been proposed that much of the information encoding how a protein folds is contained locally in the peptide chain. Here we present a large-scale simulation study designed to examine the extent to which conformations of peptide fragments in water predict native conformations in proteins. We perform replica exchange molecular dynamics (REMD) simulations of 872 8-mer, 12-mer, and 16-mer peptide fragments from 13 proteins using the AMBER 96 force field and the OBC implicit solvent model. To analyze the simulations, we compute various contact-based metrics, such as contact probability, and then apply Bayesian classifier methods to infer which metastable contacts are likely to be native vs. non-native. We find that a simple measure, the observed contact probability, is largely more predictive of a peptide's native structure in the protein than combinations of metrics or multi-body components. Our best classification model is a logistic regression model that can achieve up to 63% correct classifications for 8-mers, 71% for 12-mers, and 76% for 16-mers. We validate these results on fragments of a protein outside our training set. We conclude that local structure provides information to solve some but not all of the conformational search problem. These results help improve our understanding of folding mechanisms, and have implications for improving physics-based conformational sampling and structure prediction using all-atom molecular simulations. PMID:19197352

  10. Structural aspects of a protein-surfactant assembly: native and reduced States of human serum albumin.

    PubMed

    Anand, Uttam; Ray, Sutapa; Ghosh, Subhadip; Banerjee, Rajat; Mukherjee, Saptarshi

    2015-04-01

    The inherently present seventeen disulfide bonds of the circulatory protein, human serum albumin (HSA) provide the necessary structural stability. Various spectroscopic approaches were used to investigate the effect of reduction of these disulfide bonds and its binding with the anionic surfactant, sodium dodecyl sulfate (SDS). Based on several spectroscopic analyses, our investigations highlight the following interesting aspects: (1) HSA on reduction loses not only its tertiary structure but also a significant amount of secondary structure as well. However, the reduced state of the protein is not like the molten-globule, (2) this structural loss of the protein due to reduction is more prominent than that caused by higher SDS concentrations alone and can certainly be attributed to the role of disulfide bonds, (3) lower surfactant concentrations provide marginal structural rigidity to the native state of the protein, whereas, higher concentrations of SDS induces secondary structure to the reduced state of HSA, (4) the binding of SDS with both the native and reduced states of HSA, occurred in three distinct stages which was followed by a saturation stage. However, the nature of such binding is different for both the states as investigated by using the Stern-Volmer equations and estimating the thermodynamic parameters. Besides, in contrast to the native state, the reduced state of HSA shows that the lone tryptophan residue gets more buried. However, there occurs a sudden decrement in the lifetime of the tryptophan and the hydrodynamic diameter increases by twofold. PMID:25821118

  11. The unfolded state of the C-terminal domain of the ribosomal protein L9 contains both native and non-native structure.

    PubMed

    Shan, Bing; Eliezer, David; Raleigh, Daniel P

    2009-06-01

    Interest in the structural and dynamic properties of unfolded proteins has increased in recent years owing to continued interest in protein folding and misfolding. Knowledge of the unfolded state under native conditions is particularly important for obtaining a complete picture of the protein folding process. The C-terminal domain of protein L9 is a globular alpha, beta protein with an unusual mixed parallel and antiparallel beta-strand structure. The folding kinetics and equilibrium unfolding of CTL9 strongly depend on pH, and follow a simple two state model. Both the native and the unfolded state can be significantly populated at pH 3.8 in the absence of denaturant, allowing the native state and the unfolded state to be characterized under identical conditions. Backbone (15)N, (13)C, (1)H and side-chain (13)C(beta), (1)H(beta) chemical shifts, amide proton NOEs, and (15)N R(2) relaxation rates were obtained for the two conformational states at pH 3.8. All the data indicate that the pH 3.8 native state is well folded and is similar to the native state at neutral pH. There is significant residual structure in the pH 3.8 unfolded state. The regions corresponding to the two native state alpha-helices show strong preference to populate helical phi and psi angles. The segment that connects alpha-helix 2 and beta-strand 2 has a significant tendency to form non-native alpha-helical structure. Comparison with the pH 2.0 unfolded state and the urea unfolded state indicates that the tendency to adopt both native and non-native helical structure is stronger at pH 3.8, demonstrating that the unfolded state of CTL9 under native-like conditions is more structured. The implications for the folding of CTL9 are discussed. PMID:19301913

  12. Amyloidogenesis of Natively Unfolded Proteins

    PubMed Central

    Uversky, Vladimir N.

    2009-01-01

    Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation, which shares many structural properties with the pre-molten globule state, a partially folded intermediate first found during the equilibrium and kinetic (un)folding studies of several globular proteins and later described as one of the structural forms of natively unfolded proteins. The flexibility of this structural form is essential for the conformational rearrangements driving the formation of the core cross-beta structure of the amyloid fibril. Obviously, molecular mechanisms describing amyloidogenesis of ordered and natively unfolded proteins are different. For ordered protein to fibrillate, its unique and rigid structure has to be destabilized and partially unfolded. On the other hand, fibrillogenesis of a natively unfolded protein involves the formation of partially folded conformation; i.e., partial folding rather than unfolding. In this review recent findings are surveyed to illustrate some unique features of the natively unfolded proteins amyloidogenesis. PMID:18537543

  13. Protein denaturation in vacuo: intrinsic unfolding pathways associated with the native tertiary structure of lysozyme

    NASA Astrophysics Data System (ADS)

    Arteca, Gustavo A.; Tapia, O.

    Using computer-simulated molecular dynamics, we study the effect of sequence mutation on the unfolding mechanism of a native fold. The system considered is the native fold of hen egg-white lysozyme, exposed to centrifugal unfolding in vacuo. This unfolding bias elicits configurational transitions that imitate the behaviour of anhydrous proteins diffusing after electrospraying from neutral-pH solutions. By changing the sequences threaded onto the native fold of lysozyme, we probe the role of disulfide bridges and the effect of a global mutation. We find that the initial denaturing steps share common characteristics for the tested sequences. Recurrent features are: (i) the presence of dumbbell conformers with significant residual secondary structure, (ii) the ubiquitous formation of hairpins and two-stranded β-sheets regardless of disulfide bridges, and (iii) an unfolding pattern where the reduction in folding complexity is highly correlated with the decrease in chain compactness. These findings appear to be intrinsic to the shape of the native fold, suggesting that similar unfolding pathways may be accessible to many protein sequences.

  14. Fluorinated amino acids: compatibility with native protein structures and effects on protein-protein interactions.

    PubMed

    Salwiczek, Mario; Nyakatura, Elisabeth K; Gerling, Ulla I M; Ye, Shijie; Koksch, Beate

    2012-03-21

    Fluorinated analogues of the canonical α-L-amino acids have gained widespread attention as building blocks that may endow peptides and proteins with advantageous biophysical, chemical and biological properties. This critical review covers the literature dealing with investigations of peptides and proteins containing fluorinated analogues of the canonical amino acids published over the course of the past decade including the late nineties. It focuses on side-chain fluorinated amino acids, the carbon backbone of which is identical to their natural analogues. Each class of amino acids--aliphatic, aromatic, charged and polar as well as proline--is presented in a separate section. General effects of fluorine on essential properties such as hydrophobicity, acidity/basicity and conformation of the specific side chains and the impact of these altered properties on stability, folding kinetics and activity of peptides and proteins are discussed (245 references). PMID:22130572

  15. Improvement of Structure-Based Potentials for Protein Folding by Native and Nonnative Hydrogen Bonds

    PubMed Central

    Enciso, Marta; Rey, Antonio

    2011-01-01

    Pure Gō models (where every native interaction equally stabilizes the folded state) have widely proved their convenience in the computational investigation of protein folding. However, a chemistry-based description of the real interactions also provides a desirable tune in the analysis of the folding process, and thus some hybrid Gō potentials that combine both aspects have been proposed. Among all the noncovalent interactions that contribute to protein folding, hydrogen bonds are the only ones with a partial covalent character. This feature makes them directional and, thus, more difficult to model as part of the coarse-grained descriptions that are typically employed in Gō models. Thanks to a simplified but rigorous representation of backbone hydrogen bonds that we have recently proposed, we present in this article a combined potential (Gō + backbone hydrogen bond) to study the thermodynamics of protein folding in the frame of very simple simulation models. We show that the explicit inclusion of hydrogen bonds leads to a systematic improvement in the description of protein folding. We discuss a representative set of examples (from two-state folders to downhill proteins, with different types of native structures) that reveal a relevant agreement with experimental data. PMID:21943429

  16. Structural and Thermodynamical Insights into the Recognition of Native Proteins by Anti-Peptide Antibodies

    PubMed Central

    Armstrong, A. A.; Hildreth, James E. K.; Amzel, L. M.

    2013-01-01

    The mechanism by which antibodies elicited against protein-derived peptides achieve cross-reactivity with their cognate proteins remains unknown. To address this question we have carried out the complete thermodynamic characterization of the association of a monoclonal antibody (260.33.12) raised against a peptide (SNpep) derived from staphylococcal nuclease (SNase) with both eliciting peptide and cognate protein. Although both ligands bind with similar affinity (Kd = 0.42 µM and 0.30 µM for protein and peptide, respectively), protein and peptide binding have highly different thermodynamic signatures: peptide binding is characterized by a large enthalpic contribution (ΔH = −7.7 kcal/mol) whereas protein binding is dominated by a large entropic contribution (-TΔS = −7.2 kcal/mol). The structure of the SNpep:Fab complex, determined by x-ray diffraction, reveals that the bound conformation of the peptide differs from the conformation of the corresponding loop region in crystal structures of free SNase. The energy difference, estimated by molecular dynamics simulations between native SNase and a model in which the Ω-loop is built in the conformation of the Fab-bound peptide, shows that the energetic cost of adopting this conformation is compatible with the enthalpic cost of binding the protein vis-à-vis the peptide. These results are compatible with a mechanism by which the anti-peptide antibody recognizes the cognate protein: high affinity is maintained upon binding a nonnative conformation by offsetting enthalpic penalties with reduced entropic losses. These findings provide potentially useful guidelines for the identification of linear epitopes within protein sequences that are well suited for the development of synthetic peptide vaccines. PMID:23473830

  17. The cold denatured state of the C-terminal domain of protein L9 is compact and contains both native and non-native structure.

    PubMed

    Shan, Bing; McClendon, Sebastian; Rospigliosi, Carla; Eliezer, David; Raleigh, Daniel P

    2010-04-01

    Cold denaturation is a general property of globular proteins, and the process provides insight into the origins of the cooperativity of protein folding and the nature of partially folded states. Unfortunately, studies of protein cold denaturation have been hindered by the fact that the cold denatured state is normally difficult to access experimentally. Special conditions such as addition of high concentrations of denaturant, encapsulation into reverse micelles, the formation of emulsified solutions, high pressure, or extremes of pH have been applied, but these can perturb the unfolded state of proteins. The cold denatured state of the C-terminal domain of the ribosomal protein L9 can be populated under native-like conditions by taking advantage of a destabilizing point mutation which leads to cold denaturation at temperatures above 0 degrees C. This state is in slow exchange with the native state on the NMR time scale. Virtually complete backbone (15)N, (13)C, and (1)H as well as side-chain (13)C(beta) and (1)H(beta) chemical shift assignments were obtained for the cold denatured state at pH 5.7, 12 degrees C. Chemical shift analysis, backbone N-H residual dipolar couplings, amide proton NOEs, and R(2) relaxation rates all indicate that the cold denatured state of CTL9 (the C-terminal domain of the ribosomal protein L9) not only contains significant native-like secondary structure but also non-native structure. The regions corresponding to the two native alpha-helices show a strong tendency to populate helical Phi and Psi angles. The segment which connects alpha-helix 2 and beta-strand 2 (residues 107-124) in the native state exhibits a significant preference to form non-native helical structure in the cold denatured state. The structure observed in the cold denatured state of the I98A mutant is similar to that observed in the pH 3.8 unfolded state of wild type CTL9 at 25 degrees C, suggesting that it is a robust feature of the denatured state ensemble of this

  18. X-ray Structure of Native Scorpion Toxin BmBKTx1 by Racemic Protein Crystallography Using Direct Methods

    SciTech Connect

    Mandal, Kalyaneswar; Pentelute, Brad L.; Tereshko, Valentina; Kossiakoff, Anthony A.; Kent, Stephen B.H.

    2009-04-08

    Racemic protein crystallography, enabled by total chemical synthesis, has allowed us to determine the X-ray structure of native scorpion toxin BmBKTx1; direct methods were used for phase determination. This is the first example of a protein racemate that crystallized in space group I41/a.

  19. Native electrospray ionization and electron-capture dissociation for comparison of protein structure in solution and the gas phase

    PubMed Central

    Zhang, Hao; Cui, Weidong

    2013-01-01

    The importance of protein and protein-complex structure motivates improvements in speed and sensitivity of structure determination in the gas phase and comparison with that in solution or solid state. An opportunity for the gas phase measurement is mass spectrometry (MS) combined with native electrospray ionization (ESI), which delivers large proteins and protein complexes in their near-native states to the gas phase. In this communication, we describe the combination of native ESI, electron-capture dissociation (ECD), and top-down MS for exploring the structures of ubiquitin and cytochrome c in the gas phase and their relation to those in the solid-state and solution. We probe structure by comparing the protein's flexible regions, as predicted by the B-factor in X-ray crystallography, with the ECD fragments. The underlying hypothesis is that maintenance of structure gives fragments that can be predicted from B-factors. This strategy may be applicable in general when X-ray structures are available and extendable to the study of intrinsically disordered proteins. PMID:24363606

  20. Whole Protein Native Fitness Potentials

    NASA Astrophysics Data System (ADS)

    Faraggi, Eshel; Kloczkowski, Andrzej

    2013-03-01

    Protein structure prediction can be separated into two tasks: sample the configuration space of the protein chain, and assign a fitness between these hypothetical models and the native structure of the protein. One of the more promising developments in this area is that of knowledge based energy functions. However, standard approaches using pair-wise interactions have shown shortcomings demonstrated by the superiority of multi-body-potentials. These shortcomings are due to residue pair-wise interaction being dependent on other residues along the chain. We developed a method that uses whole protein information filtered through machine learners to score protein models based on their likeness to native structures. For all models we calculated parameters associated with the distance to the solvent and with distances between residues. These parameters, in addition to energy estimates obtained by using a four-body-potential, DFIRE, and RWPlus were used as training for machine learners to predict the fitness of the models. Testing on CASP 9 targets showed that our method is superior to DFIRE, RWPlus, and the four-body potential, which are considered standards in the field.

  1. A Systematic Analysis of the Structures of Heterologously Expressed Proteins and Those from Their Native Hosts in the RCSB PDB Archive.

    PubMed

    Zhou, Ren-Bin; Lu, Hui-Meng; Liu, Jie; Shi, Jian-Yu; Zhu, Jing; Lu, Qin-Qin; Yin, Da-Chuan

    2016-01-01

    Recombinant expression of proteins has become an indispensable tool in modern day research. The large yields of recombinantly expressed proteins accelerate the structural and functional characterization of proteins. Nevertheless, there are literature reported that the recombinant proteins show some differences in structure and function as compared with the native ones. Now there have been more than 100,000 structures (from both recombinant and native sources) publicly available in the Protein Data Bank (PDB) archive, which makes it possible to investigate if there exist any proteins in the RCSB PDB archive that have identical sequence but have some difference in structures. In this paper, we present the results of a systematic comparative study of the 3D structures of identical naturally purified versus recombinantly expressed proteins. The structural data and sequence information of the proteins were mined from the RCSB PDB archive. The combinatorial extension (CE), FATCAT-flexible and TM-Align methods were employed to align the protein structures. The root-mean-square distance (RMSD), TM-score, P-value, Z-score, secondary structural elements and hydrogen bonds were used to assess the structure similarity. A thorough analysis of the PDB archive generated five-hundred-seventeen pairs of native and recombinant proteins that have identical sequence. There were no pairs of proteins that had the same sequence and significantly different structural fold, which support the hypothesis that expression in a heterologous host usually could fold correctly into their native forms. PMID:27517583

  2. A Systematic Analysis of the Structures of Heterologously Expressed Proteins and Those from Their Native Hosts in the RCSB PDB Archive

    PubMed Central

    Zhou, Ren-Bin; Lu, Hui-Meng; Liu, Jie; Shi, Jian-Yu; Zhu, Jing; Lu, Qin-Qin; Yin, Da-Chuan

    2016-01-01

    Recombinant expression of proteins has become an indispensable tool in modern day research. The large yields of recombinantly expressed proteins accelerate the structural and functional characterization of proteins. Nevertheless, there are literature reported that the recombinant proteins show some differences in structure and function as compared with the native ones. Now there have been more than 100,000 structures (from both recombinant and native sources) publicly available in the Protein Data Bank (PDB) archive, which makes it possible to investigate if there exist any proteins in the RCSB PDB archive that have identical sequence but have some difference in structures. In this paper, we present the results of a systematic comparative study of the 3D structures of identical naturally purified versus recombinantly expressed proteins. The structural data and sequence information of the proteins were mined from the RCSB PDB archive. The combinatorial extension (CE), FATCAT-flexible and TM-Align methods were employed to align the protein structures. The root-mean-square distance (RMSD), TM-score, P-value, Z-score, secondary structural elements and hydrogen bonds were used to assess the structure similarity. A thorough analysis of the PDB archive generated five-hundred-seventeen pairs of native and recombinant proteins that have identical sequence. There were no pairs of proteins that had the same sequence and significantly different structural fold, which support the hypothesis that expression in a heterologous host usually could fold correctly into their native forms. PMID:27517583

  3. Electron transfer dissociation provides higher-order structural information of native and partially unfolded protein complexes.

    PubMed

    Lermyte, Frederik; Sobott, Frank

    2015-08-01

    Top-down sequencing approaches are becoming ever more popular for protein characterization, due to the ability to distinguish and characterize different protein isoforms. Under non-denaturing conditions, electron transfer dissociation (ETD) can furthermore provide important information on the exposed surface of proteins or complexes, thereby contributing to the characterization of their higher-order structure. Here, we investigate this approach using top-down ETD of tetrameric hemoglobin, concanavalin A, and alcohol dehydrogenase combined with ion mobility (IM) on a commercially available quadrupole/ion mobility/time-of-flight instrument (Waters Synapt G2). By applying supplemental activation in the transfer cell (post-IM), we release ETD fragments and attain good sequence coverage in the exposed terminal regions of the protein. We investigate the correlation between observed sites of fragmentation with regions of solvent accessibility, as derived from the crystal structure. Ion acceleration prior to ETD is also used to cause collision-induced unfolding (CIU) of the complexes without monomer ejection, as evidenced by the IM profiles. These partially unfolded tetramers show efficient fragmentation in some regions which are not sequenced under more gentle MS conditions. We show that by increasing CIU in small increments and monitoring the changes in the fragmentation pattern, it is possible to follow the initial steps of gas-phase protein unfolding. Fragments from partially unfolded protein complexes are released immediately after electron transfer, prior to IM (they do not share the drift time of their precursor), and observed without the need for supplemental activation. This is further evidence that the higher-order structure in these protein regions has been disrupted. PMID:26081219

  4. Native and non-native structure in a protein-folding intermediate: spectroscopic studies of partially reduced IGF-I and an engineered alanine model.

    PubMed

    Hua, Q X; Narhi, L; Jia, W; Arakawa, T; Rosenfeld, R; Hawkins, N; Miller, J A; Weiss, M A

    1996-06-01

    The structure of a metastable folding intermediate of human insulin-like growth factor I (IGF-I) and an engineered model are investigated by circular dichroism and two-dimensional 1H NMR spectroscopy. The intermediate, which contains two of three native disulfide bonds, was trapped by acid quenching and isolated by reverse-phase HPLC. The reduced cysteine residues were mapped to residues 47 and 52 (corresponding to A6-A11 in insulin). In the native state this disulfide bridge anchors an adjoining amphipathic alpha-helix (helix 2; residues 42 to 49) against the hydrophobic core. Comparison of CD and 1H-NMR spectra demonstrates that the acid-quenched intermediate is partially folded and contains elements of native secondary and tertiary structure. Spectra are similar to those of an equilibrium model in which the reduced cysteine residues are replaced by alanine. Complete 1H-NMR sequential assignment of the alanine model has been obtained and demonstrates that removal of the disulfide bond is associated with local unfolding of the adjoining alpha-helix. Native secondary structure (including helices 1 and 3) is otherwise retained and defines a folded subdomain. Long-range nuclear Overhauser effects (NOE) within this subdomain are similar to those of native IGF-I; no non-native NOE is observed. Our results support the hypothesis that folding of the insulin motif is directed by a subset of native structural elements and that these elements form at an early step in the pathway. Formation of helix 2, despite its prominence in the native state, is likely to represent a late step. Hydrophobic collapse of this segment appears to precede helix formation. PMID:8656430

  5. How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?

    NASA Astrophysics Data System (ADS)

    Chen, Shu-Hua; Russell, David H.

    2015-09-01

    Here, we critically evaluate the effects of changes in the ion internal energy (Eint) on ion-neutral collision cross sections (CCS) of ions of two structurally diverse proteins, specifically the [M + 6H]6+ ion of ubiquitin (ubq6+), the [M + 5H]5+ ion of the intrinsically disordered protein (IDP) apo-metallothionein-2A (MT), and its partially- and fully-metalated isoform, the [CdiMT]5+ ion. The ion-neutral CCS for ions formed by "native-state" ESI show a strong dependence on Eint. Collisional activation is used to increase Eint prior to the ions entering and within the traveling wave (TW) ion mobility analyzer. Comparisons of experimental CCSs with those generated by molecular dynamics (MD) simulation for solution-phase ions and solvent-free ions as a function of temperature provide new insights about conformational preferences and retention of solution conformations. The Eint-dependent CCSs, which reveal increased conformational diversity of the ion population, are discussed in terms of folding/unfolding of solvent-free ions. For example, ubiquitin ions that have low internal energies retain native-like conformations, whereas ions that are heated by collisional activation possess higher internal energies and yield a broader range of CCS owing to increased conformational diversity due to losses of secondary and tertiary structures. In contrast, the CCS profile for the IDP apoMT is consistent with kinetic trapping of an ion population composed of a wide range of conformers, and as the Eint is increased, these structurally labile conformers unfold to an elongated conformation.

  6. How Closely Related Are Conformations of Protein Ions Sampled by IM-MS to Native Solution Structures?

    PubMed

    Chen, Shu-Hua; Russell, David H

    2015-09-01

    Here, we critically evaluate the effects of changes in the ion internal energy (E(int)) on ion-neutral collision cross sections (CCS) of ions of two structurally diverse proteins, specifically the [M + 6H](6+) ion of ubiquitin (ubq(6+)), the [M + 5H](5+) ion of the intrinsically disordered protein (IDP) apo-metallothionein-2A (MT), and its partially- and fully-metalated isoform, the [CdiMT](5+) ion. The ion-neutral CCS for ions formed by "native-state" ESI show a strong dependence on E(int). Collisional activation is used to increase E(int) prior to the ions entering and within the traveling wave (TW) ion mobility analyzer. Comparisons of experimental CCSs with those generated by molecular dynamics (MD) simulation for solution-phase ions and solvent-free ions as a function of temperature provide new insights about conformational preferences and retention of solution conformations. The E(int)-dependent CCSs, which reveal increased conformational diversity of the ion population, are discussed in terms of folding/unfolding of solvent-free ions. For example, ubiquitin ions that have low internal energies retain native-like conformations, whereas ions that are heated by collisional activation possess higher internal energies and yield a broader range of CCS owing to increased conformational diversity due to losses of secondary and tertiary structures. In contrast, the CCS profile for the IDP apoMT is consistent with kinetic trapping of an ion population composed of a wide range of conformers, and as the E(int) is increased, these structurally labile conformers unfold to an elongated conformation. PMID:26115967

  7. Protein Thermodynamics from the 3D Topological Structure of the Native State

    NASA Astrophysics Data System (ADS)

    Wood, Gregory; Dallakayan, Sargis; Jacobs, Donald

    2004-03-01

    Thermodynamic stability is calculated from the new Distance Constraint Model (DCM)[1]. Microscopic interactions are treated as constraints to which entropy and energies are assigned. From the 3D structure, an ensemble of mechanical frameworks are constructed representing distinct topologies of fluctuating constraints. For each framework, total energy is additive over all constraints while total entropy is additive over a select set of independent constraints. Independent constraints are identified via a graph theoretical algorithm, Floppy Inclusion and Rigid Substructure Topography (FIRST) [2]. Using Monte Carlo sampling a free energy landscape is calculated in constraint space. Excellent fits to heat capacity data for ubiquitin are achieved. Work supported by NIH GM48680-0952. [1] D. J. Jacobs, S. Dallakyan, G. G. Wood and A. Heckathorne, cond-mat/0309207 (to appear in PRE) [2] D. J. Jacobs, A. Rader, L. A. Kuhn and M. F. Thorpe, Proteins 44 150 (2001)

  8. In situ structural characterization of a recombinant protein in native Escherichia coli membranes with solid-state magic-angle-spinning NMR.

    PubMed

    Fu, Riqiang; Wang, Xingsheng; Li, Conggang; Santiago-Miranda, Adriana N; Pielak, Gary J; Tian, Fang

    2011-08-17

    The feasibility of using solid-state magic-angle-spinning NMR spectroscopy for in situ structural characterization of the LR11 (sorLA) transmembrane domain (TM) in native Escherichia coli membranes is presented. LR11 interacts with the human amyloid precursor protein (APP), a central player in the pathology of Alzheimer's disease. The background signals from E. coli lipids and membrane proteins had only minor effects on the LR11 TM resonances. Approximately 50% of the LR11 TM residues were assigned by using (13)C PARIS data. These assignments allowed comparisons of the secondary structure of the LR11 TM in native membrane environments and commonly used membrane mimics (e.g., micelles). In situ spectroscopy bypasses several obstacles in the preparation of membrane proteins for structural analysis and offers the opportunity to investigate how membrane heterogeneity, bilayer asymmetry, chemical gradients, and macromolecular crowding affect the protein structure. PMID:21774553

  9. Scale Invariant Fluctuations of Proteins Native States

    NASA Astrophysics Data System (ADS)

    Tang, Qian-Yuan; Zhang, Yang-Yang; Wang, Jun; Wang, Wei; Chialvo, Dante R.

    Long-range correlations in biological systems often hints for the presence of universal mechanism at work. Here we study protein native dynamics by analyzing a large set of structure ensembles determined by solution NMR. For proteins of diverse sizes, the average distance-dependent cross-correlation functions ϕ (r) and its correlation length ξϕ are analyzed. The analysis uncovered the presence of nontrivial scaling in the proteins' equilibrium dynamics around native states. We show that the correlation length is proportional to the gyration radius of the molecule, implying that the motion of any residue could influence all the others, up to the entire molecule. In addition, it is found that certain shapes are favored, such that for any given protein size the folding process ``chooses'' the shape with the maximum susceptibility. These results suggest that the proteins native state is critical in the same sense with other slowly built self-organized critical systems, which once posed near the minimum of the energy landscape, preserve their dynamic flexibility. Supported by Natural Science Foundation of China (Grants 11334004, 11174133, 81421091) and National Basic Research Program of China (Grant 2013CB834100).

  10. Protein-inhibitor flexible docking by a multicanonical sampling: native complex structure with the lowest free energy and a free-energy barrier distinguishing the native complex from the others.

    PubMed

    Kamiya, Narutoshi; Yonezawa, Yasushige; Nakamura, Haruki; Higo, Junichi

    2008-01-01

    Flexible docking between a protein (lysozyme) and an inhibitor (tri-N-acetyl-D-glucosamine, tri-NAG) was carried out by an enhanced conformational sampling method, multicanonical molecular dynamics simulation. We used a flexible all-atom model to express lysozyme, tri-NAG, and water molecules surrounding the two bio-molecules. The advantages of this sampling method are as follows: the conformation of system is widely sampled without trapping at energy minima, a thermally equilibrated conformational ensemble at an arbitrary temperature can be reconstructed from the simulation trajectory, and the thermodynamic weight can be assigned to each sampled conformation. During the simulation, exchanges between the binding and free (i.e., unbinding) states of the protein and the inhibitor were repeatedly observed. The conformational ensemble reconstructed at 300 K involved various conformational clusters. The main outcome of the current study is that the most populated conformational cluster (i.e., the cluster of the lowest free energy) was assigned to the native complex structure (i.e., the X-ray complex structure). The simulation also produced non-native complex structures, where the protein and the inhibitor bound with different modes from that of the native complex structure, as well as the unbinding structures. A free-energy barrier (i.e., activation free energy) was clearly detected between the native complex structures and the other structures. The thermal fluctuations of tri-NAG in the lowest free-energy complex correlated well with the X-ray B-factors of tri-NAG in the X-ray complex structure. The existence of the free-energy barrier ensures that the lowest free-energy structure can be discriminated naturally from the other structures. In other words, the multicanonical molecular dynamics simulation can predict the native complex structure without any empirical objective function. The current study also manifested that the flexible all-atom model and the physico

  11. Strong improvement of interfacial properties can result from slight structural modifications of proteins: the case of native and dry-heated lysozyme.

    PubMed

    Desfougères, Yann; Saint-Jalmes, Arnaud; Salonen, Anniina; Vié, Véronique; Beaufils, Sylvie; Pezennec, Stéphane; Desbat, Bernard; Lechevalier, Valérie; Nau, Françoise

    2011-12-20

    Identification of the key physicochemical parameters of proteins that determine their interfacial properties is still incomplete and represents a real stake challenge, especially for food proteins. Many studies have thus consisted in comparing the interfacial behavior of different proteins, but it is difficult to draw clear conclusions when the molecules are completely different on several levels. Here the adsorption process of a model protein, the hen egg-white lysozyme, and the same protein that underwent a thermal treatment in the dry state, was characterized. The consequences of this treatment have been previously studied: net charge and hydrophobicity increase and lesser protein stability, but no secondary and tertiary structure modification (Desfougères, Y.; Jardin, J.; Lechevalier, V.; Pezennec, S.; Nau, F. Biomacromolecules 2011, 12, 156-166). The present study shows that these slight modifications dramatically increase the interfacial properties of the protein, since the adsorption to the air-water interface is much faster and more efficient (higher surface pressure). Moreover, a thick and strongly viscoelastic multilayer film is created, while native lysozyme adsorbs in a fragile monolayer film. Another striking result is that completely different behaviors were observed between two molecular species, i.e., native and native-like lysozyme, even though these species could not be distinguished by usual spectroscopic methods. This suggests that the air-water interface could be considered as a useful tool to reveal very subtle differences between protein molecules. PMID:22040020

  12. Rapana thomasiana grosse (gastropoda) haemocyanin: spectroscopic studies of the structure in solution and the conformational stability of the native protein and its structural subunits.

    PubMed

    Dolashka, P; Genov, N; Parvanova, K; Voelter, W; Geiger, M; Stoeva, S

    1996-04-01

    1. The stability towards pH changes, thermal and chemical (guanidine hydrochloride) denaturation of the oxy- and apo-forms of the native Rapana thomasiana haemocyanin and its structural subunits, RHSS1 and RHSS2, has been investigated using fluorescence and CD spectroscopy. The association of the subunits into haemocyanin aggregates increases considerably the melting temperature and the free energy of stabilization in water. The guanidine hydrochloride denaturation of the aggregated oxygen-transporting protein depends slightly on the protein concentration. The denaturation of the individual subunits is concentration-independent. Rapana haemocyanin is 5.9-7.5 kJ/mol more stable than the constituent polypeptide chains. 2. Upon excitation of the native haemocyanin and the subunits at 295 or 280 nm the fluorescence emission is determined by tryptophyl residues 'buried' deeply in the hydrophobic interior of the protein globules. This is confirmed by quenching experiments with acrylamide, caesium and iodide ions. The efficiency of the radiationless energy transfer between the phenol (donor) and indole (acceptor) fluorophores in the three species, native haemocyanin, RHSS1 and RHSS2, has been determined. An efficient 'interchain' energy transfer between tyrosyl and tryptophyl residues from different polypeptide chains occurs in the non-dissociated form of the haemocyanin. 3. The tryptophan emission of the oxyhaemocyanin, oxy-RHSS1 and oxy-RHSS 2 is strongly quenched by the copper-dioxygen complex at the active site and the respective quantum yields of fluorescence of the oxygenated species are 4-7 times lower than those of the apo-forms. Protonated imidazole groups quench the fluorescence of neighbouring exited indole rings, probably by charge-transfer complex formation. PMID:8670098

  13. Analysis of human follistatin structure: identification of two discontinuous N-terminal sequences coding for activin A binding and structural consequences of activin binding to native proteins.

    PubMed

    Wang, Q; Keutmann, H T; Schneyer, A L; Sluss, P M

    2000-09-01

    A primary physiological function of follistatin is the binding and neutralization of activin, a transforming growth factor-beta family growth factor, and loss of function mutations are lethal. Despite the critical biological importance of follistatin's neutralization of activin, the structural basis of activin's binding to follistatin is poorly understood. The purposes of these studies were 1) to identify the primary sequence(s) within the N-terminal domain of the follistatin coding for activin binding, and 2) to determine whether activin binding to the native protein causes changes in other structural domains of follistatin. Synthetic peptide mimotopes identified within a 63-residue N-terminal domain two discontinuous sequences capable of binding labeled activin A. The first is located in a region (amino acids 3-26) of follistatin, a site previously identified by directed mutagenesis as important for activin binding. The second epitope, predicted to be located between amino acids 46 and 59, is newly identified. Although the sequences 3-26 and 46-59 code for activin binding, native follistatin only binds activin if disulfide bonding is intact. Furthermore, pyridylethylation of Cys residues followed by N-terminal sequencing and amino acid analysis revealed that all of the Cys residues in follistatin are involved in disulfide bonds and lack reactive free sulfhydryl groups. Specific ligands were used to probe the structural effects of activin binding on the other domains of the full-length molecule, comprised largely of the three 10-Cys follistatin module domains. No effects on ligand binding to follistatin-like module I or II were observed after the binding of activin A to native protein. In contrast, activin binding diminished recognition of domain III and enhanced that of the C domain by their respective monoclonal antibody probes, indicating an alteration of the antigenic structures of these regions. Thus, subsequent to activin binding, interactions are likely to

  14. Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability

    PubMed Central

    Camilloni, Carlo; Sala, Benedetta Maria; Sormanni, Pietro; Porcari, Riccardo; Corazza, Alessandra; De Rosa, Matteo; Zanini, Stefano; Barbiroli, Alberto; Esposito, Gennaro; Bolognesi, Martino; Bellotti, Vittorio; Vendruscolo, Michele; Ricagno, Stefano

    2016-01-01

    A wide range of human diseases is associated with mutations that, destabilizing proteins native state, promote their aggregation. However, the mechanisms leading from folded to aggregated states are still incompletely understood. To investigate these mechanisms, we used a combination of NMR spectroscopy and molecular dynamics simulations to compare the native state dynamics of Beta-2 microglobulin (β2m), whose aggregation is associated with dialysis-related amyloidosis, and its aggregation-resistant mutant W60G. Our results indicate that W60G low aggregation propensity can be explained, beyond its higher stability, by an increased average protection of the aggregation-prone residues at its surface. To validate these findings, we designed β2m variants that alter the aggregation-prone exposed surface of wild-type and W60G β2m modifying their aggregation propensity. These results allowed us to pinpoint the role of dynamics in β2m aggregation and to provide a new strategy to tune protein aggregation by modulating the exposure of aggregation-prone residues. PMID:27150430

  15. Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability.

    PubMed

    Camilloni, Carlo; Sala, Benedetta Maria; Sormanni, Pietro; Porcari, Riccardo; Corazza, Alessandra; De Rosa, Matteo; Zanini, Stefano; Barbiroli, Alberto; Esposito, Gennaro; Bolognesi, Martino; Bellotti, Vittorio; Vendruscolo, Michele; Ricagno, Stefano

    2016-01-01

    A wide range of human diseases is associated with mutations that, destabilizing proteins native state, promote their aggregation. However, the mechanisms leading from folded to aggregated states are still incompletely understood. To investigate these mechanisms, we used a combination of NMR spectroscopy and molecular dynamics simulations to compare the native state dynamics of Beta-2 microglobulin (β2m), whose aggregation is associated with dialysis-related amyloidosis, and its aggregation-resistant mutant W60G. Our results indicate that W60G low aggregation propensity can be explained, beyond its higher stability, by an increased average protection of the aggregation-prone residues at its surface. To validate these findings, we designed β2m variants that alter the aggregation-prone exposed surface of wild-type and W60G β2m modifying their aggregation propensity. These results allowed us to pinpoint the role of dynamics in β2m aggregation and to provide a new strategy to tune protein aggregation by modulating the exposure of aggregation-prone residues. PMID:27150430

  16. Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability

    NASA Astrophysics Data System (ADS)

    Camilloni, Carlo; Sala, Benedetta Maria; Sormanni, Pietro; Porcari, Riccardo; Corazza, Alessandra; De Rosa, Matteo; Zanini, Stefano; Barbiroli, Alberto; Esposito, Gennaro; Bolognesi, Martino; Bellotti, Vittorio; Vendruscolo, Michele; Ricagno, Stefano

    2016-05-01

    A wide range of human diseases is associated with mutations that, destabilizing proteins native state, promote their aggregation. However, the mechanisms leading from folded to aggregated states are still incompletely understood. To investigate these mechanisms, we used a combination of NMR spectroscopy and molecular dynamics simulations to compare the native state dynamics of Beta-2 microglobulin (β2m), whose aggregation is associated with dialysis-related amyloidosis, and its aggregation-resistant mutant W60G. Our results indicate that W60G low aggregation propensity can be explained, beyond its higher stability, by an increased average protection of the aggregation-prone residues at its surface. To validate these findings, we designed β2m variants that alter the aggregation-prone exposed surface of wild-type and W60G β2m modifying their aggregation propensity. These results allowed us to pinpoint the role of dynamics in β2m aggregation and to provide a new strategy to tune protein aggregation by modulating the exposure of aggregation-prone residues.

  17. Folding and Stabilization of Native-Sequence-Reversed Proteins.

    PubMed

    Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

    2016-01-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

  18. Folding and Stabilization of Native-Sequence-Reversed Proteins

    PubMed Central

    Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong

    2016-01-01

    Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844

  19. How special is the biochemical function of native proteins?

    PubMed Central

    Skolnick, Jeffrey; Gao, Mu; Zhou, Hongyi

    2016-01-01

    Native proteins perform an amazing variety of biochemical functions, including enzymatic catalysis, and can engage in protein-protein and protein-DNA interactions that are essential for life. A key question is how special are these functional properties of proteins. Are they extremely rare, or are they an intrinsic feature? Comparison to the properties of compact conformations of artificially generated compact protein structures selected for thermodynamic stability but not any type of function, the artificial (ART) protein library, demonstrates that a remarkable number of the properties of native-like proteins are recapitulated. These include the complete set of small molecule ligand-binding pockets and most protein-protein interfaces. ART structures are predicted to be capable of weakly binding metabolites and cover a significant fraction of metabolic pathways, with the most enriched pathways including ancient ones such as glycolysis. Native-like active sites are also found in ART proteins. A small fraction of ART proteins are predicted to have strong protein-protein and protein-DNA interactions. Overall, it appears that biochemical function is an intrinsic feature of proteins which nature has significantly optimized during evolution. These studies raise questions as to the relative roles of specificity and promiscuity in the biochemical function and control of cells that need investigation. PMID:26962440

  20. A beta-complex statistical four body contact potential combined with a hydrogen bond statistical potential recognizes the correct native structure from protein decoy sets.

    PubMed

    Sánchez-González, Gilberto; Kim, Jae-Kwan; Kim, Deok-Soo; Garduño-Juárez, Ramón

    2013-08-01

    We present a new four-body knowledge-based potential for recognizing the native state of proteins from their misfolded states. This potential was extracted from a large set of protein structures determined by X-ray crystallography using BetaMol, a software based on the recent theory of the beta-complex (β-complex) and quasi-triangulation of the Voronoi diagram of spheres. This geometric construct reflects the size difference among atoms in their full Euclidean metric; property not accounted for in a typical 3D Delaunay triangulation. The ability of this potential to identify the native conformation over a large set of decoys was evaluated. Experiments show that this potential outperforms a potential constructed with a classical Delaunay triangulation in decoy discrimination tests. The addition of a statistical hydrogen bond potential to our four-body potential allows a significant improvement in the decoy discrimination, in such a way that we are able to predict successfully the native structure in 90% of cases. PMID:23568277

  1. How amide hydrogens exchange in native proteins

    PubMed Central

    Persson, Filip; Halle, Bertil

    2015-01-01

    Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N–H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N–H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion. PMID:26195754

  2. How amide hydrogens exchange in native proteins.

    PubMed

    Persson, Filip; Halle, Bertil

    2015-08-18

    Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N-H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N-H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion. PMID:26195754

  3. Rapid Generation of Amyloid from Native Proteins In vitro

    PubMed Central

    Dorta-Estremera, Stephanie M; Li, Jingjing; Cao, Wei

    2013-01-01

    Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion. PMID:24335677

  4. IFACEwat: the interfacial water-implemented re-ranking algorithm to improve the discrimination of near native structures for protein rigid docking

    PubMed Central

    2014-01-01

    Background Protein-protein docking is an in silico method to predict the formation of protein complexes. Due to limited computational resources, the protein-protein docking approach has been developed under the assumption of rigid docking, in which one of the two protein partners remains rigid during the protein associations and water contribution is ignored or implicitly presented. Despite obtaining a number of acceptable complex predictions, it seems to-date that most initial rigid docking algorithms still find it difficult or even fail to discriminate successfully the correct predictions from the other incorrect or false positive ones. To improve the rigid docking results, re-ranking is one of the effective methods that help re-locate the correct predictions in top high ranks, discriminating them from the other incorrect ones. In this paper, we propose a new re-ranking technique using a new energy-based scoring function, namely IFACEwat - a combined Interface Atomic Contact Energy (IFACE) and water effect. The IFACEwat aims to further improve the discrimination of the near-native structures of the initial rigid docking algorithm ZDOCK3.0.2. Unlike other re-ranking techniques, the IFACEwat explicitly implements interfacial water into the protein interfaces to account for the water-mediated contacts during the protein interactions. Results Our results showed that the IFACEwat increased both the numbers of the near-native structures and improved their ranks as compared to the initial rigid docking ZDOCK3.0.2. In fact, the IFACEwat achieved a success rate of 83.8% for Antigen/Antibody complexes, which is 10% better than ZDOCK3.0.2. As compared to another re-ranking technique ZRANK, the IFACEwat obtains success rates of 92.3% (8% better) and 90% (5% better) respectively for medium and difficult cases. When comparing with the latest published re-ranking method F2Dock, the IFACEwat performed equivalently well or even better for several Antigen/Antibody complexes

  5. Structure determination of functional membrane proteins using small-angle neutron scattering (sans) with small, mixed-lipid liposomes: native beef heart mitochondrial cytochrome c oxidase forms dimers.

    PubMed

    Rubinson, Kenneth A; Pokalsky, Christine; Krueger, Susan; Prochaska, Lawrence J

    2013-01-01

    The low-resolution three-dimensional structure of purified native beef heart mitochondrial cytochrome c oxidase (COX) in asolectin unilamellar liposomes has been measured by small-angle neutron scattering under the conditions where the protein remains fully functional. From a neutron scattering perspective, the use of mixed-lipid liposomes provided for a more homogeneous matrix than can be achieved using a single lipid. As a result, the measurements were able to be performed under conditions where the liposome scattering was essentially eliminated (contrast-matched conditions). The protein structure in the membrane was modeled as a simple parallelepiped with side lengths of (59 × 70 × 120) Å with uncertainties, respectively, (11, 12, 20 Å). The molecular mass calculated for a typical protein with this volume is estimated to be (410 ± 124) kDa, which indicates the mass of a COX dimer. The longest dimension has some uncertainty due to intermolecular scattering contributing to the data. Nevertheless, that length was estimated using an average protein density and the known dimer molecular mass. Using the same cross sectional dimensions for the structure, the length is estimated to be 120 Å. However, the measured scattering curve of the dimer in the liposome differs significantly from that calculated from the X-ray structure of the dimer in a crystal of mixed micelles (PDB 3AG1). The calculated SANS scattering from the crystal structure was fit with a parallelepiped, measuring (59 × 101 × 129) Å with fitting uncertainties, respectively, (2, 3, 3 Å). Our results suggest that COX is a functional dimer when reconstituted into mixed-lipid liposomes. PMID:23143018

  6. Assessment of semiempirical enthalpy of formation in solution as an effective energy function to discriminate native-like structures in protein decoy sets.

    PubMed

    Urquiza-Carvalho, Gabriel Aires; Fragoso, Wallace Duarte; Rocha, Gerd Bruno

    2016-08-01

    In this work, we tested the PM6, PM6-DH+, PM6-D3, and PM7 enthalpies of formation in aqueous solution as scoring functions across 33 decoy sets to discriminate native structures or good models in a decoy set. In each set these semiempirical quantum chemistry methods were compared according to enthalpic and geometric criteria. Enthalpically, we compared the methods according to how much lower was the enthalpy of each native, when compared with the mean enthalpy of its set. Geometrically, we compared the methods according to the fraction of native contacts (Q), which is a measure of geometric closeness between an arbitrary structure and the native. For each set and method, the Q of the best decoy was compared with the Q0 , which is the Q of the decoy closest to the native in the set. It was shown that the PM7 method is able to assign larger energy differences between the native structure and the decoys in a set, arguably because of a better description of dispersion interactions, however PM6-DH+ was slightly better than the rest at selecting geometrically good models in the absence of a native structure in the set. © 2016 Wiley Periodicals, Inc. PMID:27249629

  7. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  8. Structure-biological function relationship extended to mitotic arrest-deficient 2-like protein Mad2 native and mutants-new opportunity for genetic disorder control.

    PubMed

    Avram, Speranta; Milac, Adina; Mernea, Maria; Mihailescu, Dan; Putz, Mihai V; Buiu, Catalin

    2014-01-01

    Overexpression of mitotic arrest-deficient proteins Mad1 and Mad2, two components of spindle assembly checkpoint, is a risk factor for chromosomal instability (CIN) and a trigger of many genetic disorders. Mad2 transition from inactive open (O-Mad2) to active closed (C-Mad2) conformations or Mad2 binding to specific partners (cell-division cycle protein 20 (Cdc20) or Mad1) were targets of previous pharmacogenomics studies. Here, Mad2 binding to Cdc20 and the interconversion rate from open to closed Mad2 were predicted and the molecular features with a critical contribution to these processes were determined by extending the quantitative structure-activity relationship (QSAR) method to large-size proteins such as Mad2. QSAR models were built based on available published data on 23 Mad2 mutants inducing CIN-related functional changes. The most relevant descriptors identified for predicting Mad2 native and mutants action mechanism and their involvement in genetic disorders are the steric (van der Waals area and solvent accessible area and their subdivided) and energetic van der Waals energy descriptors. The reliability of our QSAR models is indicated by significant values of statistical coefficients: Cross-validated correlation q2 (0.53-0.65) and fitted correlation r2 (0.82-0.90). Moreover, based on established QSAR equations, we rationally design and analyze nine de novo Mad2 mutants as possible promoters of CIN. PMID:25411801

  9. Knowledge of Native Protein-Protein Interfaces Is Sufficient To Construct Predictive Models for the Selection of Binding Candidates.

    PubMed

    Popov, Petr; Grudinin, Sergei

    2015-10-26

    Selection of putative binding poses is a challenging part of virtual screening for protein-protein interactions. Predictive models to filter out binding candidates with the highest binding affinities comprise scoring functions that assign a score to each binding pose. Existing scoring functions are typically deduced by collecting statistical information about interfaces of native conformations of protein complexes along with interfaces of a large generated set of non-native conformations. However, the obtained scoring functions become biased toward the method used to generate the non-native conformations, i.e., they may not recognize near-native interfaces generated with a different method. The present study demonstrates that knowledge of only native protein-protein interfaces is sufficient to construct well-discriminative predictive models for the selection of binding candidates. Here we introduce a new scoring method that comprises a knowledge-based potential called KSENIA deduced from structural information about the native interfaces of 844 crystallographic protein-protein complexes. We derive KSENIA using convex optimization with a training set composed of native protein complexes and their near-native conformations obtained using deformations along the low-frequency normal modes. As a result, our knowledge-based potential has only marginal bias toward a method used to generate putative binding poses. Furthermore, KSENIA is smooth by construction, which allows it to be used along with rigid-body optimization to refine the binding poses. Using several test benchmarks, we demonstrate that our method discriminates well native and near-native conformations of protein complexes from non-native ones. Our methodology can be easily adapted to the recognition of other types of molecular interactions, such as protein-ligand, protein-RNA, etc. KSENIA will be made publicly available as a part of the SAMSON software platform at https://team.inria.fr/nano-d/software . PMID

  10. Autofluorescence based visualization of proteins from unstained native-PAGE

    NASA Astrophysics Data System (ADS)

    Manjunath, S.; Rao, Bola Sadashiva S.; Satyamoorthy, Kapaettu; Mahato, Krishna Kishore

    2015-03-01

    Proteins are the most diverse and functionally active biomolecules in the living system. In order to understand their diversity and dynamic functionality, visualization in native form without altering structural and functional properties during the separation from the complex mixtures is very much essential. In the present study, a sensitive methodology for optimal visualization of unstained or untagged proteins in native poly-acrylamide gel electrophoresis (N-PAGE) has been developed where, concentration of the acrylamide and bis-acrylamide mixture, Percentage of the gel, fixing of the N-PAGE by methanol: acetic acid: water and washing of the gel in the mili-Q water has been optimized for highest sensitivity using laser induced autofluorescence. The outcome with bovine serum albumin (BSA) in PAGE was found to be highest at acrylamide and bis-acrylamide concentrations of 29.2 and 0.8 respectively in 12% N-PAGE. After the electrophoresis run, washing of the N-PAGE immediately with miliQ water for 12 times and eliminating the methanol: acetic acid: water, fixing of the N-PAGE yielded better sensitivity of visualization. Using the above methodology 25ng of BSA protein band in PAGE was clearly identified by the technique. The currently used staining techniques for the visualization of proteins are coomassie brilliant blue and silver staining, have the sensitivity of 100ng and 5ng respectively. The current methodology was found to be more sensitive as compared to coomassie staining and less sensitive compared to silver staining respectively. The added advantage of this methodology is the faster visualization of proteins without altering their structure and functional properties.

  11. Chemical Protein Ubiquitylation with Preservation of the Native Cysteine Residues.

    PubMed

    Yang, Kun; Li, Guorui; Gong, Ping; Gui, Weijun; Yuan, Libo; Zhuang, Zhihao

    2016-06-01

    We report a cysteine-based ligation strategy for generating a monoubiquitylated protein while preserving the native cysteine residues on the acceptor protein. In monoubiquitylation of proliferating cell nuclear antigen (PCNA) this method circumvents the need to mutate the native cysteine residues on PCNA. The chemically ubiquitylated PCNA contains a noncleavable linkage of the same length as the native isopeptide linkage. It also retains the normal function of the native Ub-PCNA in stimulating the ATPase activity of replication factor C (RFC) and lesion bypass synthesis by Polη. This method may be adapted for chemical ubiquitylation of other proteins and for site-specific modification of a target protein at a specific site through sulfhydryl chemistry. PMID:27113245

  12. Pigment-protein architecture in the light-harvesting antenna complexes of purple bacteria: does the crystal structure reflect the native pigment-protein arrangement?

    PubMed

    Leupold, D; Voigt, B; Beenken, W; Stiel, H

    2000-09-01

    Structural analysis of crystallized peripheral (LH2) and core antenna complexes (LH1) of purple bacteria has revealed circular aggregates of high rotational symmetry (C8, C9 and C16, respectively). Quantum-chemical calculations indicate that in particular the waterwheel-like arrangements of pigments should show characteristic structure-sensitive spectroscopic behavior in the near infrared absorption region. Laser-spectroscopic data obtained with non-crystallized, isolated LH2 of Rhodospirillum molischianum are in line with a highly symmetric (C8) circular aggregate, but deviations have been found for LH2 of Rhodobacter sphaeroides and Rhodopseudomonas acidophila. For both the latter, C-shaped incomplete circular aggregates (as seen only recently in electron micrographs of crystallized LH1-reaction center complexes) may be a suitable preliminary model. PMID:11034303

  13. Self-assembly studies of native and recombinant fibrous proteins

    NASA Astrophysics Data System (ADS)

    Wilson, Donna Lucille

    The structure of silk proteins consists of alternating amorphous (glycine-rich) and ordered crystalline regions (poly(alanine) and poly(glycine-alanine) repeats), where the organized regions are typically beta-sheet assemblies. In collagen, the basic helical repeat (glycine-proline-hydroxyproline and variants on this repeat) drives hierarchical assembly. Three polypeptide chains form left-handed poly-proline II-like helices, these three chains then self-assemble to form a right-handed triple helix. The focus of this thesis is on these proteins and defined variations thereof to reveal features of fibrous protein self-assembly. The amino acid sequences of native silk and collagen and their respective assembly environments have been systematically manipulated. Spider silk protein, based on the consensus sequence of Nephila clavipes dragline-silk, was genetically engineered to include methionines flanking the beta-sheet forming polyalanine regions. These methionines could be selectively oxidized and reduced, altering the bulkiness and charge of a methionine-based sulfoxide group to control beta-sheet formation by steric hindrance. A second version of the sterical trigger included a recognition site for Protein Kinase A allowing for the selective phosphorylation of a serine. Patterning a monolayer of precursor "director" molecules on length scales ranging from nanometer- to micrometer-length scales simplifies the interpretation of supramolecular assembly. Utilizing the atomic force microscopy (AFM)-based technique of dip-pen nanolithography, thiolated collagen and a collagen-like peptide were patterned at 30--50 nm line widths on evaporated gold surfaces. These are the largest molecules thus far positively printed on a surface at such small-length scales. The method preserved the triple helical structure and biological activity of collagen and even fostered the formation of characteristic higher-levels of structural organization. Nanopatterns were also achieved for

  14. The Knotted Protein UCH-L1 Exhibits Partially Unfolded Forms under Native Conditions that Share Common Structural Features with Its Kinetic Folding Intermediates.

    PubMed

    Lou, Shih-Chi; Wetzel, Svava; Zhang, Hongyu; Crone, Elizabeth W; Lee, Yun-Tzai; Jackson, Sophie E; Hsu, Shang-Te Danny

    2016-06-01

    The human ubiquitin C-terminal hydrolase, UCH-L1, is an abundant neuronal deubiquitinase that is associated with Parkinson's disease. It contains a complex Gordian knot topology formed by the polypeptide chain alone. Using a combination of fluorescence-based kinetic measurements, we show that UCH-L1 has two distinct kinetic folding intermediates that are transiently populated on parallel pathways between the denatured and native states. NMR hydrogen-deuterium exchange (HDX) experiments indicate the presence of partially unfolded forms (PUFs) of UCH-L1 under native conditions. HDX measurements as a function of urea concentration were used to establish the structure of the PUFs and pulse-labelled HDX NMR was used to show that the PUFs and the folding intermediates are likely the same species. In both cases, a similar stable core encompassing most of the central β-sheet is highly structured and α-helix 3, which is partially formed, packs against it. In contrast to the stable β-sheet core, the peripheral α-helices display significant local fluctuations leading to rapid exchange. The results also suggest that the main difference between the two kinetic intermediates is structure and packing of α-helices 3 and 7 and the degree of structure in β-strand 5. Together, the fluorescence and NMR results establish that UCH-L1 neither folds through a continuum of pathways nor by a single discrete pathway. Its folding is complex, the β-sheet core forms early and is present in both intermediate states, and the rate-limiting step which is likely to involve the threading of the chain to form the 52-knot occurs late on the folding pathway. PMID:27067109

  15. Native proteins trap high-energy transit conformations

    PubMed Central

    Brereton, Andrew E.; Karplus, P. Andrew

    2015-01-01

    During protein folding and as part of some conformational changes that regulate protein function, the polypeptide chain must traverse high-energy barriers that separate the commonly adopted low-energy conformations. How distortions in peptide geometry allow these barrier-crossing transitions is a fundamental open question. One such important transition involves the movement of a non-glycine residue between the left side of the Ramachandran plot (that is, ϕ < 0°) and the right side (that is, ϕ > 0°). We report that high-energy conformations with ϕ ~ 0°, normally expected to occur only as fleeting transition states, are stably trapped in certain highly resolved native protein structures and that an analysis of these residues provides a detailed, experimentally derived map of the bond angle distortions taking place along the transition path. This unanticipated information lays to rest any uncertainty about whether such transitions are possible and how they occur, and in doing so lays a firm foundation for theoretical studies to better understand the transitions between basins that have been little studied but are integrally involved in protein folding and function. Also, the context of one such residue shows that even a designed highly stable protein can harbor substantial unfavorable interactions. PMID:26601321

  16. Thermus thermophilus encodes an archaeal-like fructose-1,6-bisphosphatase: purification of native and recombinant protein for structural studies.

    PubMed

    Soulimane, T

    2010-12-01

    Fructose-1,6-bisphosphatase is one of the key enzymes of the gluconeogenic pathway. It catalyses the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. Fructose-1,6-bisphosphatase from the extreme thermophilic bacterium Thermus thermophilus has been purified by crystallisation approach. The final well-shaped crystals have been obtained using vapour diffusion sitting-drops in the presence of PEG 400 as the precipitating agent. The initially obtained native twinned crystals diffracted up to 1.2Å resolution. Untwinned crystals used for data collection, however, were grown in the presence of thiomersal. They diffract to 1.8 Å resolution and belong to the space groups I422 with cell dimensions (i) a=b=108.8Å, c=336.3Å showing two molecules in the asymmetric unit, and (ii) a=b=113.7Å, c=151.0Å with one molecule in the asymmetric unit. The crystal structure has been solved by single anomalous dispersion using a 1.9Å resolution. For further biochemical and biophysical investigations recombinant fructose-1,6-bisphosphatase has been produced in Escherichia coli. Both native (dissolved crystals) and recombinant material have been characterised by SDS-PAGE, N-terminal sequencing and MALDI-MS. PMID:20600937

  17. Dextran Preserves Native Corneal Structure During Decellularization.

    PubMed

    Lynch, Amy P; Wilson, Samantha L; Ahearne, Mark

    2016-06-01

    Corneal decellularization has become an increasingly popular technique for generating scaffolds for corneal regeneration. Most decellularization procedures result in tissue swelling, thus limiting their application. Here, the use of a polysaccharide, dextran, to reduce swelling and conserve the native corneal structure during decellularization was investigated. Corneas were treated with 1% Triton X-100, 0.5% sodium dodecyl sulfate, and nucleases under constant rotation followed by extensive washing. To reduce swelling, decellularization solutions were supplemented with 5% dextran either throughout the whole decellularization process or during the washing cycles only. Quantitative analysis of DNA content showed a 96% reduction after decellularization regardless of the addition of dextran. Dextran resulted in a significant reduction in swelling from 3.85 ± 0.43 nm without to 1.94 ± 0.29-2.01 ± 0.37 nm (p < 0.05) remaining at similar dimensions to the native tissue (1.73 ± 0.23 nm). Tissue transparency was restored to all decellularized corneas following submersion in glycerol. Transmission electron microscopy (TEM) analysis found that dextran must be present throughout the decellularization protocol to preserve the native corneal architecture, anisotropy analysis demonstrated comparable results (0.22 ± 0.03) to the native cornea (0.24 ± 0.02), p > 0.05. Dextran can counteract the detrimental effects of decellularizing agents on the biomechanical properties of the tissue resulting in similar compressive moduli (mean before decellularization: 5.40 ± 1.18 kPa; mean after decellularization with dextran: 5.64 ± 1.34 kPa, p > 0.05). Cells remained viable in the presence of decellularized scaffolds. The findings of this study indicate that dextran not only prevents significant corneal swelling during decellularization but also enhances the maintenance of the native corneal ultrastructure. PMID:27068608

  18. Implementation of Multiple Spectroscopic Techniques to Simultaneously Observe Native and Mutated Protein Unfolding

    NASA Astrophysics Data System (ADS)

    Cull, Brennan; Ben, Kelty; Link, Justin

    A protein's natural, correctly folded structure can determine the protein's ability to carry out its function. If the unfolding process of proteins can be observed, then the relative stability can be better understood between native and mutated proteins. A global picture of the unfolding process may be completed through the studies of strategically mutated proteins using tryptophan as a probe. Horse heart cytochrome c, a thoroughly studied, model protein was used in our investigation to explore this idea. Various spectroscopic techniques such as circular dichroism (CD), absorbance, and fluorescence were simultaneously applied while slowly unfolding our protein by increasing the concentration of a chemical denaturant, guanidine hydrochloride. This provided us information about the thermodynamic properties of the protein and several mutants which can then be interpreted to gain relative stability information among mutations. Efforts to utilize these techniques on native and mutated proteins in comparison to current scientific unfolding theories will be presented in this session.

  19. Display of native proteins on Bacillus subtilis spores.

    PubMed

    Pan, Jae-Gu; Choi, Soo-Keun; Jung, Heung-Chae; Kim, Eui-Joong

    2014-09-01

    In principle, protein display is enabled by fusing target proteins to naturally secreted, surface-anchored protein motifs. In this work, we developed a method of native protein display on the Bacillus spore surface that obviates the need to construct fusion proteins to display a motif. Spore coat proteins are expressed in the mother cell compartment and are subsequently assembled and deposited on the surface of spores. Therefore, target proteins overexpressed in the mother cell compartment during the late sporulation phase were expected to be targeted and displayed on the spore surface. As a proof of principle, we demonstrated the display of carboxymethylcellulase (CMCase) in its native form on the spore surface. The target protein, CMCase, was expressed under the control of the cry1Aa promoter, which is controlled by σ(E) and σ(K) and is expressed in the mother cell compartment. The correct display was confirmed using enzyme activity assays, flow cytometry, and immunogold electron microscopy. In addition, we demonstrated the display of a β-galactosidase tetramer and confirmed its correct display using enzyme activity assays and protein characterization. This native protein display system, combined with the robust nature of Bacillus spores, will broaden the range of displayable target proteins. Consequently, the applications of display technology will be expanded, including high-throughput screening, vaccines, biosensors, biocatalysis, bioremediation, and other innovative bioprocesses. PMID:25168353

  20. Spatial Arrangement Overrules Environmental Factors to Structure Native and Non-Native Assemblages of Synanthropic Harvestmen

    PubMed Central

    Muster, Christoph; Meyer, Marc; Sattler, Thomas

    2014-01-01

    Understanding how space affects the occurrence of native and non-native species is essential for inferring processes that shape communities. However, studies considering spatial and environmental variables for the entire community – as well as for the native and non-native assemblages in a single study – are scarce for animals. Harvestmen communities in central Europe have undergone drastic turnovers during the past decades, with several newly immigrated species, and thus provide a unique system to study such questions. We studied the wall-dwelling harvestmen communities from 52 human settlements in Luxembourg and found the assemblages to be largely dominated by non-native species (64% of specimens). Community structure was analysed using Moran's eigenvector maps as spatial variables, and landcover variables at different radii (500 m, 1000 m, 2000 m) in combination with climatic parameters as environmental variables. A surprisingly high portion of pure spatial variation (15.7% of total variance) exceeded the environmental (10.6%) and shared (4%) components of variation, but we found only minor differences between native and non-native assemblages. This could result from the ecological flexibility of both, native and non-native harvestmen that are not restricted to urban habitats but also inhabit surrounding semi-natural landscapes. Nevertheless, urban landcover variables explained more variation in the non-native community, whereas coverage of semi-natural habitats (forests, rivers) at broader radii better explained the native assemblage. This indicates that some urban characteristics apparently facilitate the establishment of non-native species. We found no evidence for competitive replacement of native by invasive species, but a community with novel combination of native and non-native species. PMID:24595309

  1. Bidirectional Transformation of a Metamorphic Protein between the Water-Soluble and Transmembrane Native States.

    PubMed

    Tanaka, Koji; Caaveiro, Jose M M; Tsumoto, Kouhei

    2015-11-24

    The bidirectional transformation of a protein between its native water-soluble and integral transmembrane conformations is demonstrated for FraC, a hemolytic protein of the family of pore-forming toxins. In the presence of biological membranes, the water-soluble conformation of FraC undergoes a remarkable structural reorganization generating cytolytic transmembrane nanopores conducive to cell death. So far, the reverse transformation from the native transmembrane conformation to the native water-soluble conformation has not been reported. We describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein. PMID:26544760

  2. Dynamics of Vacuoles and H+-Pyrophosphatase Visualized by Monomeric Green Fluorescent Protein in Arabidopsis: Artifactual Bulbs and Native Intravacuolar Spherical Structures[W][OPEN

    PubMed Central

    Segami, Shoji; Makino, Sachi; Miyake, Ai; Asaoka, Mariko; Maeshima, Masayoshi

    2014-01-01

    We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H+-pyrophosphatase (H+-PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H+-PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H+-PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H+-PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H+-PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed. PMID:25118245

  3. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    PubMed

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  4. Characterization of native PfABCG protein in Plasmodium falciparum.

    PubMed

    Edaye, Sonia; Georges, Elias

    2015-09-15

    The Plasmodium falciparum genome encodes 16 members of ABC proteins, with one member of the ABCG subfamily (PfABCG). Analysis of PfABCG amino acid sequence shows equal sequence identity to hsABCG1 and G2. Using N-terminal directed antibody against a recombinant fragment of PfABCG, we show that PfABCG migrates with an apparent molecular mass of 65KDa polypeptide on SDS-PAGE. PfABCG is expressed in all four stages of the parasite erythrocytic life cycle, with lower and higher expression in ring and late trophozoite stages, respectively. The protein localizes to the plasma membrane and a novel spherical structure beneath the cell membrane. Similar localization is also observed in gametocytes where PfABCG is highly expressed. Analysis of PfABCG genomic sequences for polymorphisms and changes in protein expression between different strains of P. falciparum revealed identical nucleotide sequence among the different strains, but variable protein expression. PfABCG expression is least in HB3 chloroquine sensitive strain, while higher expression levels are seen in other chloroquine-sensitive and -resistant strains, with highest levels of expression in 7G8. The differential expression of PfABCG in three chloroquine-sensitive strains (e.g., 3D7, HB3 and D10) predicts the sensitivity of the different strains to ketotifen, an anti-histaminic drug, whereby low expression is associated with decreased sensitivity to ketotifen. Taken together, the results in this report provide the first description of native PfABCG expression and subcellular localization in asexual stages of the parasite and its localization in gametocytes. It remains to be determined if PfABCG is functionally equivalent to mammalian ABCG1, ABCG2 or both. PMID:26239803

  5. Effects of compact volume and chain stiffness on the conformations of native proteins.

    PubMed Central

    Hao, M H; Rackovsky, S; Liwo, A; Pincus, M R; Scheraga, H A

    1992-01-01

    An investigation of the statistical properties of the native conformations of proteins, observed from crystal structures, is reported. Protein conformations were analyzed in terms of a bond vector correlation function and molecular volume. It was observed that, while the volume of a protein structure varies nearly linearly with the number of residues, the bond vector correlation function exhibits a universal feature for all sizes of proteins. To interpret the nature of the bond vector correlation function of native protein structures quantitatively, Monte Carlo simulations of realistic polypeptide chains of specific but arbitrary amino acid sequence were carried out. The molecule was constrained in an ellipsoidal volume determined by its chain length, and conformations with unacceptable nonbonded contacts between different amino acid residues were excluded. The interactions within a terminally blocked single residue, which correlate two nearest-neighbor peptide groups in a chain, were taken into account by an energetically biased sampling of its phi-psi space. The simulated chain correlation functions were found to be in good agreement with those of the crystal structures of beta-sheet-type and mixed-type (alpha+beta) proteins of similar length. On the basis of these calculations, it is concluded that the observed conformations of these native proteins may arise from two basic factors: the compactness of structures under hydrophobic interactions and the intrinsic stiffness of polypeptide chains due to the interactions within each terminally blocked residue. PMID:1631164

  6. Structural Properties of the Native Ligamentum Teres

    PubMed Central

    Philippon, Marc J.; Rasmussen, Matthew T.; Turnbull, Travis Lee; Trindade, Christiano A.C.; Hamming, Mark G.; Ellman, Michael B.; Harris, Matthew; LaPrade, Robert F.; Wijdicks, Coen A.

    2014-01-01

    Background: A majority of studies investigating the role of the ligamentum teres (LT) have focused primarily on anatomical and histological descriptions. To date, however, the structural properties of the LT have yet to be fully elucidated. Purpose: To investigate the structural properties of the native LT in a human cadaveric model. Study Design: Descriptive laboratory study. Methods: A total of 12 human cadaveric hemipelvises (mean age, 53.6 years; range, 34-63 years) were dissected free of all extra-articular soft tissues to isolate the LT and its acetabular and femoral attachments. A dynamic tensile testing machine distracted each femur in line with the fibers of the LT at a displacement-controlled rate of 0.5 mm/s. The anatomic dimensions, structural properties, and modes of failure were recorded. Results: The LT achieved a mean yield load of 75 N and ultimate failure load of 204 N. The LT had mean lengths of 38.0 and 53.0 mm at its yield and failure points, respectively. The most common (75% of specimens) mechanism of failure was tearing at the fovea capitis. On average, the LT had a linear stiffness of 16 N/mm and elastic modulus of 9.24 MPa. The mean initial length and cross-sectional area were 32 mm and 59 mm2, respectively. Conclusion: The human LT had a mean ultimate failure load of 204 N. Therefore, the results of this investigation, combined with recent biomechanical and outcomes studies, suggest that special consideration should be given to preserving the structural and corresponding biomechanical integrity of the LT during surgical intervention. Clinical Relevance: The LT may be more important as a static stabilizer of the hip joint than previously recognized. Further studies are recommended to investigate the appropriate indications to perform surgical repair or reconstruction of the LT for preservation of hip stability and function. PMID:26535290

  7. Chemical cross-linking and native mass spectrometry: A fruitful combination for structural biology

    PubMed Central

    Sinz, Andrea; Arlt, Christian; Chorev, Dror; Sharon, Michal

    2015-01-01

    Mass spectrometry (MS) is becoming increasingly popular in the field of structural biology for analyzing protein three-dimensional-structures and for mapping protein–protein interactions. In this review, the specific contributions of chemical crosslinking and native MS are outlined to reveal the structural features of proteins and protein assemblies. Both strategies are illustrated based on the examples of the tetrameric tumor suppressor protein p53 and multisubunit vinculin-Arp2/3 hybrid complexes. We describe the distinct advantages and limitations of each technique and highlight synergistic effects when both techniques are combined. Integrating both methods is especially useful for characterizing large protein assemblies and for capturing transient interactions. We also point out the future directions we foresee for a combination of in vivo crosslinking and native MS for structural investigation of intact protein assemblies. PMID:25970732

  8. Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes

    NASA Astrophysics Data System (ADS)

    Lu, Jonathan; Trnka, Michael J.; Roh, Soung-Hun; Robinson, Philip J. J.; Shiau, Carrie; Fujimori, Danica Galonic; Chiu, Wah; Burlingame, Alma L.; Guan, Shenheng

    2015-12-01

    Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise.

  9. Atomic-level analysis of membrane-protein structure.

    PubMed

    Hendrickson, Wayne A

    2016-06-01

    Membrane proteins are substantially more challenging than natively soluble proteins as subjects for structural analysis. Thus, membrane proteins are greatly underrepresented in structural databases. Recently, focused consortium efforts and advances in methodology for protein production, crystallographic analysis and cryo-EM analysis have accelerated the pace of atomic-level structure determination of membrane proteins. PMID:27273628

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

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  11. Tannin-assisted aggregation of natively unfolded proteins

    NASA Astrophysics Data System (ADS)

    Zanchi, D.; Narayanan, T.; Hagenmuller, D.; Baron, A.; Guyot, S.; Cabane, B.; Bouhallab, S.

    2008-06-01

    Tannin-protein interactions are essentially physical: hydrophobic and hydrogen-bond-mediated. We explored the tannin-assisted protein aggregation on the case of β-casein, which is a natively unfolded protein known for its ability to form micellar aggregates. We used several tannins with specified length. Our SAXS results show that small tannins increase the number of proteins per micelle, but keeping their size constant. It leads to a tannin-assisted compactization of micelles. Larger tannins, with linear dimensions greater than the crown width of micelles, lead to the aggregation of micelles by a bridging effect. Experimental results can be understood within a model where tannins are treated as effective enhancers of hydrophobic attraction between specific sites in proteins.

  12. Turnip yellow mosaic virus forms infectious particles without the native beta-annulus structure and flexible coat protein N-terminus.

    PubMed

    Powell, Joshua D; Barbar, Elisar; Dreher, Theo W

    2012-01-20

    Structural studies have implicated the TYMV N-terminal amino acids of the coat protein (CP) in both static (virion stabilization) and dynamic (RNA encapsidation and disencapsidation) roles. We have deleted residues 2-5, 2-10 and 2-26 from the N-terminus and expressed the mutant CPs in E. coli to assess assembly in the absence of genomic RNA and in plant infections to assess infectivity and virion properties. In E. coli, the deletion constructs formed virus-like particles, but in decreased yield. All mutants were infectious in Chinese cabbage, producing normal symptoms but with a slight delay and decreased viral yields. Virions were progressively less stable with increasing deletion size and also more accessible to small molecules. These results show that the N-terminal 26 amino acids are not essential for viral processes in vivo, although removal of these residues decreases stability and increases porosity, both important factors for virion integrity and survival outside the host. PMID:22078163

  13. Extracting knowledge from protein structure geometry

    PubMed Central

    Røgen, Peter; Koehl, Patrice

    2013-01-01

    Protein structure prediction techniques proceed in two steps, namely the generation of many structural models for the protein of interest, followed by an evaluation of all these models to identify those that are native-like. In theory, the second step is easy, as native structures correspond to minima of their free energy surfaces. It is well known however that the situation is more complicated as the current force fields used for molecular simulations fail to recognize native states from misfolded structures. In an attempt to solve this problem we follow an alternate approach and derive a new potential from geometric knowledge extracted from native and misfolded conformers of protein structures. This new potential, MPP, has two main features that are key to its success. Firstly, it is composite in that it includes local and non local geometric information on proteins. At the short range level it captures and quantifies the mapping between the sequences and structures of short (7-mer) fragments of protein backbones through the introduction of a new local energy term. The local energy term is then augmented with a non local residue-based pairwise potential, and a solvent potential. Secondly, it is optimized to yield a maximized correlation between the energy of a structural model and its RMS to the native structure of the corresponding protein. We have shown that MPP yields high correlation values between RMS and energy and that it is able to retrieve the native structure of a protein from a set of high-resolution decoys. PMID:23280479

  14. Native topology determines force-induced unfolding pathways in globular proteins

    NASA Astrophysics Data System (ADS)

    Klimov, D. K.; Thirumalai, D.

    2000-06-01

    Single-molecule manipulation techniques reveal that stretching unravels individually folded domains in the muscle protein titin and the extracellular matrix protein tenascin. These elastic proteins contain tandem repeats of folded domains with -sandwich architecture. Herein, we propose by stretching two model sequences (S1 and S2) with four-stranded -barrel topology that unfolding forces and pathways in folded domains can be predicted by using only the structure of the native state. Thermal refolding of S1 and S2 in the absence of force proceeds in an all-or-none fashion. In contrast, phase diagrams in the force-temperature (f,T) plane and steered Langevin dynamics studies of these sequences, which differ in the native registry of the strands, show that S1 unfolds in an allor-none fashion, whereas unfolding of S2 occurs via an obligatory intermediate. Force-induced unfolding is determined by the native topology. After proving that the simulation results for S1 and S2 can be calculated by using native topology alone, we predict the order of unfolding events in Ig domain (Ig27) and two fibronectin III type domains (9FnIII and 10FnIII). The calculated unfolding pathways for these proteins, the location of the transition states, and the pulling speed dependence of the unfolding forces reflect the differences in the way the strands are arranged in the native states. We also predict the mechanisms of force-induced unfolding of the coiled-coil spectrin (a three-helix bundle protein) for all 20 structures deposited in the Protein Data Bank. Our approach suggests a natural way to measure the phase diagram in the (f,C) plane, where C is the concentration of denaturants.

  15. Serial crystallographic analysis of protein isomorphous replacement data from a mixture of native and derivative microcrystals.

    PubMed

    Zhang, Tao; Yao, Deqiang; Wang, Jiawei; Gu, Yuanxin; Fan, Haifu

    2015-12-01

    A post-experimental identification/purification procedure similar to that described in Zhang et al. [(2015), IUCrJ, 2, 322-326] has been proposed for use in the treatment of multiphase protein serial crystallography (SX) diffraction snapshots. As a proof of concept, the procedure was tested using theoretical serial femtosecond crystallography (SFX) data from a mixture containing native and derivatized crystals of a protein. Two known proteins were taken as examples. Multiphase diffraction snapshots were subjected to two rounds of indexing using the program CrystFEL [White et al. (2012). J. Appl. Cryst. 45, 335-341]. In the first round, an ab initio indexing was performed to derive a set of approximate primitive unit-cell parameters, which are roughly the average of those from the native protein and the derivative. These parameters were then used in a second round of indexing as input to CrystFEL. The results were then used to separate the diffraction snapshots into two subsets corresponding to the native and the derivative. For each test sample, integration of the two subsets of snapshots separately led to two sets of three-dimensional diffraction intensities, one belonging to the native and the other to the derivative. Based on these two sets of intensities, a conventional single isomorphous replacement (SIR) procedure solved the structure easily. PMID:26627658

  16. Native globular actin has a thermodynamically unstable quasi-stationary structure with elements of intrinsic disorder.

    PubMed

    Kuznetsova, Irina M; Povarova, Olga I; Uversky, Vladimir N; Turoverov, Konstantin K

    2016-02-01

    The native form of globular actin, G-actin, is formed in vivo as a result of complex post-translational folding processes that require ATP energy expenditure and are assisted by the 70 kDa heat shock protein, prefoldin and chaperonin containing TCP-1. G-actin is stabilized by the binding of one ATP molecule and one Ca(2+) ion (or Mg(2+) in vivo). Chemical denaturants, heating or Ca(2+) removal transform native actin (N) into 'inactivated actin' (I), a compact oligomer comprising 14-16 subunits. Viscogenic and crowding agents slow this process but do not stop it. The lack of calcium in the solution accelerates the spontaneous N → I transition. Thus, native G-actin has a kinetically stable (as a result of the high free energy barrier between the N and I states) but thermodynamically unstable structure, which, in the absence of Ca(2+) or other bivalent metal ions, spontaneously converts to the thermodynamically stable I state. It was noted that native actin has much in common with intrinsically disordered proteins: it has functionally important disordered regions; it is constantly in complex with one of its numerous partners; and it plays key roles in many cellular processes, in a manner similar to disordered hub proteins. By analyzing actin folding in vivo and unfolding in vitro, we advanced the hypothesis that proteins in a native state may have a thermodynamically unstable quasi-stationary structure. The kinetically stable native state of these proteins appears forcibly under the influence of intracellular folding machinery. The denaturation of such proteins is always irreversible because the inactivated state, for which the structure is determined by the amino acid sequence of a protein, comprises the thermodynamically stable state under physiological conditions. PMID:26460158

  17. Proteins with Highly Similar Native Folds Can Show Vastly Dissimilar Folding Behavior When Desolvated**

    PubMed Central

    Schennach, Moritz; Breuker, Kathrin

    2014-01-01

    Proteins can be exposed to vastly different environments such as the cytosol or membranes, but the delicate balance between external factors and intrinsic determinants of protein structure, stability, and folding is only poorly understood. Here we used electron capture dissociation to study horse and tuna heart Cytochromes c in the complete absence of solvent. The significantly different stability of their highly similar native folds after transfer into the gas phase, and their strikingly different folding behavior in the gas phase, can be rationalized on the basis of electrostatic interactions such as salt bridges. In the absence of hydrophobic bonding, protein folding is far slower and more complex than in solution. PMID:24259450

  18. Protein complex analysis of native brain potassium channels by proteomics.

    PubMed

    Sandoz, Guillaume; Lesage, Florian

    2008-01-01

    TREK potassium channels belong to a family of channel subunits with two-pore domains (K(2P)). TREK1 knockout mice display impaired polyunsaturated fatty acid-mediated protection against brain ischemia, reduced sensitivity to volatile anesthetics, resistance to depression and altered perception of pain. Recently, we isolated native TREK1 channels from mouse brain and identified their specific components by mass spectrometry. Among the identified partners, the A-Kinase Anchoring Protein AKAP150 binds to a regulatory domain of TREK1 and acts as a molecular switch. It transforms low activity, outwardly rectifying TREK1 currents into robust leak conductances resistant to stimulation by arachidonic acid, membrane stretch and acidification. Inhibition of the TREK1/AKAP150 channel by Gs-coupled receptors is as extensive as for TREK1 alone (but faster) whereas inhibition of TREK1/AKAP150 by Gq-coupled receptors is reduced. Furthermore, the association of AKAP150 with TREK1 channels integrates them into postsynaptic scaffolds where G protein-coupled membrane receptors and channels dock simultaneously. This chapter describes the proteomic approach used to study the composition of native TREK1 channels and point out its advantages and limitations over more classical methods (two-hybrid screenings in the yeast and bacteria or GST-pull down). PMID:18998088

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

    PubMed

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

    2016-04-01

    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. PMID:27015007

  20. Structural and spectroscopic studies of the native hemocyanin from Maia squinado and its structural subunits

    NASA Astrophysics Data System (ADS)

    Dolashka-Angelova, Pavlina; Hristova, Rumijana; Schuetz, Juergen; Stoeva, Stanka; Schwarz, Heinz; Voelter, Wolfgang

    2000-09-01

    The dodecameric hemocyanin of the crab Maia squinado contains five major electrophoretically separable polypeptide chains (structural subunits) which have been purified by FPLC ion exchange chromatography. The various proteins have been characterized by fluorescence spectroscopy, combined with fluorescence quenching studies, using acrylamide, caesium chloride and potassium iodide as tryptophan quenchers. The results show that the tryptophyl side chains of dodecameric Hc are deeply buried in hydrophobic regions of the hemocyanin aggregates and the quenching efficiency values for the native Hc in comparison with those from the constituent subunits are two to four times less. The conformational stabilities of the native dodecameric aggregate and its isolated structural subunits towards various denaturants (pH, temperature, guanidinium hydrochloride) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby, both, the oxy- and apo-forms of the protein have been considered. The critical temperatures for the structural subunits, Tc, determined by fluorescence spectroscopy, are in the region of 50-60°C, coinciding with the melting temperatures, Tm, determined by CD spectroscopy. The free energy of stabilization in water, Δ GDH 2O , toward guanidinium hydrochloride is about two times higher for the dodecamer as compared to the isolated subunits. These studies reveal that oligomerization between functional subunits has a stabilizing effect on the whole molecule and differences in the primary structures result in different stabilities of the subunits.

  1. New Supercharging Reagents Produce Highly Charged Protein Ions in Native Mass Spectrometry

    PubMed Central

    Going, Catherine C.; Xia, Zijie; Williams, Evan R.

    2015-01-01

    The effectiveness of two new supercharging reagents for producing highly charged ions by electrospray ionization (ESI) from aqueous solutions in which proteins have native structures and reactivities were investigated. In aqueous solution, 2-thiophenone and 4-hydroxymethyl-1,3-dioxolan-2-one (HD) at a concentration of 2% by volume can increase the average charge of cytochrome c and myoglobin by up to 163%, resulting in even higher charge states than those that are produced from water/methanol/acid solutions in which proteins are denatured. The greatest extent of supercharging occurs in pure water, but these supercharging reagents are also highly effective in aqueous solutions containing 200 mM ammonium acetate buffer commonly used in native mass spectrometry (MS). These reagents are less effective supercharging reagents than m-nitrobenzyl alcohol (m-NBA) and propylene carbonate (PC) when ions are formed from water/methanol/acid. The extent to which loss of the heme group from myoglobin occurs is related to the extent of supercharging. Results from guanidine melts of cytochrome c monitored with tryptophan fluorescence show that the supercharging reagents PC, sulfolane and HD are effective chemical denaturants in solution. These results provide additional evidence for the role of protein structural changes in the electrospray droplet as the primary mechanism for supercharging with these reagents in native MS. These results also demonstrate that for at least some proteins, the formation of highly charged ions from native MS is no longer a significant barrier for obtaining structural information using conventional tandem MS methods. PMID:26421324

  2. New supercharging reagents produce highly charged protein ions in native mass spectrometry.

    PubMed

    Going, Catherine C; Xia, Zijie; Williams, Evan R

    2015-11-01

    The effectiveness of two new supercharging reagents for producing highly charged ions by electrospray ionization (ESI) from aqueous solutions in which proteins have native structures and reactivities were investigated. In aqueous solution, 2-thiophenone and 4-hydroxymethyl-1,3-dioxolan-2-one (HD) at a concentration of 2% by volume can increase the average charge of cytochrome c and myoglobin by up to 163%, resulting in even higher charge states than those that are produced from water/methanol/acid solutions in which these proteins are denatured. The greatest extent of supercharging occurs in pure water, but these supercharging reagents are also highly effective in aqueous solutions containing 200 mM ammonium acetate buffer commonly used in native mass spectrometry (MS). These reagents are less effective supercharging reagents than m-nitrobenzyl alcohol (m-NBA) and propylene carbonate (PC) when ions are formed from water/methanol/acid. The extent to which loss of the heme group from myoglobin occurs is related to the extent of supercharging. Results from guanidine melts of cytochrome c monitored with tryptophan fluorescence show that the supercharging reagents PC, sulfolane and HD are effective chemical denaturants in solution. These results provide additional evidence for the role of protein structural changes in the electrospray droplet as the primary mechanism for supercharging with these reagents in native MS. These results also demonstrate that for at least some proteins, the formation of highly charged ions from native MS is no longer a significant barrier for obtaining structural information using conventional tandem MS methods. PMID:26421324

  3. Isolation of Camelid Single-Domain Antibodies Against Native Proteins Using Recombinant Multivalent Peptide Ligands.

    PubMed

    Alturki, Norah A; Henry, Kevin A; MacKenzie, C Roger; Arbabi-Ghahroudi, Mehdi

    2015-01-01

    Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (1) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (2) expression, purification, and characterization of the multivalent peptide-VT ligands; (3) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (4) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield custom peptide-VT ligands that appear to maintain the antigenic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest. PMID:26424272

  4. Sucralose Destabilization of Protein Structure.

    PubMed

    Chen, Lee; Shukla, Nimesh; Cho, Inha; Cohn, Erin; Taylor, Erika A; Othon, Christina M

    2015-04-16

    Sucralose is a commonly employed artificial sweetener that behaves very differently than its natural disaccharide counterpart, sucrose, in terms of its interaction with biomolecules. The presence of sucralose in solution is found to destabilize the native structure of two model protein systems: the globular protein bovine serum albumin and an enzyme staphylococcal nuclease. The melting temperature of these proteins decreases as a linear function of sucralose concentration. We correlate this destabilization to the increased polarity of the molecule. The strongly polar nature is manifested as a large dielectric friction exerted on the excited-state rotational diffusion of tryptophan using time-resolved fluorescence anisotropy. Tryptophan exhibits rotational diffusion proportional to the measured bulk viscosity for sucrose solutions over a wide range of concentrations, consistent with a Stokes-Einstein model. For sucralose solutions, however, the diffusion is dependent on the concentration, strongly diverging from the viscosity predictions, and results in heterogeneous rotational diffusion. PMID:26263149

  5. Structural alphabets for protein structure classification: a comparison study.

    PubMed

    Le, Quan; Pollastri, Gianluca; Koehl, Patrice

    2009-03-27

    Finding structural similarities between proteins often helps reveal shared functionality, which otherwise might not be detected by native sequence information alone. Such similarity is usually detected and quantified by protein structure alignment. Determining the optimal alignment between two protein structures, however, remains a hard problem. An alternative approach is to approximate each three-dimensional protein structure using a sequence of motifs derived from a structural alphabet. Using this approach, structure comparison is performed by comparing the corresponding motif sequences or structural sequences. In this article, we measure the performance of such alphabets in the context of the protein structure classification problem. We consider both local and global structural sequences. Each letter of a local structural sequence corresponds to the best matching fragment to the corresponding local segment of the protein structure. The global structural sequence is designed to generate the best possible complete chain that matches the full protein structure. We use an alphabet of 20 letters, corresponding to a library of 20 motifs or protein fragments having four residues. We show that the global structural sequences approximate well the native structures of proteins, with an average coordinate root mean square of 0.69 A over 2225 test proteins. The approximation is best for all alpha-proteins, while relatively poorer for all beta-proteins. We then test the performance of four different sequence representations of proteins (their native sequence, the sequence of their secondary-structure elements, and the local and global structural sequences based on our fragment library) with different classifiers in their ability to classify proteins that belong to five distinct folds of CATH. Without surprise, the primary sequence alone performs poorly as a structure classifier. We show that addition of either secondary-structure information or local information from the

  6. Differences in ecological structure, function, and native species abundance between native and invaded Hawaiian streams.

    PubMed

    Holitzki, Tara M; MacKenzie, Richard A; Wiegner, Tracy N; McDermid, Karla J

    2013-09-01

    Poeciliids, one of the most invasive species worldwide, are found on almost every continent and have been identified as an "invasive species of concern" in the United States, New Zealand, and Australia. Despite their global prevalence, few studies have quantified their impacts on tropical stream ecosystem structure, function, and biodiversity. Utilizing Hawaiian streams as model ecosystems, we documented how ecological structure, function, and native species abundance differed between poeciliid-free and poeciliid-invaded tropical streams. Stream nutrient yields, benthic biofilm biomass, densities of macroinvertebrates and fish, and community structures of benthic algae, macroinvertebrates, and fish were compared between streams with and without established poeciliid populations on the island of Hawai'i, Hawaii, USA. Sum nitrate (sigmaNO3(-) = NO3(-) + NO2(-)), total nitrogen, and total organic carbon yields were eight times, six times, and five times higher, respectively, in poeciliid streams than in poeciliid-free streams. Benthic biofilm ash-free dry mass was 1.5x higher in poeciliid streams than in poeciliid-free streams. Percentage contributions of chironomids and hydroptilid caddisflies to macroinvertebrate densities were lower in poeciliid streams compared to poeciliid-free streams, while percentage contributions of Cheumatopsyche analis caddisflies, Dugesia sp. flatworms, and oligochaetes were higher. Additionally, mean densities of native gobies were two times lower in poeciliid streams than in poeciliid-free ones, with poeciliid densities being approximately eight times higher than native fish densities. Our results, coupled with the wide distribution of invasive poeciliids across Hawaii and elsewhere in the tropics, suggest that poeciliids may negatively impact the ecosystem structure, function, and native species abundance of tropical streams they invade. This underscores the need for increased public awareness to prevent future introductions and for

  7. Residues in substrate proteins that interact with GroEL in the capture process are buried in the native state

    NASA Astrophysics Data System (ADS)

    Stan, George; Brooks, Bernard R.; Lorimer, George H.; Thirumalai, D.

    2006-03-01

    We have used a bioinformatic approach to predict the natural substrate proteins for the Escherichia coli chaperonin GroEL based on two simple criteria. Natural substrate proteins should contain binding motifs similar in sequence to the mobile loop peptide of GroES that displaces the binding motif during the chaperonin cycle. Secondly, each substrate protein should contain multiple copies of the binding motif so that the chaperonin can perform "work" on the substrate protein. To validate these criteria, we have used a database of 252 proteins that have been experimentally shown to interact with the chaperonin machinery in vivo. More than 80% are identified by these criteria. The binding motifs of all 79 proteins in the database with a known three-dimensional structure are buried (<50% solvent-accessible surface area) in the native state. Our results show that the binding motifs are inaccessible in the native state but become solvent-exposed in unfolded state, thus enabling GroEL to distinguish between unfolded and native states. The structures of the binding motif in the native states of the substrate proteins include -helices, -strands, and random coils. The diversity of secondary structures implies that there are large and varied conformational transitions in the recognition motifs after their displacement by the mobile loops of GroES. chaperonin | E. coli | natural substrates | recognition motif

  8. Influences of Membrane Mimetic Environments on Membrane Protein Structures

    PubMed Central

    Zhou, Huan-Xiang; Cross, Timothy A.

    2013-01-01

    The number of membrane protein structures in the Protein Data Bank is becoming significant and growing. Here, the transmembrane domain structures of the helical membrane proteins are evaluated to assess the influences of the membrane mimetic environments. Toward this goal, many of the biophysical properties of membranes are discussed and contrasted with those of the membrane mimetics commonly used for structure determination. Although the mimetic environments can perturb the protein structures to an extent that potentially gives rise to misinterpretation of functional mechanisms, there are also many structures that have a native-like appearance. From this assessment, an initial set of guidelines is proposed for distinguishing native-like from nonnative-like membrane protein structures. With experimental techniques for validation and computational methods for refinement and quality assessment and enhancement, there are good prospects for achieving native-like structures for these very important proteins. PMID:23451886

  9. Probing RNA Native Conformational Ensembles with Structural Constraints.

    PubMed

    Fonseca, Rasmus; van den Bedem, Henry; Bernauer, Julie

    2016-05-01

    Noncoding ribonucleic acids (RNA) play a critical role in a wide variety of cellular processes, ranging from regulating gene expression to post-translational modification and protein synthesis. Their activity is modulated by highly dynamic exchanges between three-dimensional conformational substates, which are difficult to characterize experimentally and computationally. Here, we present an innovative, entirely kinematic computational procedure to efficiently explore the native ensemble of RNA molecules. Our procedure projects degrees of freedom onto a subspace of conformation space defined by distance constraints in the tertiary structure. The dimensionality reduction enables efficient exploration of conformational space. We show that the conformational distributions obtained with our method broadly sample the conformational landscape observed in NMR experiments. Compared to normal mode analysis-based exploration, our procedure diffuses faster through the experimental ensemble while also accessing conformational substates to greater precision. Our results suggest that conformational sampling with a highly reduced but fully atomistic representation of noncoding RNA expresses key features of their dynamic nature. PMID:27028235

  10. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

    In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

  11. Localizing chemical groups while imaging single native proteins by high-resolution atomic force microscopy.

    PubMed

    Pfreundschuh, Moritz; Alsteens, David; Hilbert, Manuel; Steinmetz, Michel O; Müller, Daniel J

    2014-05-14

    Simultaneous high-resolution imaging and localization of chemical interaction sites on single native proteins is a pertinent biophysical, biochemical, and nanotechnological challenge. Such structural mapping and characterization of binding sites is of importance in understanding how proteins interact with their environment and in manipulating such interactions in a plethora of biotechnological applications. Thus far, this challenge remains to be tackled. Here, we introduce force-distance curve-based atomic force microscopy (FD-based AFM) for the high-resolution imaging of SAS-6, a protein that self-assembles into cartwheel-like structures. Using functionalized AFM tips bearing Ni(2+)-N-nitrilotriacetate groups, we locate specific interaction sites on SAS-6 at nanometer resolution and quantify the binding strength of the Ni(2+)-NTA groups to histidine residues. The FD-based AFM approach can readily be applied to image any other native protein and to locate and structurally map histidine residues. Moreover, the surface chemistry used to functionalize the AFM tip can be modified to map other chemical interaction sites. PMID:24766578

  12. Structure of mutant human oncogene protein determined

    SciTech Connect

    Baum, R.

    1989-01-16

    The protein encoded by a mutant human oncogene differs only slightly in structure from the native protein that initiates normal cell division, a finding that may complicate efforts to develop inhibitors of the mutant protein. Previously, the x-ray structure of the protein encoded by the normal c-Ha-ras gene, a protein believed to signal cells to start or stop dividing through its interaction with guanosine triphosphate (GTP), was reported. The structure of the protein encoded by a transforming c-Ha-ras oncogene, in which a valine codon replaces the normal glycine codon at position 12 in the gene, has now been determined. The differences in the structures of the mutant and normal proteins are located primarily in a loop that interacts with the /beta/-phosphate of a bound guanosine diphosphate (GDP) molecule.

  13. The effect of denaturants on protein structure.

    PubMed Central

    Dunbar, J.; Yennawar, H. P.; Banerjee, S.; Luo, J.; Farber, G. K.

    1997-01-01

    Virtually all studies of the protein-folding reaction add either heat, acid, or a chemical denaturant to an aqueous protein solution in order to perturb the protein structure. When chemical denaturants are used, very high concentrations are usually necessary to observe any change in protein structure. In a solution with such high denaturant concentrations, both the structure of the protein and the structure of the solvent around the protein can be altered. X-ray crystallography is the obvious experimental technique to probe both types of changes. In this paper, we report the crystal structures of dihydrofolate reductase with urea and of ribonuclease A with guanidinium chloride. These two classic denaturants have similar effects on the native structure of the protein. The most important change that occurs is a reduction in the overall thermal factor. These structures offer a molecular explanation for the reduction in mobility. Although the reduction is observed only with the native enzyme in the crystal, a similar decrease in mobility has also been observed in the unfolded state in solution (Makhatadze G, Privalov PL. 1992. Protein interactions with urea and guanidinium chloride: A calorimetric study. PMID:9260285

  14. Native display of complete foreign protein domains on the surface of hepatitis B virus capsids

    PubMed Central

    Kratz, Peter A.; Böttcher, Bettina; Nassal, Michael

    1999-01-01

    The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein’s primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy. PMID:10051569

  15. Analysis of low-density lipoprotein-associated proteins using the method of digitized native protein mapping.

    PubMed

    Jin, Ya; Chen, Jin; Wang, Ahui; Zhang, Jun; Chen, Shumin; Manabe, Takashi; Tan, Wen

    2016-07-01

    The method of digitized native protein mapping, combining nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS (in data-independent acquisition mode, or MS(E) ), was improved by using a new MS/MS mode, ion mobility separation enhanced-MS(E) (HDMS(E) ), and applied to analyze the area of human plasma low-density lipoprotein (LDL). An 18 mm × 4.8 mm rectangular area which included LDL on a nondenaturing micro 2D gel of human plasma was grid-cut into 72 square gel pieces and subjected to quantitative LC-MS/MS. Compared with MS(E) , HDMS(E) showed significantly higher performance, by assigning 50% more proteins and detecting each protein in more squares. A total of 253 proteins were assigned with LC-HDMS(E) and the quantity distribution of each was reconstructed as a native protein map. The maps showed that Apo B-100 was the most abundant protein in the grid-cut area, concentrated at pI ca. 5.4-6.1 and apparent mass ca. 1000 kDa, which corresponded to four gel pieces, squares 39-42. An Excel macro was prepared to search protein maps which showed protein quantity peaks localized within this concentrated region of Apo B-100. Twenty-two proteins out of the 252 matched this criterion, in which 19 proteins have been reported to be associated with LDL. This method only requires several microliters of a plasma sample and the principle of the protein separation is totally different from the commonly used ultracentrifugation. The results obtained by this method would provide new insights on the structure and function of LDL. PMID:27174546

  16. Slipknotting upon native-like loop formation in a trefoil knot protein.

    PubMed

    Noel, Jeffrey K; Sułkowska, Joanna I; Onuchic, José N

    2010-08-31

    Protein knots and slipknots, mostly regarded as intriguing oddities, are gradually being recognized as significant structural motifs. Recent experimental results show that knotting, starting from a fully extended polypeptide, has not yet been observed. Understanding the nucleation process of folding knots is thus a natural challenge for both experimental and theoretical investigation. In this study, we employ energy landscape theory and molecular dynamics to elucidate the entire folding mechanism. The full free energy landscape of a knotted protein is mapped using an all-atom structure-based protein model. Results show that, due to the topological constraint, the protein folds through a three-state mechanism that contains (i) a precise nucleation site that creates a correctly twisted native loop (first barrier) and (ii) a rate-limiting free energy barrier that is traversed by two parallel knot-forming routes. The main route corresponds to a slipknot conformation, a collapsed configuration where the C-terminal helix adopts a hairpin-like configuration while threading, and the minor route to an entropically limited plug motion, where the extended terminus is threaded as through a needle. Knot formation is a late transition state process and results show that random (nonspecific) knots are a very rare and unstable set of configurations both at and below folding temperature. Our study shows that a native-biased landscape is sufficient to fold complex topologies and presents a folding mechanism generalizable to all known knotted protein topologies: knotting via threading a native-like loop in a preordered intermediate. PMID:20702769

  17. Native mass spectrometry and ion mobility characterize the orange carotenoid protein functional domains.

    PubMed

    Zhang, Hao; Liu, Haijun; Lu, Yue; Wolf, Nathan R; Gross, Michael L; Blankenship, Robert E

    2016-06-01

    Orange Carotenoid Protein (OCP) plays a unique role in protecting many cyanobacteria from light-induced damage. The active form of OCP is directly involved in energy dissipation by binding to the phycobilisome (PBS), the major light-harvesting complex in cyanobacteria. There are two structural modules in OCP, an N-terminal domain (NTD), and a C-terminal domain (CTD), which play different functional roles during the OCP-PBS quenching cycle. Because of the quasi-stable nature of active OCP, structural analysis of active OCP has been lacking compared to its inactive form. In this report, partial proteolysis was used to generate two structural domains, NTD and CTD, from active OCP. We used multiple native mass spectrometry (MS) based approaches to interrogate the structural features of the NTD and the CTD. Collisional activation and ion mobility analysis indicated that the NTD releases its bound carotenoid without forming any intermediates and the CTD is resistant to unfolding upon collisional energy ramping. The unfolding intermediates observed in inactive intact OCP suggest that it is the N-terminal extension and the NTD-CTD loop that lead to the observed unfolding intermediates. These combined approaches extend the knowledge of OCP photo-activation and structural features of OCP functional domains. Combining native MS, ion mobility, and collisional activation promises to be a sensitive new approach for studies of photosynthetic protein-pigment complexes. PMID:26921809

  18. Normal Modes of Prion Proteins: From Native to Infectious particle◊

    PubMed Central

    Samson, Abraham O.; Levitt, Michael

    2011-01-01

    Prion proteins (PrP) are the infectious agent in transmissible spongiform encephalopathies (i.e. mad cow disease). To be infectious, prion proteins must undergo a conformational change involving a decrease of α-helical content along with an increase of β-strand structure. This conformational change was evaluated by means of elastic normal modes. Elastic normal modes show a diminution of two α-helices by one and two residues, as well as an extension of two β-strands by three residues each which could instigate the conformational change. The conformational change occurs in a region that is compatible with immunological studies, and it is observed more frequently in mutant prions which are prone to conversion, than in WT prions due to differences in their starting structures, which are amplified through normal modes. These findings are valuable for our comprehension of the conversion mechanism associated with the conformational change of prion proteins. PMID:21338080

  19. Thermally responsive silicon nanowire arrays for native/denatured-protein separation

    NASA Astrophysics Data System (ADS)

    Wang, Hongwei; Wang, Yanwei; Yuan, Lin; Wang, Lei; Yang, Weikang; Wu, Zhaoqiang; Li, Dan; Chen, Hong

    2013-03-01

    We present our findings of the selective adsorption of native and denatured proteins onto thermally responsive, native-protein resistant poly(N-isopropylacrylamide) (PNIPAAm) decorated silicon nanowire arrays (SiNWAs). The PNIPAAm-SiNWAs surface, which shows very low levels of native-protein adsorption, favors the adsorption of denatured proteins. The amount of denatured-protein adsorption is higher at temperatures above the lower critical solution temperature (LCST) of PNIPAAm. Temperature cycling surrounding the LCST, which ensures against thermal denaturation of native proteins, further increases the amount of denatured-protein adsorption. Moreover, the PNIPAAm-SiNWAs surface is able to selectively adsorb denatured protein even from mixtures of different protein species; meanwhile, the amount of native proteins in solution is kept nearly at its original level. It is believed that these results will not only enrich current understanding of protein interactions with PNIPAAm-modified SiNWAs surfaces, but may also stimulate applications of PNIPAAm-SiNWAs surfaces for native/denatured protein separation.

  20. The Native Form and Maturation Process of Hepatitis C Virus Core Protein

    PubMed Central

    Yasui, Kohichiroh; Wakita, Takaji; Tsukiyama-Kohara, Kyoko; Funahashi, Shin-Ichi; Ichikawa, Masumi; Kajita, Tadahiro; Moradpour, Darius; Wands, Jack R.; Kohara, Michinori

    1998-01-01

    The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles. PMID:9621068

  1. CoDNaS 2.0: a comprehensive database of protein conformational diversity in the native state

    PubMed Central

    Monzon, Alexander Miguel; Rohr, Cristian Oscar; Fornasari, María Silvina; Parisi, Gustavo

    2016-01-01

    CoDNaS (conformational diversity of the native state) is a protein conformational diversity database. Conformational diversity describes structural differences between conformers that define the native state of proteins. It is a key concept to understand protein function and biological processes related to protein functions. CoDNaS offers a well curated database that is experimentally driven, thoroughly linked, and annotated. CoDNaS facilitates the extraction of key information on small structural differences based on protein movements. CoDNaS enables users to easily relate the degree of conformational diversity with physical, chemical and biological properties derived from experiments on protein structure and biological characteristics. The new version of CoDNaS includes ∼70% of all available protein structures, and new tools have been added that run sequence searches, display structural flexibility profiles and allow users to browse the database for different structural classes. These tools facilitate the exploration of protein conformational diversity and its role in protein function. Database URL: http://ufq.unq.edu.ar/codnas PMID:27022160

  2. CoDNaS 2.0: a comprehensive database of protein conformational diversity in the native state.

    PubMed

    Monzon, Alexander Miguel; Rohr, Cristian Oscar; Fornasari, María Silvina; Parisi, Gustavo

    2016-01-01

    CoDNaS (conformational diversity of the native state) is a protein conformational diversity database. Conformational diversity describes structural differences between conformers that define the native state of proteins. It is a key concept to understand protein function and biological processes related to protein functions. CoDNaS offers a well curated database that is experimentally driven, thoroughly linked, and annotated. CoDNaS facilitates the extraction of key information on small structural differences based on protein movements. CoDNaS enables users to easily relate the degree of conformational diversity with physical, chemical and biological properties derived from experiments on protein structure and biological characteristics. The new version of CoDNaS includes ∼70% of all available protein structures, and new tools have been added that run sequence searches, display structural flexibility profiles and allow users to browse the database for different structural classes. These tools facilitate the exploration of protein conformational diversity and its role in protein function. Database URL:http://ufq.unq.edu.ar/codnas. PMID:27022160

  3. Protein structures in SDS micelle-protein complexes.

    PubMed Central

    Parker, W; Song, P S

    1992-01-01

    Sodium dodecyl sulfate (SDS) is used more often than any other detergent as an excellent denaturing or "unfolding" detergent. However, formation of ordered structure (alpha-helix or beta-sheet) in certain peptides is known to be induced by interaction with SDS micelles. The SDS-induced structures formed by these peptides are amphiphilic, having both a hydrophobic and a hydrophilic face. Previous work in this area has revealed that SDS induces helical folding in a wide variety of non-helical proteins. Here, we describe the interaction of several structurally unrelated proteins with SDS micelles and the correlation of these structures to helical amphiphilic regions present in the primary sequence. It is likely that the ability of native nonordered protein structures to form induced amphiphilic ordered structures is rather common. PMID:1600087

  4. Structural investigations on native collagen type I fibrils using AFM

    SciTech Connect

    Strasser, Stefan; Zink, Albert; Janko, Marek; Heckl, Wolfgang M.; Thalhammer, Stefan . E-mail: stefan.thalhammer@gsf.de

    2007-03-02

    This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.

  5. Structural and Inherent Case in the Non-Native Processing of Spanish: Constraints on Inflectional Variability

    ERIC Educational Resources Information Center

    Hopp, Holger; León Arriaga, Mayra E.

    2016-01-01

    This article reports an eye-tracking study on the native and non-native processing of case in Spanish. Twenty-four native and 27 first language (L1) German non-native speakers of Spanish were tested on their sensitivity to case marking violations involving structural case with objects of ditransitive verbs and to violations of inherent case for…

  6. Normal modes of prion proteins: from native to infectious particle.

    PubMed

    Samson, Abraham O; Levitt, Michael

    2011-03-29

    Prion proteins (PrP) are the infectious agent in transmissible spongiform encephalopathies (i.e., mad cow disease). To be infectious, prion proteins must undergo a conformational change involving a decrease in α-helical content along with an increase in β-strand content. This conformational change was evaluated by means of elastic normal modes. Elastic normal modes show a diminution of two α-helices by one and two residues, as well as an extension of two β-strands by three residues each, which could instigate the conformational change. The conformational change occurs in a region that is compatible with immunological studies, and it is observed more frequently in mutant prions that are prone to conversion than in wild-type prions because of differences in their starting structures, which are amplified through normal modes. These findings are valuable for our comprehension of the conversion mechanism associated with the conformational change in prion proteins. PMID:21338080

  7. Mapping Protein Conformational Landscapes under Strongly Native Conditions with Hydrogen Exchange Mass Spectrometry.

    PubMed

    Witten, Jacob; Ruschak, Amy; Poterba, Timothy; Jaramillo, Alexis; Miranker, Andrew D; Jaswal, Sheila S

    2015-08-01

    The thermodynamic stability and kinetic barriers separating protein conformations under native conditions are critical for proper protein function and for understanding dysfunction in diseases of protein conformation. Traditional methods to probe protein unfolding and folding employ denaturants and highly non-native conditions, which may destabilize intermediate species or cause irreversible aggregation, especially at the high protein concentrations typically required. Hydrogen exchange (HX) is ideal for detecting conformational behavior under native conditions without the need for denaturants, but detection by NMR is limited to small highly soluble proteins. Mass spectrometry (MS) can, in principle, greatly extend the applicability of native-state HX to larger proteins and lower concentrations. However, quantitative analysis of HXMS profiles is currently limited by experimental and theoretical challenges. Here we address both limitations, by proposing an approach based on using standards to eliminate the systematic experimental artifacts in HXMS profiles, and developing the theoretical framework to describe HX behavior across all regimes based on the Linderstrøm-Lang formalism. We demonstrate proof of principle by a practical application to native-state HX of a globular protein. The framework and the practical tools developed advance the ability of HXMS to extract thermodynamic and kinetic conformational parameters of proteins under native conditions. PMID:26146955

  8. Genetic variation and population structure in native Americans.

    PubMed

    Wang, Sijia; Lewis, Cecil M; Jakobsson, Mattias; Ramachandran, Sohini; Ray, Nicolas; Bedoya, Gabriel; Rojas, Winston; Parra, Maria V; Molina, Julio A; Gallo, Carla; Mazzotti, Guido; Poletti, Giovanni; Hill, Kim; Hurtado, Ana M; Labuda, Damian; Klitz, William; Barrantes, Ramiro; Bortolini, Maria Cátira; Salzano, Francisco M; Petzl-Erler, Maria Luiza; Tsuneto, Luiza T; Llop, Elena; Rothhammer, Francisco; Excoffier, Laurent; Feldman, Marcus W; Rosenberg, Noah A; Ruiz-Linares, Andrés

    2007-11-01

    We examined genetic diversity and population structure in the American landmass using 678 autosomal microsatellite markers genotyped in 422 individuals representing 24 Native American populations sampled from North, Central, and South America. These data were analyzed jointly with similar data available in 54 other indigenous populations worldwide, including an additional five Native American groups. The Native American populations have lower genetic diversity and greater differentiation than populations from other continental regions. We observe gradients both of decreasing genetic diversity as a function of geographic distance from the Bering Strait and of decreasing genetic similarity to Siberians--signals of the southward dispersal of human populations from the northwestern tip of the Americas. We also observe evidence of: (1) a higher level of diversity and lower level of population structure in western South America compared to eastern South America, (2) a relative lack of differentiation between Mesoamerican and Andean populations, (3) a scenario in which coastal routes were easier for migrating peoples to traverse in comparison with inland routes, and (4) a partial agreement on a local scale between genetic similarity and the linguistic classification of populations. These findings offer new insights into the process of population dispersal and differentiation during the peopling of the Americas. PMID:18039031

  9. Genetic Variation and Population Structure in Native Americans

    PubMed Central

    Ramachandran, Sohini; Ray, Nicolas; Bedoya, Gabriel; Rojas, Winston; Parra, Maria V; Molina, Julio A; Gallo, Carla; Mazzotti, Guido; Poletti, Giovanni; Hill, Kim; Hurtado, Ana M; Labuda, Damian; Klitz, William; Barrantes, Ramiro; Bortolini, Maria Cátira; Salzano, Francisco M; Petzl-Erler, Maria Luiza; Tsuneto, Luiza T; Llop, Elena; Rothhammer, Francisco; Excoffier, Laurent; Feldman, Marcus W; Rosenberg, Noah A; Ruiz-Linares, Andrés

    2007-01-01

    We examined genetic diversity and population structure in the American landmass using 678 autosomal microsatellite markers genotyped in 422 individuals representing 24 Native American populations sampled from North, Central, and South America. These data were analyzed jointly with similar data available in 54 other indigenous populations worldwide, including an additional five Native American groups. The Native American populations have lower genetic diversity and greater differentiation than populations from other continental regions. We observe gradients both of decreasing genetic diversity as a function of geographic distance from the Bering Strait and of decreasing genetic similarity to Siberians—signals of the southward dispersal of human populations from the northwestern tip of the Americas. We also observe evidence of: (1) a higher level of diversity and lower level of population structure in western South America compared to eastern South America, (2) a relative lack of differentiation between Mesoamerican and Andean populations, (3) a scenario in which coastal routes were easier for migrating peoples to traverse in comparison with inland routes, and (4) a partial agreement on a local scale between genetic similarity and the linguistic classification of populations. These findings offer new insights into the process of population dispersal and differentiation during the peopling of the Americas. PMID:18039031

  10. Multipass Membrane Protein Structure Prediction Using Rosetta

    PubMed Central

    Yarov-Yarovoy, Vladimir; Schonbrun, Jack; Baker, David

    2006-01-01

    We describe the adaptation of the Rosetta de novo structure prediction method for prediction of helical transmembrane protein structures. The membrane environment is modeled by embedding the protein chain into a model membrane represented by parallel planes defining hydrophobic, interface, and polar membrane layers for each energy evaluation. The optimal embedding is determined by maximizing the exposure of surface hydrophobic residues within the membrane and minimizing hydrophobic exposure outside of the membrane. Protein conformations are built up using the Rosetta fragment assembly method and evaluated using a new membrane-specific version of the Rosetta low-resolution energy function in which residue–residue and residue–environment interactions are functions of the membrane layer in addition to amino acid identity, distance, and density. We find that lower energy and more native-like structures are achieved by sequential addition of helices to a growing chain, which may mimic some aspects of helical protein biogenesis after translocation, rather than folding the whole chain simultaneously as in the Rosetta soluble protein prediction method. In tests on 12 membrane proteins for which the structure is known, between 51 and 145 residues were predicted with root-mean-square deviation <4Å from the native structure. PMID:16372357

  11. Sucralose Destabilization of Protein Structure

    NASA Astrophysics Data System (ADS)

    Cho, Inha; Chen, Lee; Shukla, Nimesh; Othon, Christina

    2015-03-01

    Sucralose is a commonly employed artificial sweetener. Sucralose behaves very differently than its natural disaccharide counterpart, sucrose, in terms of its interaction with biomolecules. The presence of sucralose in solution is found to destabilize the native structure of the globular protein Bovine Serum Albumin (BSA). The melting temperature decreases as a linear function of sucralose concentration. We correlate this destabilization with the increased polarity of the sucralose molecule as compared to sucrose. The strongly polar nature is observed as a large dielectric friction exerted on the excited state rotational diffusion of tryptophan using time-resolved fluorescence anisotropy. Tryptophan exhibits rotational diffusion proportional to the measured bulk viscosity for sucrose solutions over a wide range of concentrations, consistent with a Stokes-Einstein diffusional model. For sucralose solutions however, the diffusion is linearly dependent with the concentration, strongly diverging from the viscosity predictions. The polar nature of sucralose causes a dramatically different interaction with biomolecules than natural disaccharide molecules. Connecticut Space Grant Consortium.

  12. Release of Native-like Gaseous Proteins from Electrospray Droplets via the Charged Residue Mechanism: Insights from Molecular Dynamics Simulations.

    PubMed

    McAllister, Robert G; Metwally, Haidy; Sun, Yu; Konermann, Lars

    2015-10-01

    The mechanism whereby gaseous protein ions are released from charged solvent droplets during electrospray ionization (ESI) remains a matter of debate. Also, it is unclear to what extent electrosprayed proteins retain their solution structure. Molecular dynamics (MD) simulations offer insights into the temporal evolution of protein systems. Surprisingly, there have been no all-atom simulations of the protein ESI process to date. The current work closes this gap by investigating the behavior of protein-containing aqueous nanodroplets that carry excess positive charge. We focus on "native ESI", where proteins initially adopt their biologically active solution structures. ESI proceeds while the protein remains entrapped within the droplet. Protein release into the gas phase occurs upon solvent evaporation to dryness. Droplet shrinkage is accompanied by ejection of charge carriers (Na(+) for the conditions chosen here), keeping the droplet at ∼85% of the Rayleigh limit throughout its life cycle. Any remaining charge carriers bind to the protein as the final solvent molecules evaporate. The outcome of these events is largely independent of the initial protein charge and the mode of charge carrier binding. ESI charge states and collision cross sections of the MD structures agree with experimental data. Our results confirm the Rayleigh/charged residue model (CRM). Field emission of excess Na(+) plays an ancillary role by governing the net charge of the shrinking droplet. Models that envision protein ejection from the droplet are not supported. Most nascent CRM ions retain native-like conformations. For unfolded proteins ESI likely proceeds along routes that are different from the native state mechanism explored here. PMID:26325619

  13. High-resolution atomic force microscopy and spectroscopy of native membrane proteins

    NASA Astrophysics Data System (ADS)

    Bippes, Christian A.; Muller, Daniel J.

    2011-08-01

    Membranes confining cells and cellular compartments are essential for life. Membrane proteins are molecular machines that equip cell membranes with highly sophisticated functionality. Examples of such functions are signaling, ion pumping, energy conversion, molecular transport, specific ligand binding, cell adhesion and protein trafficking. However, it is not well understood how most membrane proteins work and how the living cell regulates their function. We review how atomic force microscopy (AFM) can be applied for structural and functional investigations of native membrane proteins. High-resolution time-lapse AFM imaging records membrane proteins at work, their oligomeric state and their dynamic assembly. The AFM stylus resembles a multifunctional toolbox that allows the measurement of several chemical and physical parameters at the nanoscale. In the single-molecule force spectroscopy (SMFS) mode, AFM quantifies and localizes interactions in membrane proteins that stabilize their folding and modulate their functional state. Dynamic SMFS discloses fascinating insights into the free energy landscape of membrane proteins. Single-cell force spectroscopy quantifies the interactions of live cells with their environment to single-receptor resolution. In the future, technological progress in AFM-based approaches will enable us to study the physical nature of biological interactions in more detail and decipher how cells control basic processes.

  14. Efficient Synthesis of Peptide and Protein Functionalized Pyrrole-Imidazole Polyamides Using Native Chemical Ligation

    PubMed Central

    Janssen, Brian M. G.; van Ommeren, Sven P. F. I.; Merkx, Maarten

    2015-01-01

    The advancement of DNA-based bionanotechnology requires efficient strategies to functionalize DNA nanostructures in a specific manner with other biomolecules, most importantly peptides and proteins. Common DNA-functionalization methods rely on laborious and covalent conjugation between DNA and proteins or peptides. Pyrrole-imidazole (Py–Im) polyamides, based on natural minor groove DNA-binding small molecules, can bind to DNA in a sequence specific fashion. In this study, we explore the use of Py–Im polyamides for addressing proteins and peptides to DNA in a sequence specific and non-covalent manner. A generic synthetic approach based on native chemical ligation was established that allows efficient conjugation of both peptides and recombinant proteins to Py–Im polyamides. The effect of Py–Im polyamide conjugation on DNA binding was investigated by Surface Plasmon Resonance (SPR). Although the synthesis of different protein-Py–Im-polyamide conjugates was successful, attenuation of DNA affinity was observed, in particular for the protein-Py–Im-polyamide conjugates. The practical use of protein-Py–Im-polyamide conjugates for addressing DNA structures in an orthogonal but non-covalent manner, therefore, remains to be established. PMID:26053396

  15. Compaction of ribosomal protein S6 by sucrose occurs only under native conditions.

    PubMed

    Chen, LuYang; Ferreira, José A B; Costa, Sílvia M B; Cabrita, Gonçalo J M; Otzen, Daniel E; Melo, Eduardo Pinho

    2006-02-21

    The effect of osmolyte sucrose on the stability and compaction of the folded and unfolded states of ribosomal protein S6 from Thermus thermophilus was analyzed. Confirming previous results obtained with sodium sulfate and trehalose, refolding stopped-flow measurements of S6 show that sucrose favors the conversion of the unfolded state ensemble to a highly compact structure (75% as compact as the folded state). This conversion occurs when the unfolded state is suddenly placed under native conditions and the compact state accumulates in a transient off-folding pathway. This effect of sucrose on the compaction of the unfolded state ensemble is counteracted by guanidinium hydrochloride. The compact state does not accumulate at higher guanidinium concentrations and the unfolded state ensemble does not display increased compaction in the presence of 6 M guanidinium as evaluated by collisional quenching of tryptophan fluorescence. In contrast, accessibility of the tryptophan residue of folded S6 above 1 M sucrose concentration decreased as a result of an increased compaction of the folded state. Unfolding stopped-flow measurements of S6 reflect this increased compaction of the folded state, but the unfolding pathway is not affected by sucrose. Compaction of folded and unfolded S6 induced by sucrose occurs under native conditions indicating that decreased protein conformational entropy significantly contributes to the mechanism of protein stabilization by osmolytes. PMID:16475807

  16. Interaction between dimer interface residues of native and mutated SOD1 protein: a theoretical study.

    PubMed

    Keerthana, S P; Kolandaivel, P

    2015-04-01

    Cu-Zn superoxide dismutase 1 (SOD1) is a highly conserved bimetallic protein enzyme, used for the scavenging the superoxide radicals (O2 (-)) produced due to aerobic metabolism in the mitochondrial respiratory chain. Over 100 mutations have been identified and found to be in the homodimeric structure of SOD1. The enzyme has to be maintained in its dimeric state for the structural stability and enzymatic activity. From our investigation, we found that the mutations apart from the dimer interface residues are found to affect the dimer stability of protein and hence enhancing the aggregation and misfolding tendency of mutated protein. The homodimeric state of SOD1 is found to be held together by the non-covalent interactions. The molecular dynamics simulation has been used to study the hydrogen bond interactions between the dimer interface residues of the monomers in native and mutated forms of SOD1 in apo- and holo-states. The results obtained by this analysis reveal the fact that the loss of hydrogen bond interactions between the monomers of the dimer is responsible for the reduced stability of the apo- and holo-mutant forms of SOD1. The conformers with dimer interface residues in native and mutated protein obtained by the molecular dynamics simulation is subjected to quantum mechanical study using M052X/6-31G(d) level of theory. The charge transfer between N-H···O interactions in the dimer interface residues were studied. The weak interaction between the monomers of the dimer accounts for the reduced dimerization and enhanced deformation energy in the mutated SOD1 protein. PMID:25578810

  17. Interplaying roles of native topology and chain length in marginally cooperative and noncooperative folding of small protein fragments

    NASA Astrophysics Data System (ADS)

    Badasyan, Artem; Liu, Zhirong; Chan, Hue Sun

    Coarse-grained chain simulations were used to study fragments of two homologous proteins of the peripheral subunit-binding domain (PSBD) family, Bacillus stearothermophilus PSBD (E3BD) and Escherichia coli 2-oxo-glutarate dehydrogenase PSBD (BBL). To ascertain a robust rank order of folding cooperativity, native-centric intraprotein interactions were modeled by (i) a common Gō;-like potential, and (ii) native-centric potentials with desolvation barriers or (iii) many-body terms. Homologous proteins can possess substantially different folding cooperativity. Consistent with experiment, our calculations indicated that E3BD fragments fold more cooperatively than BBL fragments of approximately the same chain length. For a given fragment, native contacts deduced from Protein Data Bank structures can vary significantly depending on the number of residues that the structure encompasses in addition to those of the fragment itself, resulting in variation in model folding cooperativity predicted using different native contact sets for the same fragment. This observation underscores that folding cooperativity of these fragments can be extremely sensitive to change in chain length. Thus, a ˜40-residue protein fragment's folding cooperativity, or lack thereof, does not necessarily imply essentially identical behaviors for super- or sub-fragments with only several residues more, or less, than the given fragment. Ramifications for experimental investigations of downhill folding are discussed.

  18. Ensemble-based evaluation for protein structure models

    PubMed Central

    Jamroz, Michal; Kolinski, Andrzej; Kihara, Daisuke

    2016-01-01

    Motivation: Comparing protein tertiary structures is a fundamental procedure in structural biology and protein bioinformatics. Structure comparison is important particularly for evaluating computational protein structure models. Most of the model structure evaluation methods perform rigid body superimposition of a structure model to its crystal structure and measure the difference of the corresponding residue or atom positions between them. However, these methods neglect intrinsic flexibility of proteins by treating the native structure as a rigid molecule. Because different parts of proteins have different levels of flexibility, for example, exposed loop regions are usually more flexible than the core region of a protein structure, disagreement of a model to the native needs to be evaluated differently depending on the flexibility of residues in a protein. Results: We propose a score named FlexScore for comparing protein structures that consider flexibility of each residue in the native state of proteins. Flexibility information may be extracted from experiments such as NMR or molecular dynamics simulation. FlexScore considers an ensemble of conformations of a protein described as a multivariate Gaussian distribution of atomic displacements and compares a query computational model with the ensemble. We compare FlexScore with other commonly used structure similarity scores over various examples. FlexScore agrees with experts’ intuitive assessment of computational models and provides information of practical usefulness of models. Availability and implementation: https://bitbucket.org/mjamroz/flexscore Contact: dkihara@purdue.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27307633

  19. The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

    PubMed Central

    Suda, Kitaru; Filipek, Slawomir; Palczewski, Krzysztof; Engel, Andreas; Fotiadis, Dimitrios

    2005-01-01

    Summary Rhodopsin, the prototypical G-protein-coupled receptor, which is densely packed in the disc membranes of rod outer segments, was proposed to function as a monomer. However, a growing body of evidence indicates dimerization and oligomerization of numerous G-protein-coupled receptors, and atomic force microscopy images revealed rows of rhodopsin dimers in murine disc membranes. In this work we demonstrate by electron microscopy of negatively stained samples, blue native- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, chemical crosslinking, and by proteolysis that native bovine rhodopsin exists mainly as dimers and higher oligomers. These results corroborate the recent findings from atomic force microscopy and molecular modeling on the supramolecular structure and packing arrangement of murine rhodopsin dimers. PMID:15764373

  20. Protein Structure Databases.

    PubMed

    Laskowski, Roman A

    2016-01-01

    Web-based protein structure databases come in a wide variety of types and levels of information content. Those having the most general interest are the various atlases that describe each experimentally determined protein structure and provide useful links, analyses, and schematic diagrams relating to its 3D structure and biological function. Also of great interest are the databases that classify 3D structures by their folds as these can reveal evolutionary relationships which may be hard to detect from sequence comparison alone. Related to these are the numerous servers that compare folds-particularly useful for newly solved structures, and especially those of unknown function. Beyond these are a vast number of databases for the more specialized user, dealing with specific families, diseases, structural features, and so on. PMID:27115626

  1. Charge site assignment in native proteins by ultraviolet photodissociation (UVPD) mass spectrometry.

    PubMed

    Morrison, Lindsay J; Brodbelt, Jennifer S

    2016-01-01

    Characterization of all gas-phase charge sites of natively sprayed proteins and peptides is demonstrated using 193 nm UVPD. The high sequence coverage offered by UVPD is exploited for the accurate determination of charge sites in protein systems up to 18 kDa, allowing charge site to be studied as a function of protein conformation and the presence of disulfide bonds. Charging protons are found on both basic sidechains and on the amide backbone of less basic amino acids such as serine, glutamine, and proline. UVPD analysis was performed on the 3+ charge state of melittin, the 5+ to 8+ charge states of ubiquitin, and the 8+ charge state of reduced and oxidized β-lactoglobulin. The location of charges in gas-phase proteins is known to impact structure; molecular modeling of different charge site motifs of 3+ melittin demonstrates how placement of protons in simulations can dramatically impact the predicted structure of the molecule. The location of positive charge sites in ubiquitin and β-lactoglobulin are additionally found to depend on the presence or absence of salt-bridges, columbic repulsion across the length of the peptide, and protein conformation. Charge site isomers are demonstrated for ubiquitin and β-lactoglobulin but found to be much less numerous than previously predicted. PMID:26596460

  2. Insights into Lysine Deacetylation of Natively Folded Substrate Proteins by Sirtuins.

    PubMed

    Knyphausen, Philipp; de Boor, Susanne; Kuhlmann, Nora; Scislowski, Lukas; Extra, Antje; Baldus, Linda; Schacherl, Magdalena; Baumann, Ulrich; Neundorf, Ines; Lammers, Michael

    2016-07-01

    Sirtuins are NAD(+)-dependent lysine deacylases, regulating a variety of cellular processes. The nuclear Sirt1, the cytosolic Sirt2, and the mitochondrial Sirt3 are robust deacetylases, whereas the other sirtuins have preferences for longer acyl chains. Most previous studies investigated sirtuin-catalyzed deacylation on peptide substrates only. We used the genetic code expansion concept to produce natively folded, site-specific, and lysine-acetylated Sirt1-3 substrate proteins, namely Ras-related nuclear, p53, PEPCK1, superoxide dismutase, cyclophilin D, and Hsp10, and analyzed the deacetylation reaction. Some acetylated proteins such as Ras-related nuclear, p53, and Hsp10 were robustly deacetylated by Sirt1-3. However, other reported sirtuin substrate proteins such as cyclophilin D, superoxide dismutase, and PEPCK1 were not deacetylated. Using a structural and functional approach, we describe the ability of Sirt1-3 to deacetylate two adjacent acetylated lysine residues. The dynamics of this process have implications for the lifetime of acetyl modifications on di-lysine acetylation sites and thus constitute a new mechanism for the regulation of proteins by acetylation. Our studies support that, besides the primary sequence context, the protein structure is a major determinant of sirtuin substrate specificity. PMID:27226597

  3. Label-free detection of native proteins by surface-enhanced Raman spectroscopy using iodide-modified nanoparticles.

    PubMed

    Xu, Li-Jia; Zong, Cheng; Zheng, Xiao-Shan; Hu, Pei; Feng, Jia-Min; Ren, Bin

    2014-02-18

    Proteins perform vital functional and structural duties in living systems, and the in-depth investigation of protein in its native state is one of the most important challenges in the postgenomic era. Surface-enhanced Raman spectroscopy (SERS) can provide the intrinsic fingerprint information of samples with ultrahigh sensitivity but suffers from the reproducibility and reliability issues. In this paper, we proposed an iodide-modified Ag nanoparticles method (Ag IMNPs) for label-free detection of proteins. The silver nanoparticles provide the huge enhancement to boost the Raman signal of proteins, and the coated iodide layer offers a barrier to prevent the direct interaction between the proteins and the metal surface, helping to keep the native structures of proteins. With this method, highly reproducible and high-quality SERS signals of five typical proteins (lysozyme, avidin, bovine serum albumin, cytochrome c, and hemoglobin) have been obtained, and the SERS features of the proteins without chromophore were almost identical to the respective normal Raman spectra. This unique feature allows the qualitative identification of them by simply taking the intensity ratio of the Raman peaks of tryptophan to phenylalanine residues. We further demonstrated that the method can also be used for label-free multiplex analysis of protein mixture as well as to study the dynamic process of protein damage stimulated by hydrogen peroxide. This method proves to be very promising for further applications in proteomics and biomedical research. PMID:24460183

  4. Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.

    PubMed

    Su, C; Wang, F; Ciolek, D; Pan, Y C

    1994-11-15

    Electrophoresis of proteins and protein-protein complexes in polyacrylamide gels under native conditions using a horizontal gel apparatus is described. The advantage of this system is that it permits the detection of both negatively and positively charged proteins as well as protein-protein complexes in the same gel. During electrophoresis, a continuous gel sandwiched between two glass plates is placed horizontally on the platform and submerged in a reservoir buffer. The sample wells are made along the center of the gel, allowing positively and negatively charged proteins to migrate toward the cathode and anode, respectively. Several proteins with varying molecular weights and isoelectric point (pI) values and pairs of proteins capable of forming protein-protein complexes were chosen as model systems to illustrate the methodology. The effects of several parameters on the performance of the gel system including protein molecular weight, pI, and gel concentration were also examined and the results obtained by this method are comparable to those obtained by the vertical system. Following electrophoresis, both negatively and positively charged proteins as well as protein-protein complexes can be transferred by electroblotting onto polyvinylidene difluoride membranes for further analyses. PMID:7695108

  5. Structural and Functional Analysis of the Native Peripherin-ROM1 Complex Isolated from Photoreceptor Cells*

    PubMed Central

    Kevany, Brian M.; Tsybovsky, Yaroslav; Campuzano, Iain D. G.; Schnier, Paul D.; Engel, Andreas; Palczewski, Krzysztof

    2013-01-01

    Peripherin and its homologue ROM1 are retina-specific members of the tetraspanin family of integral membrane proteins required for morphogenesis and maintenance of photoreceptor outer segments, regions that collect light stimuli. Over 100 pathogenic mutations in peripherin cause inherited rod- and cone-related dystrophies in humans. Peripherin and ROM1 interact in vivo and are predicted to form a core heterotetrameric complex capable of creating higher order oligomers. However, structural analysis of tetraspanin proteins has been hampered by their resistance to crystallization. Here we present a simplified methodology for high yield purification of peripherin-ROM1 from bovine retinas that permitted its biochemical and biophysical characterization. Using size exclusion chromatography and blue native gel electrophoresis, we confirmed that the core native peripherin-ROM1 complex exists as a tetramer. Peripherin, but not ROM1, is glycosylated and we examined the glycosylation site and glycan composition of ROM1 by liquid chromatographic tandem mass spectrometry. Mass spectrometry was used to analyze the native complex in detergent micelles, demonstrating its tetrameric state. Our electron microscopy-generated structure solved to 18 Å displayed the tetramer as an elongated structure with an apparent 2-fold symmetry. Finally, we demonstrated that peripherin-ROM1 tetramers induce membrane curvature when reconstituted in lipid vesicles. These results provide critical insights into this key retinal component with a poorly defined function. PMID:24196967

  6. Molecular determinants of expansivity of native globular proteins: a pressure perturbation calorimetry study.

    PubMed

    Vasilchuk, Daniel; Pandharipande, Pranav P; Suladze, Saba; Sanchez-Ruiz, Jose M; Makhatadze, George I

    2014-06-12

    There is a growing interest in understanding how hydrostatic pressure (P) impacts the thermodynamic stability (ΔG) of globular proteins. The pressure dependence of stability is defined by the change in volume upon denaturation, ΔV = (∂ΔG/∂P)T. The temperature dependence of change in volume upon denaturation itself is defined by the changes in thermal expansivity (ΔE), ΔE = (∂ΔV/∂T)P. The pressure perturbation calorimetry (PPC) allows direct experimental measurement of the thermal expansion coefficient, α = E/V, of a protein in the native, αN(T), and unfolded, αU(T), states as a function of temperature. We have shown previously that αU(T) is a nonlinear function of temperature but can be predicted well from the amino acid sequence using α(T) values for individual amino acids (J. Phys. Chem. B 2010, 114, 16166-16170). In this work, we report PPC results on a diverse set of nine proteins and discuss molecular factors that can potentially influence the thermal expansion coefficient, αN(T), and the thermal expansivity, EN(T), of proteins in the native state. Direct experimental measurements by PPC show that αN(T) and EN(T) functions vary significantly for different proteins. Using comparative analysis and site-directed mutagenesis, we have eliminated the role of various structural or thermodynamic properties of these proteins such as the number of amino acid residues, secondary structure content, packing density, electrostriction, dynamics, or thermostability. We have also shown that αN(T) and EN,sp(T) functions for a given protein are rather insensitive to the small changes in the amino acid sequence, suggesting that αN(T) and EN(T) functions might be defined by a topology of a given protein fold. This conclusion is supported by the similarity of αN(T) and EN(T) functions for six resurrected ancestral thioredoxins that vary in sequence but have very similar tertiary structure. PMID:24849138

  7. Functional NifD-K fusion protein in Azotobacter vinelandii is a homodimeric complex equivalent to the native heterotetrameric MoFe protein

    SciTech Connect

    Lahiri, Surobhi; Pulakat, Lakshmi; Gavini, Nara . E-mail: gavini@biology.msstate.edu

    2005-11-18

    The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous {alpha} and {beta} subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length {lambda}CI of the pBT bait vector and also NifD-K (fusion) with the N-terminal {alpha}-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the {beta}-{beta} interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the {alpha}-RNAP of the pTRG vector and {lambda}CI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the {beta}-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the {beta}-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the {beta

  8. The Native Structure and Composition of the Cruciferin Complex in Brassica napus

    PubMed Central

    Nietzel, Thomas; Dudkina, Natalya V.; Haase, Christin; Denolf, Peter; Semchonok, Dmitry A.; Boekema, Egbert J.; Braun, Hans-Peter; Sunderhaus, Stephanie

    2013-01-01

    Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300–390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space. PMID:23192340

  9. Advances in methods for native expression and purification of RNA for structural studies

    PubMed Central

    Batey, Robert T.

    2014-01-01

    Many RNAs present unique challenges in obtaining material suitable for structural or biophysical characterization. These issues include synthesis of chemically and conformationally homogeneous RNAs, refolding RNA purified using denaturing preparation techniques, and avoiding chemical damage. To address these challenges, new methodologies in RNA expression and purification have been developed seeking to emulate those commonly used for protein purifications. In this review, recent developments in the preparation of high-quality RNA for structural biology and biophysical applications are discussed, with an emphasis on native methods. PMID:24607442

  10. Protein Structure Recognition: From Eigenvector Analysis to Structural Threading Method

    SciTech Connect

    Haibo Cao

    2003-12-12

    In this work, they try to understand the protein folding problem using pair-wise hydrophobic interaction as the dominant interaction for the protein folding process. They found a strong correlation between amino acid sequences and the corresponding native structure of the protein. Some applications of this correlation were discussed in this dissertation include the domain partition and a new structural threading method as well as the performance of this method in the CASP5 competition. In the first part, they give a brief introduction to the protein folding problem. Some essential knowledge and progress from other research groups was discussed. This part includes discussions of interactions among amino acids residues, lattice HP model, and the design ability principle. In the second part, they try to establish the correlation between amino acid sequence and the corresponding native structure of the protein. This correlation was observed in the eigenvector study of protein contact matrix. They believe the correlation is universal, thus it can be used in automatic partition of protein structures into folding domains. In the third part, they discuss a threading method based on the correlation between amino acid sequences and ominant eigenvector of the structure contact-matrix. A mathematically straightforward iteration scheme provides a self-consistent optimum global sequence-structure alignment. The computational efficiency of this method makes it possible to search whole protein structure databases for structural homology without relying on sequence similarity. The sensitivity and specificity of this method is discussed, along with a case of blind test prediction. In the appendix, they list the overall performance of this threading method in CASP5 blind test in comparison with other existing approaches.

  11. Protein-protein structure prediction by scoring molecular dynamics trajectories of putative poses.

    PubMed

    Sarti, Edoardo; Gladich, Ivan; Zamuner, Stefano; Correia, Bruno E; Laio, Alessandro

    2016-09-01

    The prediction of protein-protein interactions and their structural configuration remains a largely unsolved problem. Most of the algorithms aimed at finding the native conformation of a protein complex starting from the structure of its monomers are based on searching the structure corresponding to the global minimum of a suitable scoring function. However, protein complexes are often highly flexible, with mobile side chains and transient contacts due to thermal fluctuations. Flexibility can be neglected if one aims at finding quickly the approximate structure of the native complex, but may play a role in structure refinement, and in discriminating solutions characterized by similar scores. We here benchmark the capability of some state-of-the-art scoring functions (BACH-SixthSense, PIE/PISA and Rosetta) in discriminating finite-temperature ensembles of structures corresponding to the native state and to non-native configurations. We produce the ensembles by running thousands of molecular dynamics simulations in explicit solvent starting from poses generated by rigid docking and optimized in vacuum. We find that while Rosetta outperformed the other two scoring functions in scoring the structures in vacuum, BACH-SixthSense and PIE/PISA perform better in distinguishing near-native ensembles of structures generated by molecular dynamics in explicit solvent. Proteins 2016; 84:1312-1320. © 2016 Wiley Periodicals, Inc. PMID:27253756

  12. A homology/ab initio hybrid algorithm for sampling near-native protein conformations.

    PubMed

    Dhingra, Priyanka; Jayaram, Bhyravabhotla

    2013-08-15

    One of the major challenges for protein tertiary structure prediction strategies is the quality of conformational sampling algorithms, which can effectively and readily search the protein fold space to generate near-native conformations. In an effort to advance the field by making the best use of available homology as well as fold recognition approaches along with ab initio folding methods, we have developed Bhageerath-H Strgen, a homology/ab initio hybrid algorithm for protein conformational sampling. The methodology is tested on the benchmark CASP9 dataset of 116 targets. In 93% of the cases, a structure with TM-score ≥ 0.5 is generated in the pool of decoys. Further, the performance of Bhageerath-H Strgen was seen to be efficient in comparison with different decoy generation methods. The algorithm is web enabled as Bhageerath-H Strgen web tool which is made freely accessible for protein decoy generation (http://www.scfbio-iitd.res.in/software/Bhageerath-HStrgen1.jsp). PMID:23728619

  13. Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations.

    PubMed

    Matveev, Vladimir V

    2010-01-01

    According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity."One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity."Josiah Willard Gibbs (1839-1903). PMID:20534114

  14. Native aggregation as a cause of origin of temporary cellular structures needed for all forms of cellular activity, signaling and transformations

    PubMed Central

    2010-01-01

    According to the hypothesis explored in this paper, native aggregation is genetically controlled (programmed) reversible aggregation that occurs when interacting proteins form new temporary structures through highly specific interactions. It is assumed that Anfinsen's dogma may be extended to protein aggregation: composition and amino acid sequence determine not only the secondary and tertiary structure of single protein, but also the structure of protein aggregates (associates). Cell function is considered as a transition between two states (two states model), the resting state and state of activity (this applies to the cell as a whole and to its individual structures). In the resting state, the key proteins are found in the following inactive forms: natively unfolded and globular. When the cell is activated, secondary structures appear in natively unfolded proteins (including unfolded regions in other proteins), and globular proteins begin to melt and their secondary structures become available for interaction with the secondary structures of other proteins. These temporary secondary structures provide a means for highly specific interactions between proteins. As a result, native aggregation creates temporary structures necessary for cell activity. "One of the principal objects of theoretical research in any department of knowledge is to find the point of view from which the subject appears in its greatest simplicity." Josiah Willard Gibbs (1839-1903) PMID:20534114

  15. Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein.

    PubMed

    Tailhades, Julien; Sethi, Ashish; Petrie, Emma J; Gooley, Paul R; Bathgate, Ross A; Wade, John D; Hossain, Mohammed A

    2016-01-18

    The inhibition of the G protein-coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41-residue and three-disulfide cross-bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N(α) -deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca(2+) ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production. PMID:26612092

  16. Machine-learning scoring functions for identifying native poses of ligands docked to known and novel proteins

    PubMed Central

    2015-01-01

    Background Molecular docking is a widely-employed method in structure-based drug design. An essential component of molecular docking programs is a scoring function (SF) that can be used to identify the most stable binding pose of a ligand, when bound to a receptor protein, from among a large set of candidate poses. Despite intense efforts in developing conventional SFs, which are either force-field based, knowledge-based, or empirical, their limited docking power (or ability to successfully identify the correct pose) has been a major impediment to cost-effective drug discovery. Therefore, in this work, we explore a range of novel SFs employing different machine-learning (ML) approaches in conjunction with physicochemical and geometrical features characterizing protein-ligand complexes to predict the native or near-native pose of a ligand docked to a receptor protein's binding site. We assess the docking accuracies of these new ML SFs as well as those of conventional SFs in the context of the 2007 PDBbind benchmark dataset on both diverse and homogeneous (protein-family-specific) test sets. Further, we perform a systematic analysis of the performance of the proposed SFs in identifying native poses of ligands that are docked to novel protein targets. Results and conclusion We find that the best performing ML SF has a success rate of 80% in identifying poses that are within 1 Å root-mean-square deviation from the native poses of 65 different protein families. This is in comparison to a success rate of only 70% achieved by the best conventional SF, ASP, employed in the commercial docking software GOLD. In addition, the proposed ML SFs perform better on novel proteins that they were never trained on before. We also observed steady gains in the performance of these scoring functions as the training set size and number of features were increased by considering more protein-ligand complexes and/or more computationally-generated poses for each complex. PMID:25916860

  17. Analysis of Correlations between Energy and Residue Fluctuations in Native Proteins and Determination of Specific Sites for Binding

    NASA Astrophysics Data System (ADS)

    Haliloglu, Turkan; Erman, Burak

    2009-02-01

    The Gaussian network model is used to derive the correlations between energy and residue fluctuations in native proteins. Residues are identified that respond strongly to energy fluctuations and that display correlations with the remaining residues of the protein at the highest modes. We postulate that these residues are located at specific sites for drug binding. We test the validity of this postulate on a data set of 33 structurally distinct proteins in the unbound state. Detailed results are presented for drug binding to the HIV protease.

  18. Elucidating the chemical structure of native 1-deoxysphingosine.

    PubMed

    Steiner, Regula; Saied, Essa M; Othman, Alaa; Arenz, Christoph; Maccarone, Alan T; Poad, Berwyck L J; Blanksby, Stephen J; von Eckardstein, Arnold; Hornemann, Thorsten

    2016-07-01

    The 1-deoxysphingolipids (1-deoxySLs) are formed by an alternate substrate usage of the enzyme, serine-palmitoyltransferase, and are devoid of the C1-OH-group present in canonical sphingolipids. Pathologically elevated 1-deoxySL levels are associated with the rare inherited neuropathy, HSAN1, and diabetes type 2 and might contribute to β cell failure and the diabetic sensory neuropathy. In analogy to canonical sphingolipids, it was assumed that 1-deoxySLs also bear a (4E) double bond, which is normally introduced by sphingolipid delta(4)-desaturase 1. This, however, was never confirmed. We therefore supplemented HEK293 cells with isotope-labeled D3-1-deoxysphinganine and compared the downstream formed D3-1-deoxysphingosine (1-deoxySO) to a commercial synthetic SPH m18:1(4E)(3OH) standard. Both compounds showed the same m/z, but differed in their RPLC retention time and atmospheric pressure chemical ionization in-source fragmentation, suggesting that the two compounds are structural isomers. Using dimethyl disulfide derivatization followed by MS(2) as well as differential-mobility spectrometry combined with ozone-induced dissociation MS, we identified the carbon-carbon double bond in native 1-deoxySO to be located at the (Δ14) position. Comparing the chromatographic behavior of native 1-deoxySO to chemically synthesized SPH m18:1(14Z) and (14E) stereoisomers assigned the native compound to be SPH m18:1(14Z). This indicates that 1-deoxySLs are metabolized differently than canonical sphingolipids. PMID:27165858

  19. Structure based prediction of protein folding intermediates.

    PubMed

    Xie, D; Freire, E

    1994-09-01

    The complete unfolding of a protein involves the disruption of non-covalent intramolecular interactions within the protein and the subsequent hydration of the backbone and amino acid side-chains. The magnitude of the thermodynamic parameters associated with this process is known accurately for a growing number of globular proteins for which high-resolution structures are also available. The existence of this database of structural and thermodynamic information has facilitated the development of statistical procedures aimed at quantifying the relationships existing between protein structure and the thermodynamic parameters of folding/unfolding. Under some conditions proteins do not unfold completely, giving rise to states (commonly known as molten globules) in which the molecule retains some secondary structure and remains in a compact configuration after denaturation. This phenomenon is reflected in the thermodynamics of the process. Depending on the nature of the residual structure that exists after denaturation, the observed enthalpy, entropy and heat capacity changes will deviate in a particular and predictable way from the values expected for complete unfolding. For several proteins, these deviations have been shown to exhibit similar characteristics, suggesting that their equilibrium folding intermediates exhibit some common structural features. Employing empirically derived structure-energetic relationships, it is possible to identify in the native structure of the protein those regions with the higher probability of being structured in equilibrium partly folded states. In this work, a thermodynamic search algorithm aimed at identifying the structural determinants of the molten globule state has been applied to six globular proteins; alpha-lactalbumin, barnase, IIIGlc, interleukin-1 beta, phage T4 lysozyme and phage 434 repressor. Remarkably, the structural features of the predicted equilibrium intermediates coincide to a large extent with the known

  20. Addressing the Role of Conformational Diversity in Protein Structure Prediction

    PubMed Central

    Parisi, Gustavo; Fornasari, Maria Silvina

    2016-01-01

    Computational modeling of tertiary structures has become of standard use to study proteins that lack experimental characterization. Unfortunately, 3D structure prediction methods and model quality assessment programs often overlook that an ensemble of conformers in equilibrium populates the native state of proteins. In this work we collected sets of publicly available protein models and the corresponding target structures experimentally solved and studied how they describe the conformational diversity of the protein. For each protein, we assessed the quality of the models against known conformers by several standard measures and identified those models ranked best. We found that model rankings are defined by both the selected target conformer and the similarity measure used. 70% of the proteins in our datasets show that different models are structurally closest to different conformers of the same protein target. We observed that model building protocols such as template-based or ab initio approaches describe in similar ways the conformational diversity of the protein, although for template-based methods this description may depend on the sequence similarity between target and template sequences. Taken together, our results support the idea that protein structure modeling could help to identify members of the native ensemble, highlight the importance of considering conformational diversity in protein 3D quality evaluations and endorse the study of the variability of the native structure for a meaningful biological analysis. PMID:27159429

  1. Addressing the Role of Conformational Diversity in Protein Structure Prediction.

    PubMed

    Palopoli, Nicolas; Monzon, Alexander Miguel; Parisi, Gustavo; Fornasari, Maria Silvina

    2016-01-01

    Computational modeling of tertiary structures has become of standard use to study proteins that lack experimental characterization. Unfortunately, 3D structure prediction methods and model quality assessment programs often overlook that an ensemble of conformers in equilibrium populates the native state of proteins. In this work we collected sets of publicly available protein models and the corresponding target structures experimentally solved and studied how they describe the conformational diversity of the protein. For each protein, we assessed the quality of the models against known conformers by several standard measures and identified those models ranked best. We found that model rankings are defined by both the selected target conformer and the similarity measure used. 70% of the proteins in our datasets show that different models are structurally closest to different conformers of the same protein target. We observed that model building protocols such as template-based or ab initio approaches describe in similar ways the conformational diversity of the protein, although for template-based methods this description may depend on the sequence similarity between target and template sequences. Taken together, our results support the idea that protein structure modeling could help to identify members of the native ensemble, highlight the importance of considering conformational diversity in protein 3D quality evaluations and endorse the study of the variability of the native structure for a meaningful biological analysis. PMID:27159429

  2. Modeling Protein Aggregate Assembly and Structure

    NASA Astrophysics Data System (ADS)

    Guo, Jun-tao; Hall, Carol K.; Xu, Ying; Wetzel, Ronald

    One might say that "protein science" got its start in the domestic arts, built around the abilities of proteins to aggregate in response to environmental stresses such as heating (boiled eggs), heating and cooling (gelatin), and pH (cheese). Characterization of proteins in the late nineteenth century likewise focused on the ability of proteins to precipitate in response to certain salts and to aggregate in response to heating. Investigations by Chick and Martin (Chick and Martin, 1910) showed that the inactivating response of proteins to heat or solvent treatment is a two-step process involving separate denaturation and precipitation steps. Monitoring the coagulation and flocculation responses of proteins to heat and other stresses remained a major approach to understanding protein structure for decades, with solubility, or susceptibility to aggregation, serving as a kind of benchmark against which results of other methods, such as viscosity, chemical susceptibility, immune activity, crystallizability, and susceptibility to proteolysis, were compared (Mirsky and Pauling, 1936;Wu, 1931). Toward the middle of the last century, protein aggregation studies were largely left behind, as improved methods allowed elucidation of the primary sequence of proteins, reversible unfolding studies, and ultimately high-resolution structures. Curiously, the field of protein science, and in particular protein folding, is now gravitating back to a closer look at protein aggregation and protein aggregates. Unfortunately, the means developed during the second half of the twentieth century for studying native, globular proteins have not proved immediately amenable to the study of aggregate structures. Great progress is being made, however, to modify classical methods, including NMR and X-ray diffraction, as well as to develop newer techniques, that together should continue to expand our picture of aggregate structure (Kheterpal and Wetzel, 2006; Wetzel, 1999).

  3. Variation in the helical structure of native collagen.

    PubMed

    Orgel, Joseph P R O; Persikov, Anton V; Antipova, Olga

    2014-01-01

    The structure of collagen has been a matter of curiosity, investigation, and debate for the better part of a century. There has been a particularly productive period recently, during which much progress has been made in better describing all aspects of collagen structure. However, there remain some questions regarding its helical symmetry and its persistence within the triple-helix. Previous considerations of this symmetry have sometimes confused the picture by not fully recognizing that collagen structure is a highly complex and large hierarchical entity, and this affects and is effected by the super-coiled molecules that make it. Nevertheless, the symmetry question is not trite, but of some significance as it relates to extracellular matrix organization and cellular integration. The correlation between helical structure in the context of the molecular packing arrangement determines which parts of the amino acid sequence of the collagen fibril are buried or accessible to the extracellular matrix or the cell. In this study, we concentrate primarily on the triple-helical structure of fibrillar collagens I and II, the two most predominant types. By comparing X-ray diffraction data collected from type I and type II containing tissues, we point to evidence for a range of triple-helical symmetries being extant in the molecules native environment. The possible significance of helical instability, local helix dissociation and molecular packing of the triple-helices is discussed in the context of collagen's supramolecular organization, all of which must affect the symmetry of the collagen triple-helix. PMID:24586843

  4. Multiparametric high-resolution imaging of native proteins by force-distance curve-based AFM.

    PubMed

    Pfreundschuh, Moritz; Martinez-Martin, David; Mulvihill, Estefania; Wegmann, Susanne; Muller, Daniel J

    2014-05-01

    A current challenge in the life sciences is to understand how the properties of individual molecular machines adjust in order to meet the functional requirements of the cell. Recent developments in force-distance (FD) curve-based atomic force microscopy (FD-based AFM) enable researchers to combine sub-nanometer imaging with quantitative mapping of physical, chemical and biological properties. Here we present a protocol to apply FD-based AFM to the multiparametric imaging of native proteins under physiological conditions. We describe procedures for experimental FD-based AFM setup, high-resolution imaging of proteins in the native unperturbed state with simultaneous quantitative mapping of multiple parameters, and data interpretation and analysis. The protocol, which can be completed in 1-3 d, enables researchers to image proteins and protein complexes in the native unperturbed state and to simultaneously map their biophysical and biochemical properties at sub-nanometer resolution. PMID:24743419

  5. What makes a protein a protein? Hydrophobic core designs that specify stability and structural properties.

    PubMed Central

    Munson, M.; Balasubramanian, S.; Fleming, K. G.; Nagi, A. D.; O'Brien, R.; Sturtevant, J. M.; Regan, L.

    1996-01-01

    Here we describe how the systematic redesign of a protein's hydrophobic core alters its structure and stability. We have repacked the hydrophobic core of the four-helix-bundle protein, Rop, with altered packing patterns and various side chain shapes and sizes. Several designs reproduce the structure and native-like properties of the wild-type, while increasing the thermal stability. Other designs, either with similar sizes but different shapes, or with decreased sizes of the packing residues, destabilize the protein. Finally, overpacking the core with the larger side chains causes a loss of native-like structure. These results allow us to further define the roles of tight residue packing and the burial of hydrophobic surface area in the construction of native-like proteins. PMID:8844848

  6. Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

    PubMed

    Irani, Zahra Azimzadeh; Kerkhoven, Eduard J; Shojaosadati, Seyed Abbas; Nielsen, Jens

    2016-05-01

    Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. Biotechnol. Bioeng. 2016;113: 961-969. © 2015 Wiley Periodicals, Inc. PMID:26480251

  7. New approach to protein fold recognition based on Delaunay tessellation of protein structure

    SciTech Connect

    Zheng, W.; Cho, S.J.; Vaisman, I.I.; Tropsha, A.

    1996-12-31

    We propose new algorithms for sequence-structure compatibility (fold recognition) searches in multidimensional sequence-structure space. Individual amino acid residues in protein structures are represented by their C{sup {alpha}} atoms; thus each protein is described as a collection of points in three-dimensional space. Delaunay tessellation of a protein generates an aggregate of space-filling, irregular tetrahedra, or Delaunay simplices. Statistical analysis of quadruplet residue compositions of all Delaunay simplices in a representative dataset of protein structures leads to a novel four body contact residue potential expressed as log likelihood factor q. The q factors are calculated for native 20 letter amino acid alphabet and several reduced alphabets. Two sequence structure compatibility functions are computed as (i) the sum of q factors for all Delaunay simplices in a given protein, or (ii) 3D-1D Delaunay tessellation profiles where the individual residue profile value is calculated as the sum of q factors for all simplices that share this vertex residue. Both threading functions have been implemented in structure-recognizes-sequence and sequence-recognizes-structure protocols for protein fold recognition. We find that both profile and total score based threading functions can distinguish both the native fold from incorrect folds for a sequence, and the native sequence from non-native sequences for a fold. 25 refs., 4 figs., 1 tab.

  8. Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data

    PubMed Central

    Liu, Qun; Liu, Qinglian; Hendrickson, Wayne A.

    2013-01-01

    Structure determinations for biological macromolecules that have no known structural antecedents typically involve the incorporation of heavier atoms than those found natively in biological molecules. Currently, selenomethionyl proteins analyzed using single- or multi-wavelength anomalous diffraction (SAD or MAD) data predominate for such de novo analyses. Naturally occurring metal ions such as zinc or iron often suffice in MAD or SAD experiments, and sulfur SAD has been an option since it was first demonstrated using crambin 30 years ago; however, SAD analyses of structures containing only light atoms (Z max ≤ 20) have not been common. Here, robust procedures for enhancing the signal to noise in measurements of anomalous diffraction by combining data collected from several crystals at a lower than usual X-ray energy are described. This multi-crystal native SAD method was applied in five structure determinations, using between five and 13 crystals to determine substructures of between four and 52 anomalous scatterers (Z ≤ 20) and then the full structures ranging from 127 to 1200 ordered residues per asymmetric unit at resolutions from 2.3 to 2.8 Å. Tests were devised to assure that all of the crystals used were statistically equivalent. Elemental identities for Ca, Cl, S, P and Mg were proven by f′′ scattering-factor refinements. The procedures are robust, indicating that truly routine structure determination of typical native macromolecules is realised. Synchrotron beamlines that are optimized for low-energy X-ray diffraction measurements will facilitate such direct structural analysis. PMID:23793158

  9. Accurate Prediction of Docked Protein Structure Similarity.

    PubMed

    Akbal-Delibas, Bahar; Pomplun, Marc; Haspel, Nurit

    2015-09-01

    One of the major challenges for protein-protein docking methods is to accurately discriminate nativelike structures. The protein docking community agrees on the existence of a relationship between various favorable intermolecular interactions (e.g. Van der Waals, electrostatic, desolvation forces, etc.) and the similarity of a conformation to its native structure. Different docking algorithms often formulate this relationship as a weighted sum of selected terms and calibrate their weights against specific training data to evaluate and rank candidate structures. However, the exact form of this relationship is unknown and the accuracy of such methods is impaired by the pervasiveness of false positives. Unlike the conventional scoring functions, we propose a novel machine learning approach that not only ranks the candidate structures relative to each other but also indicates how similar each candidate is to the native conformation. We trained the AccuRMSD neural network with an extensive dataset using the back-propagation learning algorithm. Our method achieved predicting RMSDs of unbound docked complexes with 0.4Å error margin. PMID:26335807

  10. STRUCTURAL VIROLOGY. Conformational plasticity of a native retroviral capsid revealed by x-ray crystallography.

    PubMed

    Obal, G; Trajtenberg, F; Carrión, F; Tomé, L; Larrieux, N; Zhang, X; Pritsch, O; Buschiazzo, A

    2015-07-01

    Retroviruses depend on self-assembly of their capsid proteins (core particle) to yield infectious mature virions. Despite the essential role of the retroviral core, its high polymorphism has hindered high-resolution structural analyses. Here, we report the x-ray structure of the native capsid (CA) protein from bovine leukemia virus. CA is organized as hexamers that deviate substantially from sixfold symmetry, yet adjust to make two-dimensional pseudohexagonal arrays that mimic mature retroviral cores. Intra- and interhexameric quasi-equivalent contacts are uncovered, with flexible trimeric lateral contacts among hexamers, yet preserving very similar dimeric interfaces making the lattice. The conformation of each capsid subunit in the hexamer is therefore dictated by long-range interactions, revealing how the hexamers can also assemble into closed core particles, a relevant feature of retrovirus biology. PMID:26044299

  11. Monoclonal antibodies to native noncollagenous bone-specific proteins.

    PubMed Central

    Stenner, D D; Romberg, R W; Tracy, R P; Katzmann, J A; Riggs, B L; Mann, K G

    1984-01-01

    Hybridoma technology was used for preparation of murine monoclonal antibodies of high titer against bone-Gla protein and osteonectin. A procedure of immunization and hybridization similar to that already described [Katzmann, J.A., Nesheim, M.E., Hibbard, L.S. & Mann, K.G. (1981) Proc. Natl. Acad. Sci. USA 78, 162-166; and Foster, W.B., Katzmann, J.A., Miller, R.S., Nesheim, M.E. & Mann, K.G. (1982) Thromb. Res. 28, 649-661] was used. However, in contrast to earlier studies, mice were immunized with an unfractionated protein mixture that had been extracted from bone under nondenaturing conditions. The extract was labeled with 125I by the chloramine-T method. After fusion and initial hybrid growth, screening was accomplished by a solid-phase radioimmunoassay with total 125I-labeled bovine bone protein extract as the tracer. The identities of antibody-bound 125I-labeled proteins were assessed by dissolution of the solid-phase immune complex in NaDodSO4 and subsequent electrophoresis and autoradiography. Clones producing specific antibody to a single protein were selected by limiting dilution. The identity of the proteins against which the specific antibodies were produced was confirmed by immunoprecipitation, electrophoresis, and autoradiography. From two fusions, 30 positive hybrids to bone-Gla protein were identified; 7 of these were subcloned and 1 has been expanded as an ascites tumor. One hybrid population was positive for osteonectin, a Mr 15,000 peptide, and for bone-Gla protein. By limiting dilution, the osteonectin clone was selected and subsequently expanded as an ascites tumor. Titration curves made using the respective 125I-labeled purified proteins show the ascites tumors to be producing antibody of high titer (I50 = 10(-6) for anti-bone-Gla protein and (I50 = 10(-5) for antiosteonectin. Both of the antibovine antibodies are cross-reactive with the corresponding human protein. Immobilized specific anti-bone-Gla protein has been used to isolate human bone

  12. The role of negative selection in protein evolution revealed through the energetics of the native state ensemble.

    PubMed

    Hoffmann, Jordan; Wrabl, James O; Hilser, Vincent J

    2016-04-01

    Knowing the determinants of conformational specificity is essential for understanding protein structure, stability, and fold evolution. To address this issue, a novel statistical measure of energetic compatibility between sequence and structure was developed using an experimentally validated model of the energetics of the native state ensemble. This approach successfully matched sequences from a diverse subset of the human proteome to their respective folds. Unexpectedly, significant energetic compatibility between ostensibly unrelated sequences and structures was also observed. Interrogation of these matches revealed a general framework for understanding the origins of conformational specificity within a proteome: specificity is a complex function of both the ability of a sequence to adopt folds other than the native, and ability of a fold to accommodate sequences other than the native. The regional variation in energetic compatibility indicates that the compatibility is dominated by incompatibility of sequence for alternative fold segments, suggesting that evolution of protein sequences has involved substantial negative selection, with certain segments serving as "gatekeepers" that presumably prevent alternative structures. Beyond these global trends, a size dependence exists in the degree to which the energetic compatibility is determined from negative selection, with smaller proteins displaying more negative selection. This partially explains how short sequences can adopt unique folds, despite the higher probability in shorter proteins for small numbers of mutations to increase compatibility with other folds. In providing evolutionary ground rules for the thermodynamic relationship between sequence and fold, this framework imparts valuable insight for rational design of unique folds or fold switches. Proteins 2016; 84:435-447. © 2016 Wiley Periodicals, Inc. PMID:26800099

  13. Optogenetic pharmacology for control of native neuronal signaling proteins

    PubMed Central

    Kramer, Richard H; Mourot, Alexandre; Adesnik, Hillel

    2016-01-01

    The optical neuroscience revolution is transforming how we study neural circuits. By providing a precise way to manipulate endogenous neuronal signaling proteins, it also has the potential to transform our understanding of molecular neuroscience. Recent advances in chemical biology have produced light-sensitive compounds that photoregulate a wide variety of proteins underlying signaling between and within neurons. Chemical tools for optopharmacology include caged agonists and antagonists and reversibly photoswitchable ligands. These reagents act on voltage-gated ion channels and neurotransmitter receptors, enabling control of neuronal signaling with a high degree of spatial and temporal precision. By covalently attaching photoswitch molecules to genetically tagged proteins, the newly emerging methodology of optogenetic pharmacology allows biochemically precise control in targeted subsets of neurons. Now that the tools for manipulating endogenous neuronal signaling proteins are available, they can be implemented in vivo to enhance our understanding of the molecular bases of brain function and dysfunctions. PMID:23799474

  14. Optogenetic pharmacology for control of native neuronal signaling proteins.

    PubMed

    Kramer, Richard H; Mourot, Alexandre; Adesnik, Hillel

    2013-07-01

    The optical neuroscience revolution is transforming how we study neural circuits. By providing a precise way to manipulate endogenous neuronal signaling proteins, it also has the potential to transform our understanding of molecular neuroscience. Recent advances in chemical biology have produced light-sensitive compounds that photoregulate a wide variety of proteins underlying signaling between and within neurons. Chemical tools for optopharmacology include caged agonists and antagonists and reversibly photoswitchable ligands. These reagents act on voltage-gated ion channels and neurotransmitter receptors, enabling control of neuronal signaling with a high degree of spatial and temporal precision. By covalently attaching photoswitch molecules to genetically tagged proteins, the newly emerging methodology of optogenetic pharmacology allows biochemically precise control in targeted subsets of neurons. Now that the tools for manipulating endogenous neuronal signaling proteins are available, they can be implemented in vivo to enhance our understanding of the molecular bases of brain function and dysfunctions. PMID:23799474

  15. Structural Query Optimization in Native XML Databases: A Hybrid Approach

    NASA Astrophysics Data System (ADS)

    Haw, Su-Cheng; Lee, Chien-Sing

    As XML (eXtensible Mark-up Language) is gaining its popularity in data exchange over the Web, querying XML data has become an important issue to be addressed. In native XML databases (NXD), XML documents are usually modeled as trees and XML queries are typically specified in path expression. The primitive structural relationships are Parent-Child (P-C), Ancestor-Descendant (A-D), sibling and ordered query. Thus, a suitable and compact labeling scheme is crucial to identify these relationships and henceforth to process the query efficiently. We propose a novel labeling scheme consisting of < self-level:parent> to support all these relationships efficiently. Besides, we adopt the decomposition-matching-merging approach for structural query processing and propose a hybrid query optimization technique, TwigINLAB to process and optimize the twig query evaluation. Experimental results indicate that TwigINLAB can process all types of XML queries 15% better than the TwigStack algorithm in terms of execution time in most test cases.

  16. Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse

    PubMed Central

    Volpert, Marianna; Mangum, Jonathan E.; Jamsai, Duangporn; D'Sylva, Rebecca; O'Bryan, Moira K.; McIntyre, Peter

    2014-01-01

    While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences. PMID:24573035

  17. Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse

    NASA Astrophysics Data System (ADS)

    Volpert, Marianna; Mangum, Jonathan E.; Jamsai, Duangporn; D'Sylva, Rebecca; O'Bryan, Moira K.; McIntyre, Peter

    2014-02-01

    While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.

  18. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    SciTech Connect

    Zanzoni, Serena; D'Onofrio, Mariapina; Molinari, Henriette; Assfalg, Michael

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  19. Hierarchical, Self-Similar Structure in Native Squid Pen

    NASA Astrophysics Data System (ADS)

    Yang, Fei-Chi; Peters, Robert; Dies, Hannah; Rheinstadter, Maikel

    2014-03-01

    Proteins, chitin and keratin form the elementary building blocks of many biomaterials. How these molecules assemble into larger, macroscopic structures with very different properties is the fundamental question we are trying to answer. Squid pen is a transparent backbone inside the squid, which supports the mantle of the squid. The pens show a hierarchical, self-similar structure under the microscope and the AFM with fibers from 500 μm to 0.2 μm in diameter. The chitin molecules form nano-crystallites of monoclinic lattice symmetry surrounded by a protein layer, resulting in β-chitin nano-fibrils. Signals corresponding to the α-coil protein phase and β-chitin were observed in X-ray experiments in-situ. The molecular structure is highly anisotropic with 90% of the α-coils and β-chitin crystallites oriented along the fiber-axis indicating a strong correlation between the structures on millimeters down to the molecular scale. This research was funded by NSERC, NRC, CFI, and the Ontario Ministry of Economic Development and Innovation.

  20. Natively inhibited Trypanosoma brucei cathepsin B structure determined by using an X-ray laser.

    PubMed

    Redecke, Lars; Nass, Karol; DePonte, Daniel P; White, Thomas A; Rehders, Dirk; Barty, Anton; Stellato, Francesco; Liang, Mengning; Barends, Thomas R M; Boutet, Sébastien; Williams, Garth J; Messerschmidt, Marc; Seibert, M Marvin; Aquila, Andrew; Arnlund, David; Bajt, Sasa; Barth, Torsten; Bogan, Michael J; Caleman, Carl; Chao, Tzu-Chiao; Doak, R Bruce; Fleckenstein, Holger; Frank, Matthias; Fromme, Raimund; Galli, Lorenzo; Grotjohann, Ingo; Hunter, Mark S; Johansson, Linda C; Kassemeyer, Stephan; Katona, Gergely; Kirian, Richard A; Koopmann, Rudolf; Kupitz, Chris; Lomb, Lukas; Martin, Andrew V; Mogk, Stefan; Neutze, Richard; Shoeman, Robert L; Steinbrener, Jan; Timneanu, Nicusor; Wang, Dingjie; Weierstall, Uwe; Zatsepin, Nadia A; Spence, John C H; Fromme, Petra; Schlichting, Ilme; Duszenko, Michael; Betzel, Christian; Chapman, Henry N

    2013-01-11

    The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the "diffraction-before-destruction" approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals. PMID:23196907

  1. Natively Inhibited Trypanosoma brucei Cathepsin B Structure Determined by Using an X-ray Laser

    PubMed Central

    DePonte, Daniel P.; White, Thomas A.; Rehders, Dirk; Barty, Anton; Stellato, Francesco; Liang, Mengning; Barends, Thomas R.M.; Boutet, Sébastien; Williams, Garth J.; Messerschmidt, Marc; Seibert, M. Marvin; Aquila, Andrew; Arnlund, David; Bajt, Sasa; Barth, Torsten; Bogan, Michael J.; Caleman, Carl; Chao, Tzu-Chiao; Doak, R. Bruce; Fleckenstein, Holger; Frank, Matthias; Fromme, Raimund; Galli, Lorenzo; Grotjohann, Ingo; Hunter, Mark S.; Johansson, Linda C.; Kassemeyer, Stephan; Katona, Gergely; Kirian, Richard A.; Koopmann, Rudolf; Kupitz, Chris; Lomb, Lukas; Martin, Andrew V.; Mogk, Stefan; Neutze, Richard; Shoeman, Robert L.; Steinbrener, Jan; Timneanu, Nicusor; Wang, Dingjie; Weierstall, Uwe; Zatsepin, Nadia A.; Spence, John C. H.; Fromme, Petra; Schlichting, Ilme; Duszenko, Michael; Betzel, Christian; Chapman, Henry N.

    2013-01-01

    The Trypanosoma brucei cysteine protease cathepsin B (TbCatB), which is involved in host protein degradation, is a promising target to develop new treatments against sleeping sickness, a fatal disease caused by this protozoan parasite. The structure of the mature, active form of TbCatB has so far not provided sufficient information for the design of a safe and specific drug against T. brucei. By combining two recent innovations, in vivo crystallization and serial femtosecond crystallography, we obtained the room-temperature 2.1 angstrom resolution structure of the fully glycosylated precursor complex of TbCatB. The structure reveals the mechanism of native TbCatB inhibition and demonstrates that new biomolecular information can be obtained by the “diffraction-before-destruction” approach of x-ray free-electron lasers from hundreds of thousands of individual microcrystals. PMID:23196907

  2. The “Intracellular” Poly(3-Hydroxybutyrate) (PHB) Depolymerase of Rhodospirillum rubrum Is a Periplasm-Located Protein with Specificity for Native PHB and with Structural Similarity to Extracellular PHB Depolymerases†

    PubMed Central

    Handrick, René; Reinhardt, Simone; Kimmig, Philipp; Jendrossek, Dieter

    2004-01-01

    Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser42, Asp138, and His178 were identified as catalytic-triad amino acids, with Ser42 as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum. PMID:15489436

  3. Genetic diversity and structure of native and non-native populations of the endangered plant Pinus dabeshanensis.

    PubMed

    Zhang, Z Y; Wang, H; Chen, W; Pang, X M; Li, Y Y

    2016-01-01

    Owing to a severe decline in its abundance, Pinus dabeshanensis has been listed as an endangered species by the International Union for the Conservation of Nature. Although several restoration events have been undertaken since the 1960s, the natural population genetic structure of this species remains to be investigated. Herein, we examined the level of genetic diversity and structure of two native and two non-native populations using 10 microsatellite loci. A relatively high level of genetic variation (HO = 0.586 ± 0.039) and a low level of population differentiation (FST = 0.016 ± 0.011) were revealed. For forensic investigation, an assignment test was performed. To better understand the genetic differentiation between the native and non-native populations, the individuals in the transplanted and cultivated populations may have derived from populations that were not surveyed in this study. In light of our results, we discuss the real problems faced by all four populations and provide useful information for management decision-making. PMID:27323170

  4. Structural Correlates for Lexical Efficiency and Number of Languages in Non-Native Speakers of English

    ERIC Educational Resources Information Center

    Grogan, A.; Parker Jones, O.; Ali, N.; Crinion, J.; Orabona, S.; Mechias, M. L.; Ramsden, S.; Green, D. W.; Price, C. J.

    2012-01-01

    We used structural magnetic resonance imaging (MRI) and voxel based morphometry (VBM) to investigate whether the efficiency of word processing in the non-native language (lexical efficiency) and the number of non-native languages spoken (2+ versus 1) were related to local differences in the brain structure of bilingual and multilingual speakers.…

  5. Voltage sensor ring in a native structure of a membrane-embedded potassium channel

    PubMed Central

    Shi, Liang; Zheng, Hongjin; Zheng, Hui; Borkowski, Brian A.; Shi, Dan; Gonen, Tamir; Jiang, Qiu-Xing

    2013-01-01

    Voltage-gated ion channels support electrochemical activity in cells and are largely responsible for information flow throughout the nervous systems. The voltage sensor domains in these channels sense changes in transmembrane potential and control ion flux across membranes. The X-ray structures of a few voltage-gated ion channels in detergents have been determined and have revealed clear structural variations among their respective voltage sensor domains. More recent studies demonstrated that lipids around a voltage-gated channel could directly alter its conformational state in membrane. Because of these disparities, the structural basis for voltage sensing in native membranes remains elusive. Here, through electron-crystallographic analysis of membrane-embedded proteins, we present the detailed view of a voltage-gated potassium channel in its inactivated state. Contrary to all known structures of voltage-gated ion channels in detergents, our data revealed a unique conformation in which the four voltage sensor domains of a voltage-gated potassium channel from Aeropyrum pernix (KvAP) form a ring structure that completely surrounds the pore domain of the channel. Such a structure is named the voltage sensor ring. Our biochemical and electrophysiological studies support that the voltage sensor ring represents a physiological conformation. These data together suggest that lipids exert strong effects on the channel structure and that these effects may be changed upon membrane disruption. Our results have wide implications for lipid–protein interactions in general and for the mechanism of voltage sensing in particular. PMID:23401554

  6. Near-Native Protein Loop Sampling Using Nonparametric Density Estimation Accommodating Sparcity

    PubMed Central

    Day, Ryan; Lennox, Kristin P.; Sukhanov, Paul; Dahl, David B.; Vannucci, Marina; Tsai, Jerry

    2011-01-01

    Unlike the core structural elements of a protein like regular secondary structure, template based modeling (TBM) has difficulty with loop regions due to their variability in sequence and structure as well as the sparse sampling from a limited number of homologous templates. We present a novel, knowledge-based method for loop sampling that leverages homologous torsion angle information to estimate a continuous joint backbone dihedral angle density at each loop position. The φ,ψ distributions are estimated via a Dirichlet process mixture of hidden Markov models (DPM-HMM). Models are quickly generated based on samples from these distributions and were enriched using an end-to-end distance filter. The performance of the DPM-HMM method was evaluated against a diverse test set in a leave-one-out approach. Candidates as low as 0.45 Å RMSD and with a worst case of 3.66 Å were produced. For the canonical loops like the immunoglobulin complementarity-determining regions (mean RMSD <2.0 Å), the DPM-HMM method performs as well or better than the best templates, demonstrating that our automated method recaptures these canonical loops without inclusion of any IgG specific terms or manual intervention. In cases with poor or few good templates (mean RMSD >7.0 Å), this sampling method produces a population of loop structures to around 3.66 Å for loops up to 17 residues. In a direct test of sampling to the Loopy algorithm, our method demonstrates the ability to sample nearer native structures for both the canonical CDRH1 and non-canonical CDRH3 loops. Lastly, in the realistic test conditions of the CASP9 experiment, successful application of DPM-HMM for 90 loops from 45 TBM targets shows the general applicability of our sampling method in loop modeling problem. These results demonstrate that our DPM-HMM produces an advantage by consistently sampling near native loop structure. The software used in this analysis is available for download at http

  7. Splitting statistical potentials into meaningful scoring functions: Testing the prediction of near-native structures from decoy conformations

    PubMed Central

    2009-01-01

    Background Recent advances on high-throughput technologies have produced a vast amount of protein sequences, while the number of high-resolution structures has seen a limited increase. This has impelled the production of many strategies to built protein structures from its sequence, generating a considerable amount of alternative models. The selection of the closest model to the native conformation has thus become crucial for structure prediction. Several methods have been developed to score protein models by energies, knowledge-based potentials and combination of both. Results Here, we present and demonstrate a theory to split the knowledge-based potentials in scoring terms biologically meaningful and to combine them in new scores to predict near-native structures. Our strategy allows circumventing the problem of defining the reference state. In this approach we give the proof for a simple and linear application that can be further improved by optimizing the combination of Zscores. Using the simplest composite score () we obtained predictions similar to state-of-the-art methods. Besides, our approach has the advantage of identifying the most relevant terms involved in the stability of the protein structure. Finally, we also use the composite Zscores to assess the conformation of models and to detect local errors. Conclusion We have introduced a method to split knowledge-based potentials and to solve the problem of defining a reference state. The new scores have detected near-native structures as accurately as state-of-art methods and have been successful to identify wrongly modeled regions of many near-native conformations. PMID:19917096

  8. A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human Disease

    PubMed Central

    Gilmore, Brian L.; Winton, Carly E.; Demmert, Andrew C.; Tanner, Justin R.; Bowman, Sam; Karageorge, Vasilea; Patel, Kaya; Sheng, Zhi; Kelly, Deborah F.

    2015-01-01

    We present a new molecular toolkit to investigate protein assemblies natively formed in the context of human disease. The system employs tunable microchips that can be decorated with switchable adaptor molecules to select for target proteins of interest and analyze them using molecular microscopy. Implementing our new streamlined microchip approach, we could directly visualize BRCA1 gene regulatory complexes from patient-derived cancer cells for the first time. PMID:26395823

  9. Genetically Encoding an Electrophilic Amino Acid for Protein Stapling and Covalent Binding to Native Receptors

    PubMed Central

    2015-01-01

    Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability. PMID:25010185

  10. Native gel analysis of macromolecular protein complexes in cultured mammalian cells.

    PubMed

    Munawar, Nayla; Olivero, Giorgio; Jerman, Emilia; Doyle, Benjamin; Streubel, Gundula; Wynne, Kieran; Bracken, Adrian; Cagney, Gerard

    2015-11-01

    Native gel electrophoresis enables separation of cellular proteins in their non-denatured state. In experiments aimed at analysing proteins in higher order or multimeric assemblies (i.e. protein complexes) it offers some advantages over rival approaches, particularly as an interface technology with mass spectrometry. Here we separated fractions from HEK293 cells by native electrophoresis in order to survey protein complexes in the cytoplasmic, nuclear and chromatin environments, finding 689 proteins distributed among 217 previously described complexes. As expected, different fractions contained distinct combinations of macromolecular complexes, with subunits of the same complex tending to co-migrate. Exceptions to this observation could often be explained by the presence of subunits shared among different complexes. We investigated one identified complex, the Polycomb Repressor Complex 2 (PRC2), in more detail following affinity purification of the EZH2 subunit. This approach resulted in the identification of all previously reported members of PRC2. Overall, this work demonstrates that the use of native gel electrophoresis as an upstream separating step is an effective approach for analysis of the components and cellular distribution of protein complexes. PMID:26223664

  11. Elucidating the native sources of an invasive tree species, Acacia pycnantha, reveals unexpected native range diversity and structure

    PubMed Central

    Ndlovu, Joice; Richardson, David M.; Wilson, John R. U.; O'Leary, Martin; Le Roux, Johannes J.

    2013-01-01

    Background and Aims Understanding the introduction history of invasive plant species is important for their management and identifying effective host-specific biological control agents. However, uncertain taxonomy, intra- and interspecific hybridization, and cryptic speciation may obscure introduction histories, making it difficult to identify native regions to explore for host-specific agents. The overall aim of this study was to identify the native source populations of Acacia pycnantha, a tree native to south-eastern Australia and invasive in South Africa, Western Australia and Portugal. Using a phylogeographical approach also allowed an exploration of the historical processes that have shaped the genetic structure of A. pycnantha in its native range. Methods Nuclear (nDNA) and plastid DNA sequence data were used in network and tree-building analyses to reconstruct phylogeographical relationships between native and invasive A. pycnantha populations. In addition, mismatch distributions, relative rates and Bayesian analyses were used to infer recent demographic processes and timing of events in Australia that led to population structure and diversification. Key Results The plastid network indicated that Australian populations of A. pycnantha are geographically structured into two informally recognized lineages, the wetland and dryland forms, whereas the nuclear phylogeny showed little geographical structure between these two forms. Moreover, the dryland form of A. pycnantha showed close genetic similarity to the wetland form based on nDNA sequence data. Hybrid zones may explain these findings, supported here by incongruent phylogenetic placement of some of these taxa between nuclear and plastid genealogies. Conclusions It is hypothesized that habitat fragmentation due to cycles of aridity inter-dispersed with periods of abundant rainfall during the Pleistocene (approx. 100 kya) probably gave rise to native dryland and wetland forms of A. pycnantha. Although the

  12. Using entropy maximization to understand the determinants of structural dynamics beyond native contact topology.

    PubMed

    Lezon, Timothy R; Bahar, Ivet

    2010-06-01

    Comparison of elastic network model predictions with experimental data has provided important insights on the dominant role of the network of inter-residue contacts in defining the global dynamics of proteins. Most of these studies have focused on interpreting the mean-square fluctuations of residues, or deriving the most collective, or softest, modes of motions that are known to be insensitive to structural and energetic details. However, with increasing structural data, we are in a position to perform a more critical assessment of the structure-dynamics relations in proteins, and gain a deeper understanding of the major determinants of not only the mean-square fluctuations and lowest frequency modes, but the covariance or the cross-correlations between residue fluctuations and the shapes of higher modes. A systematic study of a large set of NMR-determined proteins is analyzed using a novel method based on entropy maximization to demonstrate that the next level of refinement in the elastic network model description of proteins ought to take into consideration properties such as contact order (or sequential separation between contacting residues) and the secondary structure types of the interacting residues, whereas the types of amino acids do not play a critical role. Most importantly, an optimal description of observed cross-correlations requires the inclusion of destabilizing, as opposed to exclusively stabilizing, interactions, stipulating the functional significance of local frustration in imparting native-like dynamics. This study provides us with a deeper understanding of the structural basis of experimentally observed behavior, and opens the way to the development of more accurate models for exploring protein dynamics. PMID:20585542

  13. Peptic and tryptic hydrolysis of native and heated whey protein to reduce its antigenicity.

    PubMed

    Kim, S B; Ki, K S; Khan, M A; Lee, W S; Lee, H J; Ahn, B S; Kim, H S

    2007-09-01

    This study examined the effects of enzymes on the production and antigenicity of native and heated whey protein concentrate (WPC) hydrolysates. Native and heated (10 min at 100 degrees C) WPC (2% protein solution) were incubated at 50 degrees C for 30, 60, 90, and 120 min with 0.1, 0.5, and 1% pepsin and then with 0.1, 0.5, and 1% trypsin on a protein-equivalent basis. A greater degree of hydrolysis was achieved and greater nonprotein nitrogen concentrations were obtained in heated WPC than in native WPC at all incubation times. Hydrolysis of WPC was increased with an increasing level of enzymes and higher incubation times. The highest hydrolysis (25.23%) was observed in heated WPC incubated with 1% pepsin and then with 1% trypsin for 120 min. High molecular weight bands, such as BSA, were completely eliminated from sodium dodecyl sulfate-PAGE of both native and heated WPC hydrolysates produced with pepsin for the 30-min incubation. The alpha-lactalbumin in native WPC was slightly degraded when incubated with 0.1% pepsin and then with 0.1% trypsin; however, it was almost completely hydrolyzed within 60 min of incubation with 0.5% pepsin and then with 0.5% trypsin. Incubation of native WPC with 1% pepsin and then with 1% trypsin for 30 min completely removed the BSA and alpha-lactalbumin. The beta-lactoglobulin in native WPC was not affected by the pepsin and trypsin treatments. The beta-lactoglobulin in heated WPC was partially hydrolyzed by the 0.1 and 0.5% pepsin and trypsin treatments and was completely degraded by the 1% pepsin and trypsin treatment. Antigenicity reversibly mimicked the hydrolysis of WPC and the removal of beta-lactoglobulin from hydrolysates. Antigenicity in heated and native WPC was reduced with an increasing level of enzymes. A low antigenic response was observed in heated WPC compared with native WPC. The lowest antigenicity was observed when heated WPC was incubated with 1% pepsin and then with 1% trypsin. These results suggested that

  14. Modulating non-native aggregation and electrostatic protein-protein interactions with computationally designed single-point mutations.

    PubMed

    O'Brien, C J; Blanco, M A; Costanzo, J A; Enterline, M; Fernandez, E J; Robinson, A S; Roberts, C J

    2016-06-01

    Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms. PMID:27160179

  15. Structural mechanisms of nonplanar hemes in proteins

    SciTech Connect

    Shelnutt, J.A.

    1997-05-01

    The objective is to assess the occurrence of nonplanar distortions of hemes and other tetrapyrroles in proteins and to determine the biological function of these distortions. Recently, these distortions were found by us to be conserved among proteins belonging to a functional class. Conservation of the conformation of the heme indicates a possible functional role. Researchers have suggested possible mechanisms by which heme distortions might influence biological properties; however, no heme distortion has yet been shown conclusively to participate in a structural mechanism of hemoprotein function. The specific aims of the proposed work are: (1) to characterize and quantify the distortions of the hemes in all of the more than 300 hemoprotein X-ray crystal structures in terms of displacements along the lowest-frequency normal coordinates, (2) to determine the structural features of the protein component that generate and control these nonplanar distortions by using spectroscopic studies and molecular-mechanics calculations for the native proteins, their mutants and heme-peptide fragments, and model porphyrins, (3) to determine spectroscopic markers for the various types of distortion, and, finally, (4) to discover the functional significance of the nonplanar distortions by correlating function with porphyrin conformation for proteins and model porphyrins.

  16. Structural Genomics of Protein Phosphatases

    SciTech Connect

    Almo,S.; Bonanno, J.; Sauder, J.; Emtage, S.; Dilorenzo, T.; Malashkevich, V.; Wasserman, S.; Swaminathan, S.; Eswaramoorthy, S.; et al

    2007-01-01

    The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.

  17. Autonomous induction of recombinant proteins by minimally rewiring native quorum sensing regulon of E. coli.

    PubMed

    Tsao, Chen-Yu; Hooshangi, Sara; Wu, Hsuan-Chen; Valdes, James J; Bentley, William E

    2010-05-01

    Quorum sensing (QS) enables an individual bacterium's metabolic state to be communicated to and ultimately control the phenotype of an emerging population. Harnessing the hierarchical nature of this signal transduction process may enable the exploitation of individual cell characteristics to direct or "program" entire populations of cells. We re-engineered the native QS regulon so that individual cell signals (autoinducers) are used to guide high level expression of recombinant proteins in E. coli populations. Specifically, the autoinducer-2 (AI-2) QS signal initiates and guides the overexpression of green fluorescent protein (GFP), chloramphenicol acetyl transferase (CAT) and beta-galactosidase (LacZ). The new process requires no supervision or input (e.g., sampling for optical density measurement, inducer addition, or medium exchange) and represents a low-cost, high-yield platform for recombinant protein production. Moreover, rewiring a native signal transduction circuit exemplifies an emerging class of metabolic engineering approaches that target regulatory functions. PMID:20060924

  18. Analysis of Native-Like Proteins and Protein Complexes Using Cation to Anion Proton Transfer Reactions (CAPTR)

    NASA Astrophysics Data System (ADS)

    Laszlo, Kenneth J.; Bush, Matthew F.

    2015-12-01

    Mass spectra of native-like protein complexes often exhibit narrow charge-state distributions, broad peaks, and contributions from multiple, coexisting species. These factors can make it challenging to interpret those spectra, particularly for mixtures with significant heterogeneity. Here we demonstrate the use of ion/ion proton transfer reactions to reduce the charge states of m/ z-selected, native-like ions of proteins and protein complexes, a technique that we refer to as cation to anion proton transfer reactions (CAPTR). We then demonstrate that CAPTR can increase the accuracy of charge state assignments and the resolution of interfering species in native mass spectrometry. The CAPTR product ion spectra for pyruvate kinase exhibit ~30 peaks and enable unambiguous determination of the charge state of each peak, whereas the corresponding precursor spectra exhibit ~6 peaks and the assigned charge states have an uncertainty of ±3%. 15+ bovine serum albumin and 21+ yeast enolase dimer both appear near m/ z 4450 and are completely unresolved in a mixture. After a single CAPTR event, the resulting product ions are baseline resolved. The separation of the product ions increases dramatically after each subsequent CAPTR event; 12 events resulted in a 3000-fold improvement in separation relative to the precursor ions. Finally, we introduce a framework for interpreting and predicting the figures of merit for CAPTR experiments. More generally, these results suggest that CAPTR strongly complements other mass spectrometry tools for analyzing proteins and protein complexes, particularly those in mixtures.

  19. Expression strategies for structural studies of eukaryotic membrane proteins.

    PubMed

    Lyons, Joseph A; Shahsavar, Azadeh; Paulsen, Peter Aasted; Pedersen, Bjørn Panyella; Nissen, Poul

    2016-06-01

    Integral membrane proteins in eukaryotes are central to various cellular processes and key targets in structural biology, biotechnology and drug development. However, the number of available structures for eukaryotic membrane protein belies their physiological importance. Recently, the number of available eukaryotic membrane protein structures has been steadily increasing due to the development of novel strategies in construct design, expression and structure determination. Here, we examine the major expression systems exploited for eukaryotic membrane proteins. Additionally we strive to tabulate and describe the recent expression strategies in eukaryotic membrane protein structural biology. We find that a majority of targets have been expressed in advanced host systems and modified from their wild-type form with distinct focus on conformation and thermostabilisation. However, strategies for native protein purification should also be considered where possible, particularly in light of the recent advances in single particle cryo electron microscopy. PMID:27362979

  20. Membrane protein structure from rotational diffusion☆

    PubMed Central

    Das, Bibhuti B.; Park, Sang Ho; Opella, Stanley J.

    2014-01-01

    The motional averaging of powder pattern line shapes is one of the most fundamental aspects of sold-state NMR. Since membrane proteins in liquid crystalline phospholipid bilayers undergo fast rotational diffusion, all of the signals reflect the angles of the principal axes of their dipole–dipole and chemical shift tensors with respect to the axis defined by the bilayer normal. The frequency span and sign of the axially symmetric powder patterns that result from motional averaging about a common axis provide sufficient structural restraints for the calculation of the three-dimensional structure of a membrane protein in a phospholipid bilayer environment. The method is referred to as rotationally aligned (RA) solid-state NMR and demonstrated with results on full-length, unmodified membrane proteins with one, two, and seven trans-membrane helices. RA solid-state NMR is complementary to other solid-state NMR methods, in particular oriented sample (OS) solid-state NMR of stationary, aligned samples. Structural distortions of membrane proteins from the truncations of terminal residues and other sequence modifications, and the use of detergent micelles instead of phospholipid bilayers have also been demonstrated. Thus, it is highly advantageous to determine the structures of unmodified membrane proteins in liquid crystalline phospholipid bilayers under physiological conditions. RA solid-state NMR provides a general method for obtaining accurate and precise structures of membrane proteins under near-native conditions. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. PMID:24747039

  1. Comparison of Replica Exchange Simulations of a Kinetically Trapped Protein Conformational State and its Native Form.

    PubMed

    Olson, Mark A; Legler, Patricia M; Goldman, Ellen R

    2016-03-10

    Recently an X-ray crystallographic structure of a single-domain antibody was reported with the protein chain trapped in a rare homodimeric form. One of the conformers appears to exhibit a misfolded region, and thus presumably the configurational stability is less favorable. To investigate whether simulation methods can detect any difference between the conformers and buttress the notion that one conformation is trapped on a pathway that incurs lower activation energy to unfold, adaptive temperature-based replica exchange simulations were applied to each chain to model conformational transitions. Simulation results found that the observed crystallographic difference between the two chains in the complementarity determining region CDR2 induces a stark distinction in conformational populations on the energy landscape. An appraisal of the energetic difference between the CDR2 conformations at 300 K revealed a localized order-disorder free-energy transition of roughly equivalent to two peptide hydrogen bonds in solution. It was also found that interconversion between the conformers is slower than the rate to unfold and that near an unfolding transition temperature one conformer retained a greater fraction of native-like contacts and energy over a longer time span before fully populating the denatured state, thus verifying the coexistence of a metastable conformation in the crystallographic assembly. PMID:26886055

  2. Native Electrospray Mass Spectrometry Reveals the Nature and Stoichiometry of Pigments in the FMO Photosynthetic Antenna Protein

    SciTech Connect

    Wen, Jianzhong; Zhang, Hao; Gross, Michael L; Blankenship, R. E.

    2011-05-03

    The nature and stoichiometry of pigments in the Fenna–Matthews–Olson (FMO) photosynthetic antenna protein complex were determined by native electrospray mass spectrometry. The FMO antenna complex was the first chlorophyll-containing protein that was crystallized. Previous results indicate that the FMO protein forms a trimer with seven bacteriochlorophyll a in each monomer. This model has long been a working basis to understand the molecular mechanism of energy transfer through pigment/pigment and pigment/protein coupling. Recent results have suggested, however, that an eighth bacteriochlorophyll is present in some subunits. In this report, a direct mass spectrometry measurement of the molecular weight of the intact FMO protein complex clearly indicates the existence of an eighth pigment, which is assigned as a bacteriochlorophyll a by mass analysis of the complex and HPLC analysis of the pigment. The eighth pigment is found to be easily lost during purification, which results in its partial occupancy in the mass spectra of the intact complex prepared by different procedures. The results are consistent with the recent X-ray structural models. The existence of the eighth bacteriochlorophyll a in this model antenna protein gives new insights into the functional role of the FMO protein and motivates the need for new theoretical and spectroscopic assignments of spectral features of the FMO protein.

  3. Structure Determination of Membrane Proteins by Nuclear Magnetic Resonance Spectroscopy

    NASA Astrophysics Data System (ADS)

    Opella, Stanley J.

    2013-06-01

    Many biological membranes consist of 50% or more (by weight) membrane proteins, which constitute approximately one-third of all proteins expressed in biological organisms. Helical membrane proteins function as receptors, enzymes, and transporters, among other unique cellular roles. Additionally, most drugs have membrane proteins as their receptors, notably the superfamily of G protein-coupled receptors with seven transmembrane helices. Determining the structures of membrane proteins is a daunting task because of the effects of the membrane environment; specifically, it has been difficult to combine biologically compatible environments with the requirements for the established methods of structure determination. There is strong motivation to determine the structures in their native phospholipid bilayer environment so that perturbations from nonnatural lipids and phases do not have to be taken into account. At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy.

  4. Network pattern of residue packing in helical membrane proteins and its application in membrane protein structure prediction.

    PubMed

    Pabuwal, Vagmita; Li, Zhijun

    2008-01-01

    De novo protein structure prediction plays an important role in studies of helical membrane proteins as well as structure-based drug design efforts. Developing an accurate scoring function for protein structure discrimination and validation remains a current challenge. Network approaches based on overall network patterns of residue packing have proven useful in soluble protein structure discrimination. It is thus of interest to apply similar approaches to the studies of residue packing in membrane proteins. In this work, we first carried out such analysis on a set of diverse, non-redundant and high-resolution membrane protein structures. Next, we applied the same approach to three test sets. The first set includes nine structures of membrane proteins with the resolution worse than 2.5 A; the other two sets include a total of 101 G-protein coupled receptor models, constructed using either de novo or homology modeling techniques. Results of analyses indicate the two criteria derived from studying high-resolution membrane protein structures are good indicators of a high-quality native fold and the approach is very effective for discriminating native membrane protein folds from less-native ones. These findings should be of help for the investigation of the fundamental problem of membrane protein structure prediction. PMID:18178566

  5. Mixing and Matching Detergents for Membrane Protein NMR Structure Determination

    SciTech Connect

    Columbus, Linda; Lipfert, Jan; Jambunathan, Kalyani; Fox, Daniel A.; Sim, Adelene Y.L.; Doniach, Sebastian; Lesley, Scott A.

    2009-10-21

    One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and small-angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein's hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.

  6. Native Electrospray and Electron-Capture Dissociation FTICR Mass Spectrometry for Top-Down Studies of Protein Assemblies

    SciTech Connect

    Zhang, Hao; Cui, Weidong; Wen, Jianzhong; Blankenship, Robert E.; Gross, Michael L.

    2011-07-15

    The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrometry and its coupling with native electrospray ionization (ESI) make the combination complementary to other biophysical methods of protein analysis. Protein assemblies with molecular masses up to MDa are now accessible by this approach. Most current approaches have used quadrupole/time-of-flight tandem mass spectrometry, sometimes coupled with ion mobility, to reveal stoichiometry, shape, and dissociation of protein assemblies. The amino-acid sequence of the subunits, however, still relies heavily on independent bottom-up proteomics. We describe here an approach to study protein assemblies that integrates electron-capture dissociation (ECD), native ESI, and FTICR mass spectrometry (12 T). Flexible regions of assembly subunits of yeast alcohol dehydrogenase (147 kDa), concanavalin A (103 kDa), and photosynthetic Fenna–Matthews–Olson antenna protein complex (140 kDa) can be sequenced by ECD or “activated-ion” ECD. Furthermore, noncovalent metal-binding sites can also be determined for the concanavalin A assembly. Most importantly, the regions that undergo fragmentation, either from one of the termini by ECD or from the middle of a protein, as initiated by CID, correlate well with the B-factor from X-ray crystallography of that protein. This factor is a measure of the extent an atom can move from its coordinated position as a function of temperature or crystal imperfections. The approach provides not only top-down proteomics information of the complex subunits but also structural insights complementary to those obtained by ion mobility.

  7. Protein structure mining using a structural alphabet.

    PubMed

    Tyagi, M; de Brevern, A G; Srinivasan, N; Offmann, B

    2008-05-01

    We present a comprehensive evaluation of a new structure mining method called PB-ALIGN. It is based on the encoding of protein structure as 1D sequence of a combination of 16 short structural motifs or protein blocks (PBs). PBs are short motifs capable of representing most of the local structural features of a protein backbone. Using derived PB substitution matrix and simple dynamic programming algorithm, PB sequences are aligned the same way amino acid sequences to yield structure alignment. PBs are short motifs capable of representing most of the local structural features of a protein backbone. Alignment of these local features as sequence of symbols enables fast detection of structural similarities between two proteins. Ability of the method to characterize and align regions beyond regular secondary structures, for example, N and C caps of helix and loops connecting regular structures, puts it a step ahead of existing methods, which strongly rely on secondary structure elements. PB-ALIGN achieved efficiency of 85% in extracting true fold from a large database of 7259 SCOP domains and was successful in 82% cases to identify true super-family members. On comparison to 13 existing structure comparison/mining methods, PB-ALIGN emerged as the best on general ability test dataset and was at par with methods like YAKUSA and CE on nontrivial test dataset. Furthermore, the proposed method performed well when compared to flexible structure alignment method like FATCAT and outperforms in processing speed (less than 45 s per database scan). This work also establishes a reliable cut-off value for the demarcation of similar folds. It finally shows that global alignment scores of unrelated structures using PBs follow an extreme value distribution. PB-ALIGN is freely available on web server called Protein Block Expert (PBE) at http://bioinformatics.univ-reunion.fr/PBE/. PMID:18004784

  8. Structure of giant muscle proteins

    PubMed Central

    Meyer, Logan C.; Wright, Nathan T.

    2013-01-01

    Giant muscle proteins (e.g., titin, nebulin, and obscurin) play a seminal role in muscle elasticity, stretch response, and sarcomeric organization. Each giant protein consists of multiple tandem structural domains, usually arranged in a modular fashion spanning 500 kDa to 4 MDa. Although many of the domains are similar in structure, subtle differences create a unique function of each domain. Recent high and low resolution structural and dynamic studies now suggest more nuanced overall protein structures than previously realized. These findings show that atomic structure, interactions between tandem domains, and intrasarcomeric environment all influence the shape, motion, and therefore function of giant proteins. In this article we will review the current understanding of titin, obscurin, and nebulin structure, from the atomic level through the molecular level. PMID:24376425

  9. NMR Structures of Membrane Proteins in Phospholipid Bilayers

    PubMed Central

    Radoicic, Jasmina; Lu, George J.; Opella, Stanley J.

    2014-01-01

    Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins; especially those accomplished with the proteins in phospholipid bilayer environments where they function. PMID:25032938

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

    PubMed Central

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

    2012-01-01

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

  11. Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope

    PubMed Central

    Pritchard, Laura K.; Harvey, David J.; Bonomelli, Camille

    2015-01-01

    ABSTRACT The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated, with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. In this study, we used a complementary, multistep approach involving ion mobility mass spectrometry and high-performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally, we show that unlike for gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation. IMPORTANCE Development of an HIV vaccine is desperately needed to control new infections, and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as

  12. A Historical Perspective and Overview of Protein Structure Prediction

    NASA Astrophysics Data System (ADS)

    Wooley, John C.; Ye, Yuzhen

    Carrying on many different biological functions, proteins are all composed of one or more polypeptide chains, each containing from several to hundreds or even thousands of the 20 amino acids. During the 1950s at the dawn of modern biochemistry, an essential question for biochemists was to understand the structure and function of these polypeptide chains. The sequences of protein, also referred to as their primary structures, determine the different chemical properties for different proteins, and thus continue to captivate much of the attention of biochemists. As an early step in characterizing protein chemistry, British biochemist Frederick Sanger designed an experimental method to identify the sequence of insulin (Sanger et al., 1955). He became the first person to obtain the primary structure of a protein and in 1958 won his first Nobel Price in Chemistry. This important progress in sequencing did not answer the question of whether a single (individual) protein has a distinctive shape in three dimensions (3D), and if so, what factors determine its 3D architecture. However, during the period when Sanger was studying the primary structure of proteins, American biochemist Christian Anfinsen observed that the active polypeptide chain of a model protein, bovine pancreatic ribonuclease (RNase), could fold spontaneously into a unique 3D structure, which was later called native conformation of the protein (Anfinsen et al., 1954). Anfinsen also studied the refolding of RNase enzyme and observed that an enzyme unfolded under extreme chemical environment could refold spontaneously back into its native conformation upon changing the environment back to natural conditions (Anfinsen et al., 1961). By 1962, Anfinsen had developed his theory of protein folding (which was summarized in his 1972 Nobel acceptance speech): "The native conformation is determined by the totality of interatomic interactions and hence, by the amino acid sequence, in a given environment."

  13. Can a pairwise contact potential stabilize native protein folds against decoys obtained by threading?

    PubMed

    Vendruscolo, M; Najmanovich, R; Domany, E

    2000-02-01

    We present a method to derive contact energy parameters from large sets of proteins. The basic requirement on which our method is based is that for each protein in the database the native contact map has lower energy than all its decoy conformations that are obtained by threading. Only when this condition is satisfied one can use the proposed energy function for fold identification. Such a set of parameters can be found (by perceptron learning) if Mp, the number of proteins in the database, is not too large. Other aspects that influence the existence of such a solution are the exact definition of contact and the value of the critical distance Rc, below which two residues are considered to be in contact. Another important novel feature of our approach is its ability to determine whether an energy function of some suitable proposed form can or cannot be parameterized in a way that satisfies our basic requirement. As a demonstration of this, we determine the region in the (Rc, Mp) plane in which the problem is solvable, i.e., we can find a set of contact parameters that stabilize simultaneously all the native conformations. We show that for large enough databases the contact approximation to the energy cannot stabilize all the native folds even against the decoys obtained by gapless threading. PMID:10656261

  14. Toolbox for Protein Structure Prediction.

    PubMed

    Roche, Daniel Barry; McGuffin, Liam James

    2016-01-01

    Protein tertiary structure prediction algorithms aim to predict, from amino acid sequence, the tertiary structure of a protein. In silico protein structure prediction methods have become extremely important, as in vitro-based structural elucidation is unable to keep pace with the current growth of sequence databases due to high-throughput next-generation sequencing, which has exacerbated the gaps in our knowledge between sequences and structures.Here we briefly discuss protein tertiary structure prediction, the biennial competition for the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and its role in shaping the field. We also discuss, in detail, our cutting-edge web-server method IntFOLD2-TS for tertiary structure prediction. Furthermore, we provide a step-by-step guide on using the IntFOLD2-TS web server, along with some real world examples, where the IntFOLD server can and has been used to improve protein tertiary structure prediction and aid in functional elucidation. PMID:26519323

  15. Molecular modeling and conformational analysis of native and refolded viral genome-linked protein of cardamom mosaic virus.

    PubMed

    Jebasingh, T; Jose, M; Yadunandam, A Kasin; Backiyarani, S; Srividhya, K V; Krishnaswamy, S; Usha, R

    2011-10-01

    The viral genome-linked protein (VPg) of Potyviruses is covalently attached to the 5' end of the genomic RNA. Towards biophysical characterization, the VPg coding region of Cardamom mosaic virus (CdMV) was amplified from the cDNA and expressed in E. coli. Most of the expressed VPg aggregated as inclusion bodies that were solubilized with urea and refolded with L-arginine hydrochloride. The various forms of CdMV VPg (native, denatured and refolded) were purified and the conformational variations between these forms were observed with fluorescence spectroscopy. Native and refolded CdMV VPg showed unordered secondary structure in the circular dichroism (CD) spectrum. The model of CdMV VPg was built based on the crystal structure of phosphotriesterase (from Pseudomonas diminuta), which had the maximum sequence homology with VPg to identify the arrangement of conserved amino acids in the protein to study the functional diversity of VPg. This is the first report on the VPg of CdMV, which is classified as a new member of the Macluravirus genus of the Potyviridae family. PMID:22165292

  16. Macromolecular crowding increases structural content of folded proteins.

    PubMed

    Perham, Michael; Stagg, Loren; Wittung-Stafshede, Pernilla

    2007-10-30

    Here we show that increased amount of secondary structure is acquired in the folded states of two structurally-different proteins (alpha-helical VlsE and alpha/beta flavodoxin) in the presence of macromolecular crowding agents. The structural content of flavodoxin and VlsE is enhanced by 33% and 70%, respectively, in 400 mg/ml Ficoll 70 (pH 7, 20 degrees C) and correlates with higher protein-thermal stability. In the same Ficoll range, there are only small effects on the unfolded-state structures of the proteins. This is the first in vitro assessment of crowding effects on the native-state structures at physiological conditions. Our findings imply that for proteins with low intrinsic stability, the functional structures in vivo may differ from those observed in dilute buffers. PMID:17919600

  17. Photoinduced structural changes to protein kinase A

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Thomas, Robert J.; Brancaleon, Lorenzo

    2014-03-01

    The importance of porphyrins in organisms is underscored by the ubiquitous biological and biochemical functions that are mediated by these compounds and by their potential biomedical and biotechnological applications. Protoporphyrin IX (PPIX) is the precursor to heme and has biomedical applications such as its use as a photosensitizer in phototherapy and photodetection of cancer. Among other applications, our group has demonstrated that low-irradiance exposure to laser irradiation of PPIX, Fe-PPIX, or meso-tetrakis (4-sulfonatophenyl) porphyrin (TSPP) non-covalently docked to a protein causes conformational changes in the polypeptide. Such approach can have remarkable consequences in the study of protein structure/function relationship and can be used to prompt non-native protein properties. Therefore we have investigated protein kinase A (PKA), a more relevant protein model towards the photo-treatment of cancer. PKA's enzymatic functions are regulated by the presence of cyclic adenosine monophosphate for intracellular signal transduction involved in, among other things, stimulation of transcription, tumorigenesis in Carney complex and migration of breast carcinoma cells. Since phosphorylation is a necessary step in some cancers and inflammatory diseases, inhibiting the protein kinase, and therefore phosphorylation, may serve to treat these diseases. Changes in absorption, steady-state fluorescence, and fluorescence lifetime indicate: 1) both TSPP and PPIX non-covalently bind to PKA where they maintain photoreactivity; 2) absorptive photoproduct formation occurs only when PKA is bound to TSPP and irradiated; and 3) PKA undergoes secondary structural changes after irradiation with either porphyrin bound. These photoinduced changes could affect the protein's enzymatic and signaling capabilities.

  18. Methods for optimizing the structure alphabet sequences of proteins.

    PubMed

    Dong, Qi-wen; Wang, Xiao-long; Lin, Lei

    2007-11-01

    Protein structure prediction based on fragment assemble has made great progress in recent years. Local protein structure prediction is receiving increased attention. One essential step of local protein structure prediction method is that the three-dimensional conformations must be compressed into one-dimensional series of letters of a structural alphabet. The traditional method assigns each structure fragment the structure alphabet that has the best local structure similarity. However, such locally optimal structure alphabet sequence does not guarantee to produce the globally optimal structure. This study presents two efficient methods trying to find the optimal structure alphabet sequence, which can model the native structures as accuracy as possible. First, a 28-letter structure alphabet is derived by clustering fragment in Cartesian space with fragment length of seven residues. The average quantization error of the 28 letters is 0.82 A in term of root mean square deviation. Then, two efficient methods are presented to encode the protein structures into series of structure alphabet letters, that is, the greedy and dynamic programming algorithm. They are tested on PDB database using the structure alphabet developed in Cartesian coordinates space (our structure alphabet) and in torsion angles space (the PB structure alphabet), respectively. The experimental results show that these two methods can find the approximately optimal structure alphabet sequences by searching a small fraction of the modeling space. The traditional local-optimization method achieves 26.27 A root mean square deviations between the reconstructed structures and the native one, while the modeling accuracy is improved to 3.28 A by the greedy algorithm. The results are helpful for local protein structure prediction. PMID:17493604

  19. Interactions of urea with native and unfolded proteins: a volumetric study.

    PubMed

    Son, Ikbae; Shek, Yuen Lai; Tikhomirova, Anna; Baltasar, Eduardo Hidalgo; Chalikian, Tigran V

    2014-11-26

    We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea-protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea-protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent-protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides. PMID:25365737

  20. Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology.

    PubMed

    Hackeng, T M; Griffin, J H; Dawson, P E

    1999-08-31

    The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of native chemical ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Here, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical ligation strategy for X-Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, Ile, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method's utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase A(2) and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of chemical protein synthesis. PMID:10468563

  1. Structure Determination of Membrane Proteins by Nuclear Magnetic Resonance Spectroscopy

    PubMed Central

    Opella, Stanley J.

    2014-01-01

    Many biological membranes consist of 50% or more (by weight) membrane proteins, which constitute approximately one-third of all proteins expressed in biological organisms. Helical membrane proteins function as receptors, enzymes, and transporters, among other unique cellular roles. Additionally, most drugs have membrane proteins as their receptors, notably the superfamily of G protein–coupled receptors with seven transmembrane helices. Determining the structures of membrane proteins is a daunting task because of the effects of the membrane environment; specifically, it has been difficult to combine biologically compatible environments with the requirements for the established methods of structure determination. There is strong motivation to determine the structures in their native phospholipid bilayer environment so that perturbations from nonnatural lipids and phases do not have to be taken into account. At present, the only method that can work with proteins in liquid crystalline phospholipid bilayers is solid-state NMR spectroscopy. PMID:23577669

  2. Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

    SciTech Connect

    Kiser, Philip D.; Golczak, Marcin; Lodowski, David T.; Chance, Mark R.; Palczewski, Krzysztof

    2009-12-01

    Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol in the retinal pigment epithelium (RPE). Mutations in RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis (LCA). Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Also, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To gain insight into these questions, we determined the crystal structure of native bovine RPE65 at 2.14-{angstrom} resolution. The structural, biophysical, and biochemical data presented here provide the framework needed for an in-depth understanding of the mechanism of catalytic isomerization and membrane association, in addition to the role mutations that cause LCA have in disrupting protein function.

  3. Structural origin of slow diffusion in protein folding.

    PubMed

    Chung, Hoi Sung; Piana-Agostinetti, Stefano; Shaw, David E; Eaton, William A

    2015-09-25

    Experimental, theoretical, and computational studies of small proteins suggest that interresidue contacts not present in the folded structure play little or no role in the self-assembly mechanism. Non-native contacts can, however, influence folding kinetics by introducing additional local minima that slow diffusion over the global free-energy barrier between folded and unfolded states. Here, we combine single-molecule fluorescence with all-atom molecular dynamics simulations to discover the structural origin for the slow diffusion that markedly decreases the folding rate for a designed α-helical protein. Our experimental determination of transition path times and our analysis of the simulations point to non-native salt bridges between helices as the source, which provides a quantitative glimpse of how specific intramolecular interactions influence protein folding rates by altering dynamics and not activation free energies. PMID:26404828

  4. Identifying transient protein-protein interactions in EphB2 signaling by Blue Native PAGE and Mass Spectrometry

    PubMed Central

    Darie, Costel C.; Deinhardt, Katrin; Zhang, Guoan; Cardasis, Helene S.; Chao, Moses V.; Neubert, Thomas A.

    2012-01-01

    Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands leads to activation of signal transduction pathways and to formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well-understood. We used Blue Native PAGE (BN-PAGE) and mass spectrometry (MS) to analyze the transient protein-protein interactions that resulted from stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated (pY-IPs) from the cell lysates of both unstimulated (−) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles in EphB2 signaling of proteins such as FAK, WAVEs, and Nischarin. PMID:21932443

  5. Arterivirus structural proteins and assembly

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter reviews the structural characteristics of the Arteriviridae, including the basic molecular details of all of the proteins involved, the interactions of these proteins and where they occur, and further functional characterization. Most recent available literature has been focused on equi...

  6. Colony social structure in native and invasive populations of the social wasp Vespula pensylvanica

    USGS Publications Warehouse

    Hanna, Cause; Cook, Erin D.; Thompson, Ariel R.; Dare, Lyndzey E.; Palaski, Amanda L.; Foote, David; Goodisman, Michael A. D.

    2014-01-01

    Social insects rank among the most invasive of terrestrial species. The success of invasive social insects stems, in part, from the flexibility derived from their social behaviors. We used genetic markers to investigate if the social system of the invasive wasp, Vespula pensylvanica, differed in its introduced and native habitats in order to better understand variation in social phenotype in invasive social species. We found that (1) nestmate workers showed lower levels of relatedness in introduced populations than native populations, (2) introduced colonies contained workers produced by multiple queens whereas native colonies contained workers produced by only a single queen, (3) queen mate number did not differ significantly between introduced and native colonies, and (4) workers from introduced colonies were frequently produced by queens that originated from foreign nests. Thus, overall, native and introduced colonies differed substantially in social phenotype because introduced colonies more frequently contained workers produced by multiple, foreign queens. In addition, the similarity in levels of genetic variation in introduced and native habitats, as well as observed variation in colony social phenotype in native populations, suggest that colony structure in invasive populations may be partially associated with social plasticity. Overall, the differences in social structure observed in invasive V. pensylvanica parallel those in other, distantly related invasive social insects, suggesting that insect societies often develop similar social phenotypes upon introduction into new habitats.

  7. MAGGIE Component 1: Identification and Purification of Native and Recombinant Multiprotein Complexes and Modified Proteins from Pyrococcus furiosus

    SciTech Connect

    Adams, Michael W.; W. W. Adams, Michael

    2014-01-07

    Virtualy all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes (PCs), the composition of which is largely unknown. Structural genomics efforts have demonstrated that less than 25% of the genes in a given prokaryotic genome will yield stable, soluble proteins when expressed using a one-ORF-at-a-time approach. We proposed that much of the remaining 75% of the genes encode proteins that are part of multiprotein complexes or are modified post-translationally, for example, with metals. The problem is that PCs and metalloproteins (MPs) cannot be accurately predicted on a genome-wide scale. The only solution to this dilemma is to experimentally determine PCs and MPs in biomass of a model organism and to develop analytical tools that can then be applied to the biomass of any other organism. In other words, organisms themselves must be analyzed to identify their PCs and MPs: “native proteomes” must be determined. This information can then be utilized to design multiple ORF expression systems to produce recombinant forms of PCs and MPs. Moreover, the information and utility of this approach can be enhanced by using a hyperthermophile, one that grows optimally at 100°C, as a model organism. By analyzing the native proteome at close to 100 °C below the optimum growth temperature, we will trap reversible and dynamic complexes, thereby enabling their identification, purification, and subsequent characterization. The model organism for the current study is Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100°C. It is grown up to 600-liter scale and kg quantities of biomass are available. In this project we identified native PCs and MPs using P. furiosus biomass (with MS/MS analyses to identify proteins by component 4). In addition, we provided samples of abundant native PCs and MPs for structural characterization (using SAXS by component 5). We also designed and evaluated generic bioinformatics and

  8. Structural evolution during protein denaturation as induced by different methods.

    PubMed

    Chodankar, S; Aswal, V K; Kohlbrecher, J; Vavrin, R; Wagh, A G

    2008-03-01

    Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) have been used to study conformational changes in protein bovine serum albumin (BSA) due to perturbation in its native structure as induced by varying temperature and pressure, and in presence of protein denaturating agents urea and surfactant. BSA has prolate ellipsoidal shape at ambient temperature and we observe no effect of temperature on its structure up to a temperature of about 60 degrees C . At temperatures beyond 60 degrees C , protein denaturation leads to aggregation. The protein solution exhibits a fractal structure at temperatures above 64 degrees C , and its fractal dimension increases with temperature. This is an indication of aggregation followed by gelation that evolves with increasing temperature. It is known for some of the proteins (e.g., Staphylococcal Nuclease) that pressure of 200 MPa can unfold the protein, whereas BSA does not show any protein unfolding even up to the pressure of 450 MPa . In presence of urea, the BSA protein unfolds for urea concentrations greater than 4M and acquires a random coil configuration. We make use of the dilution method to show the reversibility of protein unfolding with urea. The addition of surfactant denaturates the protein by the formation of micellelike aggregates of surfactants along the unfolded polypeptide chains of the protein. We show such structure of the protein-surfactant complex can be stabilized at higher temperatures, which is not the case for pure protein. PMID:18517416

  9. Structural evolution during protein denaturation as induced by different methods

    NASA Astrophysics Data System (ADS)

    Chodankar, S.; Aswal, V. K.; Kohlbrecher, J.; Vavrin, R.; Wagh, A. G.

    2008-03-01

    Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) have been used to study conformational changes in protein bovine serum albumin (BSA) due to perturbation in its native structure as induced by varying temperature and pressure, and in presence of protein denaturating agents urea and surfactant. BSA has prolate ellipsoidal shape at ambient temperature and we observe no effect of temperature on its structure up to a temperature of about 60°C . At temperatures beyond 60°C , protein denaturation leads to aggregation. The protein solution exhibits a fractal structure at temperatures above 64°C , and its fractal dimension increases with temperature. This is an indication of aggregation followed by gelation that evolves with increasing temperature. It is known for some of the proteins (e.g., Staphylococcal Nuclease) that pressure of 200MPa can unfold the protein, whereas BSA does not show any protein unfolding even up to the pressure of 450MPa . In presence of urea, the BSA protein unfolds for urea concentrations greater than 4M and acquires a random coil configuration. We make use of the dilution method to show the reversibility of protein unfolding with urea. The addition of surfactant denaturates the protein by the formation of micellelike aggregates of surfactants along the unfolded polypeptide chains of the protein. We show such structure of the protein-surfactant complex can be stabilized at higher temperatures, which is not the case for pure protein.

  10. Structural, energetic, and dynamic responses of the native state ensemble of staphylococcal nuclease to cavity-creating mutations.

    PubMed

    Roche, Julien; Caro, Jose A; Dellarole, Mariano; Guca, Ewelina; Royer, Catherine A; García-Moreno, Bertrand E; Garcia, Angel E; Roumestand, Christian

    2013-06-01

    The effects of cavity-creating mutations on the structural flexibility, local and global stability, and dynamics of the folded state of staphylococcal nuclease (SNase) were examined with NMR spectroscopy, MD simulations, H/D exchange, and pressure perturbation. Effects on global thermodynamic stability correlated well with the number of heavy atoms in the vicinity of the mutated residue. Variants with substitutions in the C-terminal domain and the interface between α and β subdomains showed large amide chemical shift variations relative to the parent protein, moderate, widespread, and compensatory perturbations of the H/D protection factors and increased local dynamics on a nanosecond time scale. The pressure sensitivity of the folded states of these variants was similar to that of the parent protein. Such observations point to the capacity of the folded proteins to adjust to packing defects in these regions. In contrast, cavity creation in the β-barrel subdomain led to minimal perturbation of the structure of the folded state, However, significant pressure dependence of the native state amide resonances, along with strong effects on native state H/D exchange are consistent with increased probability of population of excited state(s) for these variants. Such contrasted responses to the creation of cavities could not be anticipated from global thermodynamic stability or crystal structures; they depend on the local structural and energetic context of the substitutions. PMID:23239146

  11. Direct analysis of native and chimeric GATA specific DNA binding proteins from Aspergillus nidulans.

    PubMed Central

    Peters, D G; Caddick, M X

    1994-01-01

    In Aspergillus nidulans the regulatory gene areA is responsible for mediating nitrogen metabolite repression. The areA product (AREA) represents an example of the GATA family of DNA binding proteins, which are characterised by the presence of a GATA domain consisting of a zinc finger within a highly conserved region of 52 amino acids. Among the other transcription factors included in this family is the principal erythroid transcription factor, GATA-1, which contains two GATA domains. In order to demonstrate high specificity binding of native AREA to DNA containing the sequence -GATA-, and investigate the presence in A.nidulans of other proteins with related specificities, we have used gel mobility shift assays. Both AREA-dependent and independent complexes have been identified. Two strains bearing chimeric genes were also characterised. In these, the region encoding the native GATA domain of AREA was replaced by sequences from murine GATA-1 cDNA encoding either the equivalent C-terminal domain or both the N and C-terminal domains. Strains bearing the areA::NC-GATA construct, which includes the sequence encoding both the N and C-terminal domains of GATA-1, leads to a pronounced increase in one of two AREA-dependent complexes and implicates the N-terminal domain of GATA-1 in mediating protein-protein interactions. Images PMID:7816601

  12. On the role of physics and evolution in dictating protein structure and function.

    PubMed

    Skolnick, Jeffrey; Gao, Mu; Zhou, Hongyi

    2014-08-01

    How many of the structural and functional properties of proteins are inherent? Computer simulations provide a powerful tool to address this question. A series of studies on QS, quasi-spherical, compact polypeptides which lack any secondary structure; ART, artificial, proteins comprised of compact homopolypeptides with protein-like secondary structure; and PDB, native, single domain proteins shows that essentially all native global folds, pockets and protein-protein interfaces are in the ART library. This suggests that many protein properties are inherent and that evolution is involved in fine-tuning. The completeness of the space of ligand binding pockets and protein-protein interfaces suggests that promiscuous interactions are intrinsic to proteins and that the capacity to perform the biochemistry of life at low level does not require evolution. If so, this has profound consequences for the origin of life. PMID:25484448

  13. On the role of physics and evolution in dictating protein structure and function

    PubMed Central

    Skolnick, Jeffrey; Gao, Mu; Zhou, Hongyi

    2014-01-01

    How many of the structural and functional properties of proteins are inherent? Computer simulations provide a powerful tool to address this question. A series of studies on QS, quasi-spherical, compact polypeptides which lack any secondary structure; ART, artificial, proteins comprised of compact homopolypeptides with protein-like secondary structure; and PDB, native, single domain proteins shows that essentially all native global folds, pockets and protein-protein interfaces are in the ART library. This suggests that many protein properties are inherent and that evolution is involved in fine-tuning. The completeness of the space of ligand binding pockets and protein-protein interfaces suggests that promiscuous interactions are intrinsic to proteins and that the capacity to perform the biochemistry of life at low level does not require evolution. If so, this has profound consequences for the origin of life. PMID:25484448

  14. Prediction of membrane protein structures with complex topologies using limited constraints

    PubMed Central

    Barth, P.; Wallner, B.; Baker, D.

    2009-01-01

    Reliable structure-prediction methods for membrane proteins are important because the experimental determination of high-resolution membrane protein structures remains very difficult, especially for eukaryotic proteins. However, membrane proteins are typically longer than 200 aa and represent a formidable challenge for structure prediction. We have developed a method for predicting the structures of large membrane proteins by constraining helix–helix packing arrangements at particular positions predicted from sequence or identified by experiments. We tested the method on 12 membrane proteins of diverse topologies and functions with lengths ranging between 190 and 300 residues. Enforcing a single constraint during the folding simulations enriched the population of near-native models for 9 proteins. In 4 of the cases in which the constraint was predicted from the sequence, 1 of the 5 lowest energy models was superimposable within 4 Å on the native structure. Near-native structures could also be selected for heme-binding and pore-forming domains from simulations in which pairs of conserved histidine-chelating hemes and one experimentally determined salt bridge were constrained, respectively. These results suggest that models within 4 Å of the native structure can be achieved for complex membrane proteins if even limited information on residue-residue interactions can be obtained from protein structure databases or experiments. PMID:19190187

  15. Structure Prediction of Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Hu, Xiche

    Membrane proteins play a central role in many cellular and physiological processes. It is estimated that integral membrane proteins make up about 20-30% of the proteome (Krogh et al., 2001b; Stevens and Arkin, 2000; von Heijne, 1999). They are essential mediators of material and information transfer across cell membranes. Their functions include active and passive transport of molecules into and out of cells and organelles; transduction of energy among various forms (light, electrical, and chemical energy); as well as reception and transduction of chemical and electrical signals across membranes (Avdonin, 2005; Bockaert et al., 2002; Pahl, 1999; Rehling et al., 2004; Stack et al., 1995). Identifying these transmembrane (TM) proteins and deciphering their molecular mechanisms, then, is of great importance, particularly as applied to biomedicine. Membrane proteins are the targets of a large number of pharmacologically and toxicologically active substances, and are directly involved in their uptake, metabolism, and clearance (Bettler et al., 1998; Cohen, 2002; Heusser and Jardieu, 1997; Tibes et al., 2005; Xu et al., 2005). Despite the importance of membrane proteins, the knowledge of their high-resolution structures and mechanisms of action has lagged far behind in comparison to that of water-soluble proteins: less than 1% of all three-dimensional structures deposited in the Protein Data Bank are of membrane proteins. This unfortunate disparity stems from difficulties in overexpression and the crystallization of membrane proteins (Grisshammer and Tate, 1995; Michel, 1991).

  16. MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection.

    PubMed

    Blanié, Sophie; Gelfi, Jacqueline; Bertagnoli, Stéphane; Camus-Bouclainville, Christelle

    2010-01-01

    Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-kappaB in the nucleus of TNFalpha-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein. PMID:20211013

  17. De Novo Protein Structure Prediction

    NASA Astrophysics Data System (ADS)

    Hung, Ling-Hong; Ngan, Shing-Chung; Samudrala, Ram

    An unparalleled amount of sequence data is being made available from large-scale genome sequencing efforts. The data provide a shortcut to the determination of the function of a gene of interest, as long as there is an existing sequenced gene with similar sequence and of known function. This has spurred structural genomic initiatives with the goal of determining as many protein folds as possible (Brenner and Levitt, 2000; Burley, 2000; Brenner, 2001; Heinemann et al., 2001). The purpose of this is twofold: First, the structure of a gene product can often lead to direct inference of its function. Second, since the function of a protein is dependent on its structure, direct comparison of the structures of gene products can be more sensitive than the comparison of sequences of genes for detecting homology. Presently, structural determination by crystallography and NMR techniques is still slow and expensive in terms of manpower and resources, despite attempts to automate the processes. Computer structure prediction algorithms, while not providing the accuracy of the traditional techniques, are extremely quick and inexpensive and can provide useful low-resolution data for structure comparisons (Bonneau and Baker, 2001). Given the immense number of structures which the structural genomic projects are attempting to solve, there would be a considerable gain even if the computer structure prediction approach were applicable to a subset of proteins.

  18. Structural Basis of Protein Oxidation Resistance: A Lysozyme Study

    PubMed Central

    Girod, Marion; Enjalbert, Quentin; Brunet, Claire; Antoine, Rodolphe; Lemoine, Jérôme; Lukac, Iva; Radman, Miroslav; Krisko, Anita; Dugourd, Philippe

    2014-01-01

    Accumulation of oxidative damage in proteins correlates with aging since it can cause irreversible and progressive degeneration of almost all cellular functions. Apparently, native protein structures have evolved intrinsic resistance to oxidation since perfectly folded proteins are, by large most robust. Here we explore the structural basis of protein resistance to radiation-induced oxidation using chicken egg white lysozyme in the native and misfolded form. We study the differential resistance to oxidative damage of six different parts of native and misfolded lysozyme by a targeted tandem/mass spectrometry approach of its tryptic fragments. The decay of the amount of each lysozyme fragment with increasing radiation dose is found to be a two steps process, characterized by a double exponential evolution of their amounts: the first one can be largely attributed to oxidation of specific amino acids, while the second one corresponds to further degradation of the protein. By correlating these results to the structural parameters computed from molecular dynamics (MD) simulations, we find the protein parts with increased root-mean-square deviation (RMSD) to be more susceptible to modifications. In addition, involvement of amino acid side-chains in hydrogen bonds has a protective effect against oxidation Increased exposure to solvent of individual amino acid side chains correlates with high susceptibility to oxidative and other modifications like side chain fragmentation. Generally, while none of the structural parameters alone can account for the fate of peptides during radiation, together they provide an insight into the relationship between protein structure and susceptibility to oxidation. PMID:24999730

  19. FlexAID: Revisiting Docking on Non-Native-Complex Structures.

    PubMed

    Gaudreault, Francis; Najmanovich, Rafael J

    2015-07-27

    Small-molecule protein docking is an essential tool in drug design and to understand molecular recognition. In the present work we introduce FlexAID, a small-molecule docking algorithm that accounts for target side-chain flexibility and utilizes a soft scoring function, i.e. one that is not highly dependent on specific geometric criteria, based on surface complementarity. The pairwise energy parameters were derived from a large dataset of true positive poses and negative decoys from the PDBbind database through an iterative process using Monte Carlo simulations. The prediction of binding poses is tested using the widely used Astex dataset as well as the HAP2 dataset, while performance in virtual screening is evaluated using a subset of the DUD dataset. We compare FlexAID to AutoDock Vina, FlexX, and rDock in an extensive number of scenarios to understand the strengths and limitations of the different programs as well as to reported results for Glide, GOLD, and DOCK6 where applicable. The most relevant among these scenarios is that of docking on flexible non-native-complex structures where as is the case in reality, the target conformation in the bound form is not known a priori. We demonstrate that FlexAID, unlike other programs, is robust against increasing structural variability. FlexAID obtains equivalent sampling success as GOLD and performs better than AutoDock Vina or FlexX in all scenarios against non-native-complex structures. FlexAID is better than rDock when there is at least one critical side-chain movement required upon ligand binding. In virtual screening, FlexAID results are lower on average than those of AutoDock Vina and rDock. The higher accuracy in flexible targets where critical movements are required, intuitive PyMOL-integrated graphical user interface and free source code as well as precompiled executables for Windows, Linux, and Mac OS make FlexAID a welcome addition to the arsenal of existing small-molecule protein docking methods. PMID

  20. Nonspecific native elution of proteins and mumps virus in immunoaffinity chromatography.

    PubMed

    Brgles, Marija; Sviben, Dora; Forčić, Dubravko; Halassy, Beata

    2016-05-20

    Immunoaffinity chromatography, based on the antigen-antibody recognition, enables specific purification of any antigen (protein, virus) by its antibody. The problem with immunoaffinity chromatography is the harsh elution conditions required for disrupting strong antigen-antibody interactions, such as low pH buffers, which are often deleterious for the immobilized protein and the protein to be isolated since they can also disrupt the intramolecular forces. Therefore, immunoaffinity chromatography can only be partially used for protein and virus purification. Here we report on a nonspecific elution in immunoaffinity chromatography using native conditions by elution with amino acid solution at physiological pH for which we suppose possible competing mechanism of action. Elution potential of various amino acid solutions was tested using immunoaffinity columns specific for ovalbumin and mumps virus, and protein G affinity column. Results have shown that the most successful elution solutions were those containing imidazole and arginine of high molarity. Imidazole represents aromatic residues readily found at the antigen-antibody interaction surface and arginine is most frequently found on protein surface in general. Therefore, results on their eluting power in immunoaffinity chromatography, which increases with increasing molarity, are in line with the competing mechanism of action. Virus immunoaffinity chromatography resulted in removal on nonviable virus particles, which is important for research and biotechnology purposes. In addition, amino acids are proven stabilizers for proteins and viruses making approach presented in this work a very convenient purification method. PMID:27090389

  1. Protein structure prediction and potential energy landscape analysis using continuous global minimization

    SciTech Connect

    Dill, K.A.; Phillips, A.T.; Rosen, J.B.

    1997-12-01

    Proteins require specific three-dimensional conformations to function properly. These {open_quotes}native{close_quotes} conformations result primarily from intramolecular interactions between the atoms in the macromolecule, and also intermolecular interactions between the macromolecule and the surrounding solvent. Although the folding process can be quite complex, the instructions guiding this process are specified by the one-dimensional primary sequence of the protein or nucleic acid: external factors, such as helper (chaperone) proteins, present at the time of folding have no effect on the final state of the protein. Many denatured proteins spontaneously refold into functional conformations once denaturing conditions are removed. Indeed, the existence of a unique native conformation, in which residues distant in sequence but close in proximity exhibit a densely packed hydrophobic core, suggests that this three-dimensional structure is largely encoded within the sequential arrangement of these specific amino acids. In any case, the native structure is often the conformation at the global minimum energy. In addition to the unique native (minimum energy) structure, other less stable structures exist as well, each with a corresponding potential energy. These structures, in conjunction with the native structure, make up an energy landscape that can be used to characterize various aspects of the protein structure. 22 refs., 10 figs., 2 tabs.

  2. Interactions of Intact Unfractionated Heparin with Its Client Proteins Can Be Probed Directly Using Native Electrospray Ionization Mass Spectrometry.

    PubMed

    Zhao, Yunlong; Abzalimov, Rinat R; Kaltashov, Igor A

    2016-02-01

    Heparin and related members of the glycosaminoglycan (GAG) family are highly polyanionic linear saccharides that play important roles in a variety of physiological processes ranging from blood coagulation to embryo- and oncogenesis, tissue regeneration, and immune response regulation. These diverse functions are executed via a variety of mechanisms, including protein sequestration, activation, and facilitation of their interactions with cell-surface receptors, but deciphering the specific molecular mechanisms is frequently impossible due to the extremely high degree of GAG heterogeneity. As a result, the vast majority of studies of heparin (or related GAGs) interactions with its client proteins use synthetically produced heparin mimetics with defined structure or short heparin fragments. In this work we use native electrospray ionization mass spectrometry (ESI MS) in combination with limited charge reduction in the gas phase to obtain meaningful information on noncovalent complexes formed by intact unfractionated heparin and antithrombin-III, interaction which is central to preventing blood clotting. Complexes of different stoichiometries are observed ranging from 1:1 to 1:3 (heparin/protein ratio). In addition to binding stoichiometry, the measurements allow the range of heparin chain lengths to be obtained for each complex and the contribution of each complex to the total ionic signal to be calculated. Incorporation of ion mobility measurements in the experimental workflow allows the total analysis time to be shortened very significantly and the charge state assignment for the charge-reduced species to be verified. The possibility to study interactions of intact unfractionated heparin with a client protein carried out directly by native ESI MS without the need to use relatively homogeneous surrogates demonstrated in this work opens up a host of new exciting opportunities and goes a long way toward ameliorating the persistent but outdated view of the

  3. Structural and functional analysis of native peroxiredoxin 2 in human red blood cells.

    PubMed

    Ogasawara, Yuki; Ohminato, Takuya; Nakamura, Yusuke; Ishii, Kazuyuki

    2012-07-01

    Peroxiredoxin 2, a typical 2-Cys peroxiredoxin, is the third most abundant protein in erythrocytes. It is understood that the physiologically functional state of peroxiredoxin 2 is the monomer, and that its role in scavenging low levels of H(2)O(2) results in the formation of disulfide-linked dimers, which are reversibly reduced to monomers by the thioredoxin-thioredoxin reductase system. Additionally, peroxiredoxins are highly susceptible to sulfinic acid formation through reactions with various peroxides. This overoxidized form, which is thought to convert peroxiredoxins into molecular chaperones and to be accompanied by a transition to polymeric forms, can be reversed by sulfiredoxins. However, physiological conformational changes and the antioxidant role of erythrocyte peroxiredoxin 2 are still unclear because there is low sulfiredoxin and thioredoxin-thioredoxin reductase activity in erythrocytes. In this study, we examined the structural and redox states of peroxiredoxin 2 in fresh hemolysates and estimated the activities of native and overoxidized peroxiredoxin 2 purified from red blood cells to clear the physiological roles of peroxiredoxin 2 in erythrocyte. Our findings demonstrate that native peroxiredoxin 2 exists as high molecular weight (>160 kDa) oligomers and that decamers or higher order molecular weight oligomers (260-460 kDa) have peroxidase activity. We further showed that peroxiredoxin 2 oligomers, which were predominantly composed of monomers in the reduced form, exert a chaperone activity equal to that of overoxidized peroxiredoxin 2 polymers. These results provide the novel insight that redox-active peroxiredoxin 2 functions in human red blood cells as high molecular weight oligomers that possess peroxidase and chaperone activities. PMID:22537912

  4. Native Contact Density and Nonnative Hydrophobic Effects in the Folding of Bacterial Immunity Proteins

    PubMed Central

    Chen, Tao; Chan, Hue Sun

    2015-01-01

    The bacterial colicin-immunity proteins Im7 and Im9 fold by different mechanisms. Experimentally, at pH 7.0 and 10°C, Im7 folds in a three-state manner via an intermediate but Im9 folding is two-state-like. Accordingly, Im7 exhibits a chevron rollover, whereas the chevron arm for Im9 folding is linear. Here we address the biophysical basis of their different behaviors by using native-centric models with and without additional transferrable, sequence-dependent energies. The Im7 chevron rollover is not captured by either a pure native-centric model or a model augmented by nonnative hydrophobic interactions with a uniform strength irrespective of residue type. By contrast, a more realistic nonnative interaction scheme that accounts for the difference in hydrophobicity among residues leads simultaneously to a chevron rollover for Im7 and an essentially linear folding chevron arm for Im9. Hydrophobic residues identified by published experiments to be involved in nonnative interactions during Im7 folding are found to participate in the strongest nonnative contacts in this model. Thus our observations support the experimental perspective that the Im7 folding intermediate is largely underpinned by nonnative interactions involving large hydrophobics. Our simulation suggests further that nonnative effects in Im7 are facilitated by a lower local native contact density relative to that of Im9. In a one-dimensional diffusion picture of Im7 folding with a coordinate- and stability-dependent diffusion coefficient, a significant chevron rollover is consistent with a diffusion coefficient that depends strongly on native stability at the conformational position of the folding intermediate. PMID:26016652

  5. Structure-Based Prediction of Protein-Folding Transition Paths.

    PubMed

    Jacobs, William M; Shakhnovich, Eugene I

    2016-09-01

    We propose a general theory to describe the distribution of protein-folding transition paths. We show that transition paths follow a predictable sequence of high-free-energy transient states that are separated by free-energy barriers. Each transient state corresponds to the assembly of one or more discrete, cooperative units, which are determined directly from the native structure. We show that the transition state on a folding pathway is reached when a small number of critical contacts are formed between a specific set of substructures, after which folding proceeds downhill in free energy. This approach suggests a natural resolution for distinguishing parallel folding pathways and provides a simple means to predict the rate-limiting step in a folding reaction. Our theory identifies a common folding mechanism for proteins with diverse native structures and establishes general principles for the self-assembly of polymers with specific interactions. PMID:27602721

  6. Protein interfacial structure and nanotoxicology

    NASA Astrophysics Data System (ADS)

    White, John W.; Perriman, Adam W.; McGillivray, Duncan J.; Lin, Jhih-Min

    2009-02-01

    Here we briefly recapitulate the use of X-ray and neutron reflectometry at the air-water interface to find protein structures and thermodynamics at interfaces and test a possibility for understanding those interactions between nanoparticles and proteins which lead to nanoparticle toxicology through entry into living cells. Stable monomolecular protein films have been made at the air-water interface and, with a specially designed vessel, the substrate changed from that which the air-water interfacial film was deposited. This procedure allows interactions, both chemical and physical, between introduced species and the monomolecular film to be studied by reflectometry. The method is briefly illustrated here with some new results on protein-protein interaction between β-casein and κ-casein at the air-water interface using X-rays. These two proteins are an essential component of the structure of milk. In the experiments reported, specific and directional interactions appear to cause different interfacial structures if first, a β-casein monolayer is attacked by a κ-casein solution compared to the reverse. The additional contrast associated with neutrons will be an advantage here. We then show the first results of experiments on the interaction of a β-casein monolayer with a nanoparticle titanium oxide sol, foreshadowing the study of the nanoparticle "corona" thought to be important for nanoparticle-cell wall penetration.

  7. A Novel Approach to Decoy Set Generation: Designing a Physical Energy Function Having Local Minima with Native Structure Characteristics

    PubMed Central

    Keasar, Chen; Levitt, Michael

    2009-01-01

    We suggest a new approach to the generation of candidate structures (decoys) for ab initio prediction of protein structures. Our method is based on random sampling of conformation space and subsequent local energy minimization. At the core of this approach lies the design of a novel type of energy function. This energy function has local minima with native structure characteristics and wide basins of attraction. The current work presents our motivation for deriving such an energy function and also tests the derived energy function. Our approach is novel in that it takes advantage of the inherently rough energy landscape of proteins, which is generally considered a major obstacle for protein structure prediction. When local minima have wide basins of attraction, the protein’s conformation space can be greatly reduced by the convergence of large regions of the space into single points, namely the local minima corresponding to these funnels. We have implemented this concept by an iterative process. The potential is first used to generate decoy sets and then we study these sets of decoys to guide further development of the potential. A key feature of our potential is the use of cooperative multi-body interactions that mimic the role of the entropic and solvent contributions to the free energy. The validity and value of our approach is demonstrated by applying it to 14 diverse, small proteins. We show that, for these proteins, the size of conformation space is considerably reduced by the new energy function. In fact, the reduction is so substantial as to allow efficient conformational sampling. As a result we are able to find a significant number of near-native conformations in random searches performed with limited computational resources. PMID:12742025

  8. Immobilised native plant cysteine proteases: packed-bed reactor for white wine protein stabilisation.

    PubMed

    Benucci, Ilaria; Lombardelli, Claudio; Liburdi, Katia; Acciaro, Giuseppe; Zappino, Matteo; Esti, Marco

    2016-02-01

    This research presents a feasibility study of using a continuous packed-bed reactor (PBR), containing immobilised native plant cysteine proteases, as a specific and mild alternative technique relative to the usual bentonite fining for white wine protein stabilisation. The operational parameters for a PBR containing immobilised bromelain (PBR-br) or immobilised papain (PBR-pa) were optimised using model wine fortified with synthetic substrate (Bz-Phe-Val-Arg-pNA). The effectiveness of PBR-br, both in terms of hazing potential and total protein decrease, was significantly higher than PBR-pa, in all the seven unfined, white wines used. Among the wines tested, Sauvignon Blanc, given its total protein content as well as its very high intrinsic instability, was selected as a control wine to evaluate the effect of the treatment on wine as to its soluble protein profile, phenolic composition, mineral component, and sensory properties. The treatment in a PBR containing immobilised bromelain appeared effective in decreasing both wine hazing potential and total protein amount, while it did not significantly affect the phenol compounds, the mineral component nor the sensory quality of wine. The enzymatic treatment in PBR was shown to be a specific and mild technique for use as an alternative to bentonite fining for white wine protein stabilisation. PMID:27162393

  9. Caring for our own: the role of institutionalized support structures in Native American nursing student success.

    PubMed

    Cech, Erin A; Metz, Anneke M; Babcock, Tracy; Smith, Jessi L

    2011-09-01

    In this project, the authors asked 19 Native American baccalaureate nursing students to discuss their experiences with a formal institutionalized student support program called "Caring for Our Own: A Reservation/University Partnership Program." The authors investigated the importance of different types of support structures within this program, as viewed by Native American nursing students. They distinguished between four institutionalized support structures: tangible, informational, emotional, and belonging. The authors found that students consider tangible support (such as stipends) to be comparatively less important than other types of support, particularly emotional and belonging support. Responses also revealed the importance of a fifth type of institutionalized support-motivational. The authors further discuss how these institutionalized support structures might lead to successful outcomes for Native American nursing students. PMID:21598863

  10. The structure of Lactobacillus brevis surface layer reassembled on liposomes differs from native structure as revealed by SAXS.

    PubMed

    Kontro, Inkeri; Wiedmer, Susanne K; Hynönen, Ulla; Penttilä, Paavo A; Palva, Airi; Serimaa, Ritva

    2014-08-01

    The reassembly of the S-layer protein SlpA of Lactobacillus brevis ATCC 8287 on positively charged liposomes was studied by small angle X-ray scattering (SAXS) and zeta potential measurements. SlpA was reassembled on unilamellar liposomes consisting of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-3-trimethylammonium-propane, prepared by extrusion through membranes with pore sizes of 50nm and 100nm. Similarly extruded samples without SlpA were used as a reference. The SlpA-containing samples showed clear diffraction peaks in their SAXS intensities. The lattice constants were calculated from the diffraction pattern and compared to those determined for SlpA on native cell wall fragments. Lattice constants for SlpA reassembled on liposomes (a=9.29nm, b=8.03nm, and γ=84.9°) showed a marked change in the lattice constants b and γ when compared to those determined for SlpA on native cell wall fragments (a=9.41nm, b=6.48nm, and γ=77.0°). The latter are in good agreement with values previously determined by electron microscopy. This indicates that the structure formed by SlpA is stable on the bacterial cell wall, but SlpA reassembles into a different structure on cationic liposomes. From the (10) reflection, the lower limit of crystallite size of SlpA on liposomes was determined to be 92nm, corresponding to approximately ten aligned lattice planes. PMID:24796504

  11. Quality assessment of modeled protein structure using physicochemical properties.

    PubMed

    Rana, Prashant Singh; Sharma, Harish; Bhattacharya, Mahua; Shukla, Anupam

    2015-04-01

    Physicochemical properties of proteins always guide to determine the quality of the protein structure, therefore it has been rigorously used to distinguish native or native-like structure from other predicted structures. In this work, we explore nine machine learning methods with six physicochemical properties to predict the Root Mean Square Deviation (RMSD), Template Modeling (TM-score), and Global Distance Test (GDT_TS-score) of modeled protein structure in the absence of its true native state. Physicochemical properties namely total surface area, euclidean distance (ED), total empirical energy, secondary structure penalty (SS), sequence length (SL), and pair number (PN) are used. There are a total of 95,091 modeled structures of 4896 native targets. A real coded Self-adaptive Differential Evolution algorithm (SaDE) is used to determine the feature importance. The K-fold cross validation is used to measure the robustness of the best predictive method. Through the intensive experiments, it is found that Random Forest method outperforms over other machine learning methods. This work makes the prediction faster and inexpensive. The performance result shows the prediction of RMSD, TM-score, and GDT_TS-score on Root Mean Square Error (RMSE) as 1.20, 0.06, and 0.06 respectively; correlation scores are 0.96, 0.92, and 0.91 respectively; R(2) are 0.92, 0.85, and 0.84 respectively; and accuracy are 78.82% (with ± 0.1 err), 86.56% (with ± 0.1 err), and 87.37% (with ± 0.1 err) respectively on the testing data set. The data set used in the study is available as supplement at http://bit.ly/RF-PCP-DataSets. PMID:25524475

  12. Method for protein structure alignment

    DOEpatents

    Blankenbecler, Richard; Ohlsson, Mattias; Peterson, Carsten; Ringner, Markus

    2005-02-22

    This invention provides a method for protein structure alignment. More particularly, the present invention provides a method for identification, classification and prediction of protein structures. The present invention involves two key ingredients. First, an energy or cost function formulation of the problem simultaneously in terms of binary (Potts) assignment variables and real-valued atomic coordinates. Second, a minimization of the energy or cost function by an iterative method, where in each iteration (1) a mean field method is employed for the assignment variables and (2) exact rotation and/or translation of atomic coordinates is performed, weighted with the corresponding assignment variables.

  13. A simple model for calculating the kinetics of protein folding from three-dimensional structures.

    PubMed

    Muñoz, V; Eaton, W A

    1999-09-28

    An elementary statistical mechanical model was used to calculate the folding rates for 22 proteins from their known three-dimensional structures. In this model, residues come into contact only after all of the intervening chain is in the native conformation. An additional simplifying assumption is that native structure grows from localized regions that then fuse to form the complete native molecule. The free energy function for this model contains just two contributions-conformational entropy of the backbone and the energy of the inter-residue contacts. The matrix of inter-residue interactions is obtained from the atomic coordinates of the three-dimensional structure. For the 18 proteins that exhibit two-state equilibrium and kinetic behavior, profiles of the free energy versus the number of native peptide bonds show two deep minima, corresponding to the native and denatured states. For four proteins known to exhibit intermediates in folding, the free energy profiles show additional deep minima. The calculated rates of folding the two-state proteins, obtained by solving a diffusion equation for motion on the free energy profiles, reproduce the experimentally determined values surprisingly well. The success of these calculations suggests that folding speed is largely determined by the distribution and strength of contacts in the native structure. We also calculated the effect of mutations on the folding kinetics of chymotrypsin inhibitor 2, the most intensively studied two-state protein, with some success. PMID:10500173

  14. Protein Profile in Corpus Luteum during Pregnancy in Korean Native Cows

    PubMed Central

    Chung, H. J.; Kim, K. W.; Han, D. W.; Lee, H. C.; Yang, B. C.; Chung, H. K.; Shim, M. R.; Choi, M. S.; Jo, E. B.; Jo, Y. M.; Oh, M. Y.; Jo, S. J.; Hong, S. K.; Park, J. K.; Chang, W. K.

    2012-01-01

    Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism, but the profile of proteins associated with progesterone synthesis in cyclic and pregnant corpus luteum (CL) is not well-known in cattle. In Experiment 1, plasma progesterone level was monitored in cyclic cows (n = 5) and pregnant cows (n = 6; until d-90). A significant decline in the plasma progesterone level occurred at d-19 of cyclic cows. Progesterone level in abbatoir-derived luteal tissues was also determined at d 1 to 5, 6 to 13 and 14 to 20 of cyclic cows, and d-60 and -90 of pregnant cows (n = 5 each). Progesterone level in d-60 CL was not different from those in d 6 to 13 CL and d-90 CL, although the difference between d 6 to 13 and d-90 was significant. In Experiment 2, protein expression pattern in CL at d-90 (n = 4) was compared with that in CL of cyclic cows at d 6 to 13 (n = 5). Significant changes in the level of protein expression were detected in 32 protein spots by two-dimensional polyacrylamide gel electrophoresis (2-DE), and 23 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six proteins were found only in pregnant CL, while the other 17 proteins were found only in cyclic CL. Among the above 6 proteins, vimentin which is involved in the regulation of post-implantation development was included. Thus, the protein expression pattern in CL was disorientated from cyclic luteal phase to mid pregnancy, and alterations in specific CL protein expression may contribute to the maintenance of pregnancy in Korean native cows. PMID:25049514

  15. Isozyme multiplicity with anomalous dimer patterns in a class III alcohol dehydrogenase. Effects on the activity and quaternary structure of residue exchanges at "nonfunctional" sites in a native protein.

    PubMed

    Danielsson, O; Shafqat, J; Estonius, M; el-Ahmad, M; Jörnvall, H

    1996-11-19

    The isozymes of class III alcohol dehydrogenase/glutathione-dependent formaldehyde dehydrogenase from cod were characterized. They exhibited three unexpected properties of general interest. First, these dimeric isozymes, derived from two types of subunit (h and l, for high- and low-activity forms), were recovered from liver preparations in only the homodimeric ll and heterodimeric hl combinations. Dissociation and reassociation of the isolated hl form in vitro also resulted in lower yields of the hh than the ll homodimer, although class III subunits are usually freely associable over wide borders of divergence (human and Drosophila). The h and l primary structures show that both chain types are characteristic of class III enzymes, without large amino acid replacements at positions of known subunit interactions. Hence, the hh dimer partial restriction indicates nontraditional alterations at h-subunit interfaces. The structure provides a possible explanation, in the form of h-chain modifications that may influence the anchoring of a loop at positions of two potentially deamidative beta-aspartyl shifts at distant Asn-Gly structures. Second the ll and hl forms differ in enzymatic properties, having 5-fold different K(m) values for NAD+ at pH 8, different K(m) values for S-(hydroxymethyl)glutathione (10 versus 150 microM), and different specific activities (4.5 versus 41 units/mg), with ll resembling and hl deviating from human and other class III alcohol dehydrogenases. However, functional residues lining substrate and coenzyme pockets in the known conformations of homologous forms are largely identical in the two isozymes [only minor conservative exchanges of Val/Leu116, Val/Leu203, Ile/Val224, and Ile/Val269 (numbering system of the human class I enzyme)], again indicating effects from distantly positioned h-chain replacements. Third, the two isozymes differ a surprising amount in amino acid sequence (18%, the same as the piscine/ human difference), reflecting a

  16. Characterizing Protein Structure, Dynamics and Conformation in Lyophilized Solids

    PubMed Central

    Moorthy, Balakrishnan S.; Iyer, Lavanya K.; Topp, Elizabeth M.

    2015-01-01

    The long-term stability of protein therapeutics in the solid-state depends on the preservation of native structure during lyophilization and in the lyophilized powder. Proteins can reversibly or irreversibly unfold upon lyophilization, acquiring conformations susceptible to degradation during storage. Therefore, characterizing proteins in the dried state is crucial for the design of safe and efficacious formulations. This review summarizes the basic principles and applications of the analytical techniques that are commonly used to characterize protein structure, dynamics and conformation in lyophilized solids. The review also discusses the applications of recently developed mass spectrometry based methods (solid-state hydrogen deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS)) and their ability to study proteins in the solid-state at high resolution. PMID:26446463

  17. Sm-like protein Hfq: Composition of the native complex, modifications, and interactions.

    PubMed

    Obregon, Karla A; Hoch, Connor T; Sukhodolets, Maxim V

    2015-08-01

    The bacterial Sm-like protein Hfq has been linked functionally to reactions that involve RNA; however, its explicit role and primary cellular localization remain elusive. We carried out a detailed biochemical characterization of native Escherichia coli Hfq obtained through methods that preserve its posttranslational modifications. ESI-MS analyses indicate modifications in 2-3 subunits/hexamer with a molecular mass matching that of an oxidized C:18 lipid. We show that the majority of cellular Hfq cannot be extracted without detergents and that purified Hfq can be retained on hydrophobic matrices. Analyses of purified Hfq and the native Hfq complexes observed in whole-cell E. coli extracts indicate the existence of dodecameric assemblies likely stabilized by interlocking C-terminal polypeptides originating from separate Hfq hexamers and/or accessory nucleic acid. We demonstrate that cellular Hfq is redistributed between transcription complexes and an insoluble fraction that includes protein complexes harboring polynucleotide phosphorylase (PNP). This distribution pattern is consistent with a function at the interface of the apparatuses responsible for synthesis and degradation of RNA. Taken together with the results of prior studies, these results suggest that Hfq could function as an anchor/coupling factor responsible for de-solubilization of RNA and its tethering to the degradosome complex. PMID:25896386

  18. Native Liquid Extraction Surface Analysis Mass Spectrometry: Analysis of Noncovalent Protein Complexes Directly from Dried Substrates

    NASA Astrophysics Data System (ADS)

    Martin, Nicholas J.; Griffiths, Rian L.; Edwards, Rebecca L.; Cooper, Helen J.

    2015-08-01

    Liquid extraction surface analysis (LESA) mass spectrometry is a promising tool for the analysis of intact proteins from biological substrates. Here, we demonstrate native LESA mass spectrometry of noncovalent protein complexes of myoglobin and hemoglobin from a range of surfaces. Holomyoglobin, in which apomyoglobin is noncovalently bound to the prosthetic heme group, was observed following LESA mass spectrometry of myoglobin dried onto glass and polyvinylidene fluoride surfaces. Tetrameric hemoglobin [(αβ)2 4H] was observed following LESA mass spectrometry of hemoglobin dried onto glass and polyvinylidene fluoride (PVDF) surfaces, and from dried blood spots (DBS) on filter paper. Heme-bound dimers and monomers were also observed. The `contact' LESA approach was particularly suitable for the analysis of hemoglobin tetramers from DBS.

  19. Bayesian inference of protein structure from chemical shift data

    PubMed Central

    Bratholm, Lars A.; Christensen, Anders S.; Hamelryck, Thomas

    2015-01-01

    Protein chemical shifts are routinely used to augment molecular mechanics force fields in protein structure simulations, with weights of the chemical shift restraints determined empirically. These weights, however, might not be an optimal descriptor of a given protein structure and predictive model, and a bias is introduced which might result in incorrect structures. In the inferential structure determination framework, both the unknown structure and the disagreement between experimental and back-calculated data are formulated as a joint probability distribution, thus utilizing the full information content of the data. Here, we present the formulation of such a probability distribution where the error in chemical shift prediction is described by either a Gaussian or Cauchy distribution. The methodology is demonstrated and compared to a set of empirically weighted potentials through Markov chain Monte Carlo simulations of three small proteins (ENHD, Protein G and the SMN Tudor Domain) using the PROFASI force field and the chemical shift predictor CamShift. Using a clustering-criterion for identifying the best structure, together with the addition of a solvent exposure scoring term, the simulations suggests that sampling both the structure and the uncertainties in chemical shift prediction leads more accurate structures compared to conventional methods using empirical determined weights. The Cauchy distribution, using either sampled uncertainties or predetermined weights, did, however, result in overall better convergence to the native fold, suggesting that both types of distribution might be useful in different aspects of the protein structure prediction. PMID:25825683

  20. Structural insights into protein-metal ion partnerships.

    PubMed

    Barondeau, David P; Getzoff, Elizabeth D

    2004-12-01

    New metalloprotein structures continue to provide discoveries regarding protein-metal ion partnerships. Many recent structures reveal metal ion sites that control or are controlled by protein conformational change, including modulation by alternative splice variants and striking conformational changes. Only a few novel catalytic metal centers have been revealed recently, such as the surprising Ni-hook superoxide dismutase catalytic site and the cubane-like Mn(3)CaO(4) photosynthetic oxygen-evolving center. However, important new variations on old heme themes, breakthroughs in the fields of metal ion regulation and metallochaperones, and captivating insights into partnerships between proteins and minerals have also been described. Very high resolution metal site structures and metalloprotein design will be increasingly important in order to leverage the wealth of native metalloprotein structures into a deep understanding of metal ion site specificity and activity. PMID:15582401

  1. In situ structural analysis of Golgi intracisternal protein arrays.

    PubMed

    Engel, Benjamin D; Schaffer, Miroslava; Albert, Sahradha; Asano, Shoh; Plitzko, Jürgen M; Baumeister, Wolfgang

    2015-09-01

    We acquired molecular-resolution structures of the Golgi within its native cellular environment. Vitreous Chlamydomonas cells were thinned by cryo-focused ion beam milling and then visualized by cryo-electron tomography. These tomograms revealed structures within the Golgi cisternae that have not been seen before. Narrow trans-Golgi lumina were spanned by asymmetric membrane-associated protein arrays that had ∼6-nm lateral periodicity. Subtomogram averaging showed that the arrays may determine the narrow central spacing of the trans-Golgi cisternae through zipper-like interactions, thereby forcing cargo to the trans-Golgi periphery. Additionally, we observed dense granular aggregates within cisternae and intracisternal filament bundles associated with trans-Golgi buds. These native in situ structures provide new molecular insights into Golgi architecture and function. PMID:26311849

  2. In situ structural analysis of Golgi intracisternal protein arrays

    PubMed Central

    Engel, Benjamin D.; Schaffer, Miroslava; Albert, Sahradha; Asano, Shoh; Plitzko, Jürgen M.; Baumeister, Wolfgang

    2015-01-01

    We acquired molecular-resolution structures of the Golgi within its native cellular environment. Vitreous Chlamydomonas cells were thinned by cryo-focused ion beam milling and then visualized by cryo-electron tomography. These tomograms revealed structures within the Golgi cisternae that have not been seen before. Narrow trans-Golgi lumina were spanned by asymmetric membrane-associated protein arrays that had ∼6-nm lateral periodicity. Subtomogram averaging showed that the arrays may determine the narrow central spacing of the trans-Golgi cisternae through zipper-like interactions, thereby forcing cargo to the trans-Golgi periphery. Additionally, we observed dense granular aggregates within cisternae and intracisternal filament bundles associated with trans-Golgi buds. These native in situ structures provide new molecular insights into Golgi architecture and function. PMID:26311849

  3. Protein Structure Comparison and Classification

    NASA Astrophysics Data System (ADS)

    Çamoǧlu, Orhan; Singh, Ambuj K.

    The success of genome projects has generated an enormous amount of sequence data. In order to realize the full value of the data, we need to understand its functional role and its evolutionary origin. Sequence comparison methods are incredibly valuable for this task. However, for sequences falling in the twilight zone (usually between 20 and 35% sequence similarity), we need to resort to structural alignment and comparison for a meaningful analysis. Such a structural approach can be used for classification of proteins, isolation of structural motifs, and discovery of drug targets.

  4. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber.

    PubMed

    Xia, Xiao-Xia; Qian, Zhi-Gang; Ki, Chang Seok; Park, Young Hwan; Kaplan, David L; Lee, Sang Yup

    2010-08-10

    Spider dragline silk is a remarkably strong fiber that makes it attractive for numerous applications. Much has thus been done to make similar fibers by biomimic spinning of recombinant dragline silk proteins. However, success is limited in part due to the inability to successfully express native-sized recombinant silk proteins (250-320 kDa). Here we show that a 284.9 kDa recombinant protein of the spider Nephila clavipes is produced and spun into a fiber displaying mechanical properties comparable to those of the native silk. The native-sized protein, predominantly rich in glycine (44.9%), was favorably expressed in metabolically engineered Escherichia coli within which the glycyl-tRNA pool was elevated. We also found that the recombinant proteins of lower molecular weight versions yielded inferior fiber properties. The results provide insight into evolution of silk protein size related to mechanical performance, and also clarify why spinning lower molecular weight proteins does not recapitulate the properties of native fibers. Furthermore, the silk expression, purification, and spinning platform established here should be useful for sustainable production of natural quality dragline silk, potentially enabling broader applications. PMID:20660779

  5. Binding of non-native protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation.

    PubMed Central

    Ehrnsperger, M; Gräber, S; Gaestel, M; Buchner, J

    1997-01-01

    Small heat shock proteins (sHsps) are a conserved and ubiquitous protein family. Their ability to convey thermoresistance suggests their participation in protecting the native conformation of proteins. However, the underlying functional principles of their protective properties and their role in concert with other chaperone families remain enigmatic. Here, we analysed the influence of Hsp25 on the inactivation and subsequent aggregation of a model protein, citrate synthase (CS), under heat shock conditions in vitro. We show that stable binding of several non-native CS molecules to one Hsp25 oligomer leads to an accumulation of CS unfolding intermediates, which are protected from irreversible aggregation. Furthermore, a number of different proteins which bind to Hsp25 can be isolated from heat-shocked extracts of cells. Under permissive folding conditions, CS can be released from Hsp25 and, in cooperation with Hsp70, an ATP-dependent chaperone, the native state can be restored. Taken together, our findings allow us to integrate sHsps functionally in the cellular chaperone system operating under heat shock conditions. The task of sHsps in this context is to efficiently trap a large number of unfolding proteins in a folding-competent state and thus create a reservoir of non-native proteins for an extended period of time, allowing refolding after restoration of physiological conditions in cooperation with other chaperones. PMID:9029143

  6. Characterization of intact protein conjugates and biopharmaceuticals using ion-exchange chromatography with online detection by native electrospray ionization mass spectrometry and top-down tandem mass spectrometry.

    PubMed

    Muneeruddin, Khaja; Nazzaro, Mark; Kaltashov, Igor A

    2015-10-01

    Characterization of biopharmaceutical products is a challenging task, which needs to be carried out at several different levels (including both primary structure and conformation). An additional difficulty frequently arises due to the structural heterogeneity inherent to many protein-based therapeutics (e.g., extensive glycosylation or "designer" modifications such as chemical conjugation) or introduced postproduction as a result of stress (e.g., oxidation and deamidation). A combination of ion-exchange chromatography (IXC) with online detection by native electrospray ionization mass spectrometry (ESI MS) allows characterization of complex and heterogeneous therapeutic proteins and protein conjugates to be accomplished at a variety of levels without compromising their conformational integrity. The IXC/ESI MS measurements allow protein conjugates to be profiled by analyzing conjugation stoichiometry and the presence of multiple positional isomers, as well as to establish the effect of chemical modifications on the conformational integrity of each species. While mass profiling alone is not sufficient for identification of nonenzymatic post-translational modifications (PTMs) that result in a very small mass change of the eluting species (e.g., deamidation), this task can be completed using online top-down structural analysis, as demonstrated using stressed interferon-β as an example. The wealth of information that can be provided by IXC/native ESI MS and tandem mass spectrometry (MS/MS) on protein-based therapeutics will undoubtedly make it a very valuable addition to the experimental toolbox of biopharmaceutical analysis. PMID:26360183

  7. Information Structure of Native English-Speaking ESOL Teachers in Grammar Explanations

    ERIC Educational Resources Information Center

    Malupa-Kim, Miralynn Faigao

    2011-01-01

    The Problem: The purpose of this study was to identify and analyze the information structure of native-English speaking (NES) ESOL teachers in giving explanations in a grammar class at an Intensive English Program (IEP) at a university in southern California Method: This mixed-method study employed a sequential-exploratory design. Six grammar…

  8. Native Myocardial T1 as a Biomarker of Cardiac Structure in Non-Ischemic Cardiomyopathy.

    PubMed

    Shah, Ravi V; Kato, Shingo; Roujol, Sebastien; Murthy, Venkatesh; Bellm, Steven; Kashem, Abyaad; Basha, Tamer; Jang, Jihye; Eisman, Aaron S; Manning, Warren J; Nezafat, Reza

    2016-01-15

    Diffuse myocardial fibrosis is involved in the pathology of nonischemic cardiomyopathy (NIC). Recently, the application of native (noncontrast) myocardial T1 measurement has been proposed as a method for characterizing diffuse interstitial fibrosis. To determine the association of native T1 with myocardial structure and function, we prospectively studied 39 patients with NIC (defined as left ventricular ejection fraction (LVEF) ≤ 50% without cardiac magnetic resonance (CMR) evidence of previous infarction) and 27 subjects with normal LVEF without known overt cardiovascular disease. T1, T2, and extracellular volume fraction (ECV) were determined over 16 segments across the base, mid, and apical left ventricular (LV). NIC participants (57 ± 15 years) were predominantly men (74%), with a mean LVEF 34 ± 10%. Subjects with NIC had a greater native T1 (1,131 ± 51 vs 1,069 ± 29 ms; p <0.0001), a greater ECV (0.28 ± 0.04 vs 0.25 ± 0.02, p = 0.002), and a longer myocardial T2 (52 ± 8 vs 47 ± 5 ms; p = 0.02). After multivariate adjustment, a lower global native T1 time in NIC was associated with a greater LVEF (β = -0.59, p = 0.0003), greater right ventricular ejection fraction (β = -0.47, p = 0.006), and smaller left atrial volume index (β = 0.51, p = 0.001). The regional distribution of native myocardial T1 was similar in patients with and without NIC. In NIC, native myocardial T1 is elevated in all myocardial segments, suggesting a global (not regional) abnormality of myocardial tissue composition. In conclusion, native T1 may represent a rapid, noncontrast alternative to ECV for delineating myocardial tissue remodeling in NIC. PMID:26684511

  9. D2N: Distance to the native.

    PubMed

    Mishra, Avinash; Rana, Prashant Singh; Mittal, Aditya; Jayaram, B

    2014-10-01

    Root-mean-square-deviation (RMSD), of computationally-derived protein structures from experimentally determined structures, is a critical index to assessing protein-structure-prediction-algorithms (PSPAs). The development of PSPAs to obtain 0Å RMSD from native structures is considered central to computational biology. However, till date it has been quite challenging to measure how far a predicted protein structure is from its native - in the absence of a known experimental/native structure. In this work, we report the development of a metric "D2N" (distance to the native) - that predicts the "RMSD" of any structure without actually knowing the native structure. By combining physico-chemical properties and known universalities in spatial organization of soluble proteins to develop D2N, we demonstrate the ability to predict the distance of a proposed structure to within ±1.5Ǻ error with a remarkable average accuracy of 93.6% for structures below 5Ǻ from the native. We believe that this work opens up a completely new avenue towards assigning reliable structures to whole proteomes even in the absence of experimentally determined native structures. The D2N tool is freely available at http://www.scfbio-iitd.res.in/software/d2n.jsp. PMID:25062912

  10. Sequence repeats and protein structure

    NASA Astrophysics Data System (ADS)

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

    2012-11-01

    Repeats are frequently found in known protein sequences. The level of sequence conservation in tandem repeats correlates with their propensities to be intrinsically disordered. We employ a coarse-grained model of a protein with a two-letter amino acid alphabet, hydrophobic (H) and polar (P), to examine the sequence-structure relationship in the realm of repeated sequences. A fraction of repeated sequences comprises a distinct class of bad folders, whose folding temperatures are much lower than those of random sequences. Imperfection in sequence repetition improves the folding properties of the bad folders while deteriorating those of the good folders. Our results may explain why nature has utilized repeated sequences for their versatility and especially to design functional proteins that are intrinsically unstructured at physiological temperatures.

  11. Knotted proteins: A tangled tale of Structural Biology

    PubMed Central

    Faísca, Patrícia F.N.

    2015-01-01

    Knotted proteins have their native structures arranged in the form of an open knot. In the last ten years researchers have been making significant efforts to reveal their folding mechanism and understand which functional advantage(s) knots convey to their carriers. Molecular simulations have been playing a fundamental role in this endeavor, and early computational predictions about the knotting mechanism have just been confirmed in wet lab experiments. Here we review a collection of simulation results that allow outlining the current status of the field of knotted proteins, and discuss directions for future research. PMID:26380658

  12. Collapse of the native structure caused by a single amino acid exchange in human NAD(P)H:quinone oxidoreductase

    PubMed Central

    Uhl, Michael K.; Binter, Alexandra; Pulido, Sergio A.; Saf, Robert; Zangger, Klaus; Gruber, Karl; Macheroux, Peter

    2015-01-01

    Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is essential for the antioxidant defense system, stabilization of tumor suppressors (e.g. p53, p33, and p73), and activation of quinone-based chemotherapeutics. Overexpression of NQO1 in many solid tumors, coupled with its ability to convert quinone-based chemotherapeutics into potent cytotoxic compounds, have made it a very attractive target for anticancer drugs. A naturally occurring single-nucleotide polymorphism (C609T) leading to an amino acid exchange (P187S) has been implicated in the development of various cancers and poor survival rates following anthracyclin-based adjuvant chemotherapy. Despite its importance for cancer prediction and therapy, the exact molecular basis for the loss of function in NQO1 P187S is currently unknown. Therefore, we solved the crystal structure of NQO1 P187S. Surprisingly, this structure is almost identical to NQO1. Employing a combination of NMR spectroscopy and limited proteolysis experiments, we demonstrated that the single amino acid exchange destabilized interactions between the core and C-terminus, leading to depopulation of the native structure in solution. This collapse of the native structure diminished cofactor affinity and led to a less competent FAD-binding pocket, thus severely compromising the catalytic capacity of the variant protein. Hence, our findings provide a rationale for the loss of function in NQO1 P187S with a frequently occurring single-nucleotide polymorphism. PMID:25143260

  13. Molecular crowding enhances native state stability and refolding rates of globular proteins

    NASA Astrophysics Data System (ADS)

    Cheung, Margaret S.; Klimov, Dmitri; Thirumalai, D.

    2005-03-01

    The presence of macromolecules in cells geometrically restricts the available space for poplypeptide chains. To study the effects of macromolecular crowding on folding thermodynamics and kinetics, we used an off-lattice model of the all--sheet WW domain in the presence of large spherical particles whose interaction with the polypeptide chain is purely repulsive. At all volume fractions, c, of the crowding agents the stability of the native state is enhanced. Remarkably, the refolding rates, which are larger than the value at c = 0, increase nonmonotonically as c increases, reaching a maximum at . At high values of c, the depletion-induced intramolecular attraction produces compact structures with considerable structure in the denatured state. Changes in native state stability and folding kinetics at c can be quantitatively mapped onto confinement in a volume-fraction-dependent spherical pore with radius Rs ≈ (4π/3ϕc)1/3 Rc (Rc is the radius of the crowding particles) as long as ϕc is comparable with that in a spherical pore. In both situations, rate enhancement is due to destabilization of the denatured states with respect to ϕc = 0.

  14. On lattice protein structure prediction revisited.

    PubMed

    Dotu, Ivan; Cebrián, Manuel; Van Hentenryck, Pascal; Clote, Peter

    2011-01-01

    Protein structure prediction is regarded as a highly challenging problem both for the biology and for the computational communities. In recent years, many approaches have been developed, moving to increasingly complex lattice models and off-lattice models. This paper presents a Large Neighborhood Search (LNS) to find the native state for the Hydrophobic-Polar (HP) model on the Face-Centered Cubic (FCC) lattice or, in other words, a self-avoiding walk on the FCC lattice having a maximum number of H-H contacts. The algorithm starts with a tabu-search algorithm, whose solution is then improved by a combination of constraint programming and LNS. The flexible framework of this hybrid algorithm allows an adaptation to the Miyazawa-Jernigan contact potential, in place of the HP model, thus suggesting its potential for tertiary structure prediction. Benchmarking statistics are given for our method against the hydrophobic core threading program HPstruct, an exact method which can be viewed as complementary to our method. PMID:21358007

  15. A protein structure data and analysis system.

    PubMed

    Tian, Hao; Sunderraman, Rajshekhar; Weber, Irene; Wang, Haibin; Yang, Hong

    2005-01-01

    In this paper, we present the design and implementation of a protein structure data and analysis system that is only used in the lab for analyzing the proprietary data. It is capable of storing public protein data, such as the data in Protein Data Bank (PDB) [1], and life scientists' proprietary data. This toolkit is targeted at life scientists who want to maintain proprietary protein structure data (may be incomplete), to search and query publicly known protein structures and to compare their structure data with others. The comparison functions can be used to find structure differences between two proteins at atom level, especially in mutant versions of proteins. The system can also be used as a tool of choosing better protein structure template in new protein's tertiary structure prediction. The system is developed in Java and the protein data is stored in a relational database (Oracle 9i). PMID:17282836

  16. Native state dynamics and mechanical properties of human topoisomerase I within a structure-based coarse-grained model.

    PubMed

    Szklarczyk, Oliwia; Staroń, Krzysztof; Cieplak, Marek

    2009-11-01

    A coarse grained molecular dynamics model with an implicit solvent is used to elucidate properties of the human topoisomerase I. The model is defined through the native structure and it allows covering significantly longer time scales than in all atom simulations. Single residue and double residue motional characteristics are studied. The results are consistent with all atom simulations reported in the literature indicating usefulness of the model in further studies of this protein. Novel findings include broadening of the description of the dynamic behavior of the lip and hinge regions and a characterization of the motional properties of the RRM binding site of the enzyme. We also consider mechanical stretching of the protein and identify sources of the force peaks. The elastic properties of topoisomerase I are predicted to be average in comparison to other proteins, yielding a maximum force of resistance to pulling which should be of order 120 pN. The contact unraveling pattern is consistent with the understanding of the structure and function of the protein. We find supporting evidence for the hypothesis that the C-terminal domain acquires an ordered structure upon binding with the core enzyme even though it forms a molten globule when in isolation. PMID:19452556

  17. High-resolution Native-PAGE for membrane proteins capable of fluorescence detection and hydrodynamic state evaluation.

    PubMed

    Ihara, Makoto; Matsuura, Noriko; Yamashita, Atsuko

    2011-05-15

    An improved native polyacrylamide gel electrophoresis (PAGE) method capable of evaluating the hydrodynamic states of membrane proteins and allowing in-gel fluorescence detection was established. In this method, bis(alkyl) sulfosuccinate is used to provide negative charges for detergent-solubilized membrane proteins to facilitate proper electrophoretic migration without disturbing their native hydrodynamic states. The method achieved high-resolution electrophoretic separation, in good agreement with the elution profiles obtained by size exclusion chromatography. The applicability of in-gel fluorescence detection for tagged green fluorescent protein (GFP) facilitates the analysis of samples without any purification. This method might serve as a general analytical technique for assessing the folding, oligomerization, and protein complex formation of membrane proteins. PMID:21291856

  18. Overcoming bottlenecks in the membrane protein structural biology pipeline.

    PubMed

    Hardy, David; Bill, Roslyn M; Jawhari, Anass; Rothnie, Alice J

    2016-06-15

    Membrane proteins account for a third of the eukaryotic proteome, but are greatly under-represented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and EM cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as glucose neopentyl glycol (GNG), maltose neopentyl glycol (MNG) and calixarene-based detergents can improve protein stability without compromising their solubilizing properties. Styrene maleic acid lipid particles (SMALPs) focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis. Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future. PMID:27284049

  19. Bridging Experiments and Native-Centric Simulations of a Downhill Folding Protein.

    PubMed

    Naganathan, Athi N; De Sancho, David

    2015-11-25

    Experiments and atomistic simulations have independently contributed to the mechanistic understanding of protein folding. However, a coherent detailed picture explicitly combining both is currently lacking, a problem that seriously limits the amount of information that can be extracted. An alternative to atomistic models with physics-based potentials is the native-centric (i.e., Go̅ type) coarse-grained models, which for many years have been successfully employed to qualitatively understand features of protein folding energy landscapes. Again, quantitative validation of Go̅ models against experimental equilibrium unfolding curves is often not attempted. Here we use an atomistic topology-based model to study the folding mechanism of PDD, a protein that folds over a marginal thermodynamic barrier of ∼0.5 kBT at midpoint conditions. We find that the simulations are in exquisite agreement with several equilibrium experimental measurements including differential scanning calorimetry (DSC), an observable that is possibly the most challenging to reproduce from explicit-chain models. The dynamics, inferred using a detailed Markov state model, display a classical Chevron-like trend with a continuum of relaxation times under both folding and unfolding conditions, a signature feature of downhill folding. The number of populated microstates and the connectivity between them are shown to be temperature dependent with a maximum near the thermal denaturation midpoint, thus linking the macroscopic observation of a peak in the DSC profile of downhill folding proteins and the underlying microstate dynamics. The mechanistic picture derived from our analysis thus sheds light on the intricate and tunable nature of the downhill protein folding ensembles. In parallel, our work highlights the power of coarse-grained models to reproduce experiments at a quantitative level while also pointing at specific directions for their improvement. PMID:26524123

  20. Engineering tissue constructs to mimic native aortic and pulmonary valve leaflets' structures and mechanics

    NASA Astrophysics Data System (ADS)

    Masoumi, Nafiseh

    There are several disadvantages correlated with current heart valve replacement, including anticoagulation therapy for patients with mechanical valves and the low durability of bioprosthetic valves. The non-viable nature of such devices is a critical drawback especially for pediatric cases due to the inability of the graft to grow in vivo with the patients. A tissue engineered heart valve (TEHV) with remodeling and growth ability, is conceptually appealing to use in the surgical repair and could serve as a permanent replacements when operating for pediatric valvular lesions. It is critical that scaffolds for functional heart valve tissue engineering, be capable of mimicking the native leaflet's structure and mechanical properties at the time of implantation. Meanwhile, the scaffolds should be able to support cellular proliferation and native-like tissue formation as the TEHV remodels toward a scaffold-free state. Our overall hypothesis is that an "ideal" engineered construct, designed based on native leaflet's structure and mechanics, will complement a native heart valve leaflet in providing benchmarks for use in the design of clinically-applicable TEHV. This hypothesis was addressed through several experiments conducted in the present study. To establish a functional biomimetic TEHV, we developed scaffolds capable of matching the anisotropic stiffness of native leaflet while promoting native-like cell and collagen content and supporting the ECM generation. Scaffolds with various polymer contents (e.g., poly (glycerol sebacate) (PGS) and poly (epsilon-caprolactone) (PCL)) and structural designs (e.g., microfabricated and microfibrous scaffolds), were fabricated based on native leaflet's structure and mechanics. It was found that the tri-layered scaffold, designed with assembly of microfabricated PGS and microfibrous PGS/PCL was a functional leaflet capable of promoting tissue formation. Furthermore, to investigate the effect of cyclic stress and flexure

  1. Folding of a large protein at high structural resolution.

    PubMed

    Walters, Benjamin T; Mayne, Leland; Hinshaw, James R; Sosnick, Tobin R; Englander, S Walter

    2013-11-19

    Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (~100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways. PMID:24191053

  2. Folding of a large protein at high structural resolution

    PubMed Central

    Walters, Benjamin T.; Mayne, Leland; Hinshaw, James R.; Sosnick, Tobin R.; Englander, S. Walter

    2013-01-01

    Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways. PMID:24191053

  3. Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

    SciTech Connect

    Luka, Zigmund; Pakhomova, Svetlana; Loukachevitch, Lioudmila V; Newcomer, Marcia E; Wagner, Conrad

    2012-06-27

    Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.

  4. Selective High-Resolution Detection of Membrane Protein-Ligand Interaction in Native Membranes Using Trityl-Nitroxide PELDOR.

    PubMed

    Joseph, Benesh; Tormyshev, Victor M; Rogozhnikova, Olga Yu; Akhmetzyanov, Dmitry; Bagryanskaya, Elena G; Prisner, Thomas F

    2016-09-12

    The orchestrated interaction of transmembrane proteins with other molecules mediates several crucial biological processes. Detergent solubilization may significantly alter or even abolish such hetero-oligomeric interactions, which makes observing them at high resolution in their native environment technically challenging. Dipolar electron paramagnetic resonance (EPR) techniques such as pulsed electro-electron double resonance (PELDOR) can provide very precise distances within biomolecules. To concurrently determine the inter-subunit interaction and the intra-subunit conformational changes in hetero-oligomeric complexes, a combination of different spin labels is required. Orthogonal spin labeling using a triarylmethyl (TAM) label in combination with a nitroxide label is used to detect protein-ligand interactions in native lipid bilayers. This approach provides a higher sensitivity and total selectivity and will greatly facilitate the investigation of multimeric transmembrane complexes employing different spin labels in the native lipid environment. PMID:27511025

  5. Food web structure in exotic and native mangroves: A Hawaii-Puerto Rico comparison

    USGS Publications Warehouse

    Demopoulos, A.W.J.; Fry, B.; Smith, C.R.

    2007-01-01

    Plant invasions can fundamentally alter detrital inputs and the structure of detritus-based food webs. We examined the detrital pathways in mangrove food webs in native (Puerto Rican) and introduced (Hawaiian) Rhizophora mangle forests using a dual isotope approach and a mixing model. Based on trophic-level fractionation of 0-1??? for ?? 13C and 2-3??? for ?? 15N, among the invertebrates, only nematodes, oligochaetes, and nereid polychaetes from native mangroves exhibited stable isotopes consistent with a mangrove-derived diet. Certain fauna, in particular tubificid oligochaetes, had ?? 13C values consistent with the consumption of mangrove leaves, but they were depleted in 15N, suggesting their primary nitrogen source was low in 15N, and was possibly N 2-fixing bacteria. In introduced mangroves, all feeding groups appeared to rely heavily on non-mangrove sources, especially phytoplankton inputs. Mixing model results and discriminant analysis showed clear separation of introduced and native mangrove sites based on differential food source utilization within feeding groups, with stronger and more diverse use of benthic foods observed in native forests. Observed differences between native and invasive mangrove food webs may be due to Hawaiian detritivores being poorly adapted to utilizing the tannin-rich, nitrogen-poor mangrove detritus. In addition, differential utilization of mangrove detritus between native and introduced mangroves may be a consequence of forest age. We postulate that increasing mangrove forest age may promote diversification of bacterial food webs important in N and S cycling. Our results also suggest a potentially important role for sulfur bacteria in supporting the most abundant infaunal consumers, nematodes, in the most mature systems. ?? 2007 Springer-Verlag.

  6. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

    PubMed Central

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388

  7. Introduction to Protein Structure through Genetic Diseases

    ERIC Educational Resources Information Center

    Schneider, Tanya L.; Linton, Brian R.

    2008-01-01

    An illuminating way to learn about protein function is to explore high-resolution protein structures. Analysis of the proteins involved in genetic diseases has been used to introduce students to protein structure and the role that individual mutations can play in the onset of disease. Known mutations can be correlated to changes in protein…

  8. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

    PubMed Central

    Pleiner, Tino; Bates, Mark; Trakhanov, Sergei; Lee, Chung-Tien; Schliep, Jan Erik; Chug, Hema; Böhning, Marc; Stark, Holger; Urlaub, Henning; Görlich, Dirk

    2015-01-01

    Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with <2 nm epitope-label displacement. For this, we introduced cysteines at specific positions in the nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target. DOI: http://dx.doi.org/10.7554/eLife.11349.001 PMID:26633879

  9. Behavior of native microbial populations of WPC-34 and WPC-80 whey protein stored at different temperatures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Whey protein (WPC34 and 80) has been used as food ingredients and as a base for making biodegradable product. However, there is limited information on the behavior of native microflora associated with these products. WPC 34 and WPC80 were obtained from the manufacturer, and were stored at 5, 10, 15,...

  10. Novel application of Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE).

    PubMed

    Wang, Yanan; Zhang, Jing; Huang, Lingyun; He, Dacheng; Ma, Lin; Ouyang, Jin; Jiang, Fubin

    2012-01-27

    We have developed a novel application for DNA oligonucleotide-stabilized Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE). Oligonucleotide-stabilized Ag nanoclusters were used as fluorescent probes for direct detection of proteins after native PAGE. Some relatively low-abundance proteins, such as α-1-antichymotrypsin (ACT) and α-2-glycoprotein 1, zinc (ZAG) were easily detected by oligonucleotide-stabilized Ag nanocluster-based fluorescent imaging and identified by MS and MS/MS techniques, without the need of expensive antibodies or tedious immunoassay procedures. The pH condition for the oligonucleotide-stabilized Ag nanocluster solution was optimized and the possible mechanism of interaction between proteins and DNA oligonucleotide-stabilized Ag nanoclusters was analyzed. As a novel fluorescent detection method it is simple, fast, nontoxic and sensitive, and it shows great analytical potential in proteome research and in biochemistry. PMID:22249908

  11. Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases.

    PubMed

    Wang, Fan; Song, Wensi; Brancati, Giovanna; Segatori, Laura

    2011-12-16

    Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis. PMID:22006919

  12. Population genetic structure of the predatory, social wasp Vespula pensylvanica in its native and invasive range.

    PubMed

    Chau, Linh M; Hanna, Cause; Jenkins, Laurel T; Kutner, Rachel E; Burns, Elizabeth A; Kremen, Claire; Goodisman, Michael A D

    2015-12-01

    Invasive species cause extensive damage to their introduced ranges. Ocean archipelagos are particularly vulnerable to invasive taxa. In this study, we used polymorphic microsatellite markers to investigate the genetic structure of the social wasp Vespula pensylvanica in its native range of North America and its introduced range in the archipelago of Hawaii. Our goal was to gain a better understanding of the invasion dynamics of social species and the processes affecting biological invasions. We found that V. pensylvanica showed no significant genetic isolation by distance and little genetic structure over a span of 2000 km in its native range. This result suggests that V. pensylvanica can successfully disperse across large distances either through natural- or human-mediated mechanisms. In contrast to the genetic patterns observed in the native range, we found substantial genetic structure in the invasive V. pensylvanica range in Hawaii. The strong patterns of genetic differentiation within and between the Hawaiian Islands may reflect the effects of geographic barriers and invasion history on gene flow. We also found some evidence for gene flow between the different islands of Hawaii which was likely mediated through human activity. Overall, this study provides insight on how geographic barriers, invasion history, and human activity can shape population genetic structure of invasive species. PMID:27069607

  13. Competing processes of micellization and fibrillization in native and reduced casein proteins.

    PubMed

    Portnaya, Irina; Avni, Sharon; Kesselman, Ellina; Boyarski, Yoav; Sukenik, Shahar; Harries, Daniel; Dan, Nily; Cogan, Uri; Danino, Dganit

    2016-08-10

    Kappa-casein (κCN) and beta-casein (βCN) are disordered proteins present in mammalian milk. In vitro, βCN self-assembles into core-shell micelles. κCN self assembles into similar micelles, as well as into amyloid-like fibrils. Recent studies indicate that fibrillization can be suppressed by mixing βCN and κCN, but the mechanism of fibril inhibition has not been identified. Examining the interactions of native and reduced kappa-caseins (N-κCN and R-κCN) with βCN, we expose a competition between two different self-assembly processes: micellization and fibrillization. Quite surprisingly, however, we find significant qualitative and quantitative differences in the self-assembly between the native and reduced κCN forms. Specifically, thermodynamic analysis reveals exothermic demicellization for βCN and its mixtures with R-κCN, as opposed to endothermic demicellization of N-κCN and its mixtures with βCN at the same temperature. Furthermore, with time, R-κCN/βCN mixtures undergo phase separation into pure βCN micelles and R-κCN fibrils, while in the N-κCN/βCN mixtures fibril formation is considerably delayed and mixed micelles persist for longer periods of time. Fibrils formed in N-κCN/βCN mixtures are shorter and more flexible than those formed in R-κCN/βCN systems. Interestingly, in the N-κCN/βCN mixtures, the sugar moieties of N-κCN oligomers seem to organize on the mixed micelles surface in a manner similar to the organization of κCN in milk casein micelles. PMID:27468431

  14. Structural studies of human glioma pathogenesis-related protein 1

    SciTech Connect

    Asojo, Oluwatoyin A.; Koski, Raymond A.; Bonafé, Nathalie

    2011-10-01

    Structural analysis of a truncated soluble domain of human glioma pathogenesis-related protein 1, a membrane protein implicated in the proliferation of aggressive brain cancer, is presented. Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn{sup 2+} complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn{sup 2+} similarly to snake-venom CRISPs, which are involved in Zn{sup 2+}-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.

  15. Protein structure alignment beyond spatial proximity

    PubMed Central

    Wang, Sheng; Ma, Jianzhu; Peng, Jian; Xu, Jinbo

    2013-01-01

    Protein structure alignment is a fundamental problem in computational structure biology. Many programs have been developed for automatic protein structure alignment, but most of them align two protein structures purely based upon geometric similarity without considering evolutionary and functional relationship. As such, these programs may generate structure alignments which are not very biologically meaningful from the evolutionary perspective. This paper presents a novel method DeepAlign for automatic pairwise protein structure alignment. DeepAlign aligns two protein structures using not only spatial proximity of equivalent residues (after rigid-body superposition), but also evolutionary relationship and hydrogen-bonding similarity. Experimental results show that DeepAlign can generate structure alignments much more consistent with manually-curated alignments than other automatic tools especially when proteins under consideration are remote homologs. These results imply that in addition to geometric similarity, evolutionary information and hydrogen-bonding similarity are essential to aligning two protein structures. PMID:23486213

  16. Edaphic, salinity, and stand structural trends in chronosequences of native and non-native dominated riparian forests along the Colorado River, USA

    USGS Publications Warehouse

    Merritt, David M.; Shafroth, Patrick B.

    2012-01-01

    Tamarix spp. are introduced shrubs that have become among the most abundant woody plants growing along western North American rivers. We sought to empirically test the long-held belief that Tamarix actively displaces native species through elevating soil salinity via salt exudation. We measured chemical and physical attributes of soils (e.g., salinity, major cations and anions, texture), litter cover and depth, and stand structure along chronosequences dominated by Tamarix and those dominated by native riparian species (Populus or Salix) along the upper and lower Colorado River in Colorado and Arizona/California, USA. We tested four hypotheses: (1) the rate of salt accumulation in soils is faster in Tamarix-dominated stands than stands dominated by native species, (2) the concentration of salts in the soil is higher in mature stands dominated by Tamarix compared to native stands, (3) soil salinity is a function of Tamarix abundance, and (4) available nutrients are more concentrated in native-dominated stands compared to Tamarix-dominated stands. We found that salt concentration increases at a faster rate in Tamarix-dominated stands along the relatively free-flowing upper Colorado but not along the heavily-regulated lower Colorado. Concentrations of ions that are known to be preferentially exuded by Tamarix (e.g., B, Na, and Cl) were higher in Tamarix stands than in native stands. Soil salt concentrations in older Tamarix stands along the upper Colorado were sufficiently high to inhibit germination, establishment, or growth of some native species. On the lower Colorado, salinity was very high in all stands and is likely due to factors associated with floodplain development and the hydrologic effects of river regulation, such as reduced overbank flooding, evaporation of shallow ground water, higher salt concentrations in surface and ground water due to agricultural practices, and higher salt concentrations in fine-textured sediments derived from naturally saline

  17. An Interactive Introduction to Protein Structure

    ERIC Educational Resources Information Center

    Lee, W. Theodore

    2004-01-01

    To improve student understanding of protein structure and the significance of noncovalent interactions in protein structure and function, students are assigned a project to write a paper complemented with computer-generated images. The assignment provides an opportunity for students to select a protein structure that is of interest and detail…

  18. Population structure in the native range predicts the spread of introduced marine species.

    PubMed

    Gaither, Michelle R; Bowen, Brian W; Toonen, Robert J

    2013-06-01

    Forecasting invasion success remains a fundamental challenge in invasion biology. The effort to identify universal characteristics that predict which species become invasive has faltered in part because of the diversity of taxa and systems considered. Here, we use an alternative approach focused on the spread stage of invasions. FST, a measure of alternative fixation of alleles, is a common proxy for realized dispersal among natural populations, summarizing the combined influences of life history, behaviour, habitat requirements, population size, history and ecology. We test the hypothesis that population structure in the native range (FST) is negatively correlated with the geographical extent of spread of marine species in an introduced range. An analysis of the available data (29 species, nine phyla) revealed a significant negative correlation (R(2) = 0.245-0.464) between FST and the extent of spread of non-native species. Mode FST among pairwise comparisons between populations in the native range demonstrated the highest predictive power (R(2) = 0.464, p < 0.001). There was significant improvement when marker type was considered, with mtDNA datasets providing the strongest relationship (n = 21, R(2) = 0.333-0.516). This study shows that FST can be used to make qualitative predictions concerning the geographical extent to which a non-native marine species will spread once established in a new area. PMID:23595272

  19. Population structure in the native range predicts the spread of introduced marine species

    PubMed Central

    Gaither, Michelle R.; Bowen, Brian W.; Toonen, Robert J.

    2013-01-01

    Forecasting invasion success remains a fundamental challenge in invasion biology. The effort to identify universal characteristics that predict which species become invasive has faltered in part because of the diversity of taxa and systems considered. Here, we use an alternative approach focused on the spread stage of invasions. FST, a measure of alternative fixation of alleles, is a common proxy for realized dispersal among natural populations, summarizing the combined influences of life history, behaviour, habitat requirements, population size, history and ecology. We test the hypothesis that population structure in the native range (FST) is negatively correlated with the geographical extent of spread of marine species in an introduced range. An analysis of the available data (29 species, nine phyla) revealed a significant negative correlation (R2 = 0.245–0.464) between FST and the extent of spread of non-native species. Mode FST among pairwise comparisons between populations in the native range demonstrated the highest predictive power (R2 = 0.464, p < 0.001). There was significant improvement when marker type was considered, with mtDNA datasets providing the strongest relationship (n = 21, R2 = 0.333–0.516). This study shows that FST can be used to make qualitative predictions concerning the geographical extent to which a non-native marine species will spread once established in a new area. PMID:23595272

  20. Recombinant Minimalist Spider Wrapping Silk Proteins Capable of Native-Like Fiber Formation

    PubMed Central

    Xu, Lingling; Rainey, Jan K.; Meng, Qing; Liu, Xiang-Qin

    2012-01-01

    Spider silks are desirable biomaterials characterized by high tensile strength, elasticity, and biocompatibility. Spiders produce different types of silks for different uses, although dragline silks have been the predominant focus of previous studies. Spider wrapping silk, made of the aciniform protein (AcSp1), has high toughness because of its combination of high elasticity and tensile strength. AcSp1 in Argiope trifasciata contains a 200-aa sequence motif that is repeated at least 14 times. Here, we produced in E. coli recombinant proteins consisting of only one to four of the 200-aa AcSp1 repeats, designated W1 to W4. We observed that purified W2, W3 and W4 proteins could be induced to form silk-like fibers by shear forces in a physiological buffer. The fibers formed by W4 were ∼3.4 µm in diameter and up to 10 cm long. They showed an average tensile strength of 115 MPa, elasticity of 37%, and toughness of 34 J cm−3. The smaller W2 protein formed fewer fibers and required a higher protein concentration to form fibers, whereas the smallest W1 protein did not form silk-like fibers, indicating that a minimum of two of the 200-aa repeats was required for fiber formation. Microscopic examinations revealed structural features indicating an assembly of the proteins into spherical structures, fibrils, and silk-like fibers. CD and Raman spectral analysis of protein secondary structures suggested a transition from predominantly α-helical in solution to increasingly β-sheet in fibers. PMID:23209681

  1. Structure of the native (unligated) mannose-specific bulb lectin from Scilla campanulata (bluebell) at 1.7 A resolution.

    PubMed

    Wood, S D; Wright, L M; Reynolds, C D; Rizkallah, P J; Allen, A K; Peumans, W J; Van Damme, E J

    1999-07-01

    The X-ray crystal structure of native Scilla campanulata agglutinin, a mannose-specific lectin from bluebell bulbs and a member of the Liliaceae family, has been determined by molecular replacement and refined to an R value of 0.186 at 1.7 A resolution. The lectin crystallizes in space group P21212 with unit-cell parameters a = 70. 42, b = 92.95, c = 46.64 A. The unit cell contains eight protein molecules of Mr = 13143 Da (119 amino-acid residues). The asymmetric unit comprises two chemically identical molecules, A and B, related by a non-crystallographic twofold axis perpendicular to c. This dimer further associates by crystallographic twofold symmetry to form a tetramer. The fold of the polypeptide backbone closely resembles that found in the lectins from Galanthus nivalis (snowdrop) and Hippeastrum (amaryllis) and contains a threefold symmetric beta-prism made up of three antiparallel four-stranded beta-sheets. Each of the four-stranded beta-sheets (I, II and III) possesses a potential saccharide-binding site containing conserved residues; however, site II has two mutations relative to sites I and III which may prevent ligation at this site. Our study provides the first accurate and detailed description of a native (unligated) structure from this superfamily of mannose-specific bulb lectins and will allow comparisons with a number of lectin-saccharide complexes which have already been determined or are currently under investigation. PMID:10393293

  2. BeEP Server: using evolutionary information for quality assessment of protein structure models

    PubMed Central

    Palopoli, Nicolas; Lanzarotti, Esteban; Parisi, Gustavo

    2013-01-01

    The BeEP Server (http://www.embnet.qb.fcen.uba.ar/embnet/beep.php) is an online resource aimed to help in the endgame of protein structure prediction. It is able to rank submitted structural models of a protein through an explicit use of evolutionary information, a criterion differing from structural or energetic considerations commonly used in other assessment programs. The idea behind BeEP (Best Evolutionary Pattern) is to benefit from the substitution pattern derived from structural constraints present in a set of homologous proteins adopting a given protein conformation. The BeEP method uses a model of protein evolution that takes into account the structure of a protein to build site-specific substitution matrices. The suitability of these substitution matrices is assessed through maximum likelihood calculations from which position-specific and global scores can be derived. These scores estimate how well the structural constraints derived from each structural model are represented in a sequence alignment of homologous proteins. Our assessment on a subset of proteins from the Critical Assessment of techniques for protein Structure Prediction (CASP) experiment has shown that BeEP is capable of discriminating the models and selecting one or more native-like structures. Moreover, BeEP is not explicitly parameterized to find structural similarities between models and given targets, potentially helping to explore the conformational ensemble of the native state. PMID:23729471

  3. Generating folded protein structures with a lattice chain growth algorithm

    NASA Astrophysics Data System (ADS)

    Gan, Hin Hark; Tropsha, Alexander; Schlick, Tamar

    2000-10-01

    We present a new application of the chain growth algorithm to lattice generation of protein structure and thermodynamics. Given the difficulty of ab initio protein structure prediction, this approach provides an alternative to current folding algorithms. The chain growth algorithm, unlike Metropolis folding algorithms, generates independent protein structures to achieve rapid and efficient exploration of configurational space. It is a modified version of the Rosenbluth algorithm where the chain growth transition probability is a normalized Boltzmann factor; it was previously applied only to simple polymers and protein models with two residue types. The independent protein configurations, generated segment-by-segment on a refined cubic lattice, are based on a single interaction site for each amino acid and a statistical interaction energy derived by Miyazawa and Jernigan. We examine for several proteins the algorithm's ability to produce nativelike folds and its effectiveness for calculating protein thermodynamics. Thermal transition profiles associated with the internal energy, entropy, and radius of gyration show characteristic folding/unfolding transitions and provide evidence for unfolding via partially unfolded (molten-globule) states. From the configurational ensembles, the protein structures with the lowest distance root-mean-square deviations (dRMSD) vary between 2.2 to 3.8 Å, a range comparable to results of an exhaustive enumeration search. Though the ensemble-averaged dRMSD values are about 1.5 to 2 Å larger, the lowest dRMSD structures have similar overall folds to the native proteins. These results demonstrate that the chain growth algorithm is a viable alternative to protein simulations using the whole chain.

  4. Structures of Coxsackievirus A16 Capsids with Native Antigenicity: Implications for Particle Expansion, Receptor Binding, and Immunogenicity

    PubMed Central

    Ren, Jingshan; Wang, Xiangxi; Zhu, Ling; Hu, Zhongyu; Gao, Qiang; Yang, Pan; Li, Xuemei; Wang, Junzhi; Shen, Xinliang; Fry, Elizabeth E.

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) that affect more than a million children in China each year and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, the development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The structural details of the expanded forms of the capsids, which possess nonnative antigenicity, are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. Here, we remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant viruslike particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilize the native antigenic state that is different from the one used in a natural virus infection. As expected, the mature CVA16 virus is similar to EV71; however, structural and immunogenic comparisons highlight differences that may have implications for vaccine production. IMPORTANCE Hand-foot-and-mouth disease is a serious public health threat to children in Asian-Pacific countries, resulting in millions of cases. EV71 and CVA16 are the two dominant causative agents of the disease that, while usually mild, can cause severe neurological complications, leading to hundreds of deaths. EV71 vaccines do not provide protection against CVA16. A CVA16 vaccine or bivalent EV71/CVA16 vaccine is therefore urgently needed. We report atomic structures for the mature CVA16 virus, a natural empty particle, and a recombinant CVA16 virus-like particle that does not contain the viral genome. All three particles have similar

  5. Genetic Structure of Pacific Trout at the Extreme Southern End of Their Native Range.

    PubMed

    Abadía-Cardoso, Alicia; Garza, John Carlos; Mayden, Richard L; García de León, Francisco Javier

    2015-01-01

    Salmonid fishes are cold water piscivores with a native distribution spanning nearly the entire temperate and subarctic northern hemisphere. Trout in the genus Oncorhynchus are the most widespread salmonid fishes and are among the most important fish species in the world, due to their extensive use in aquaculture and valuable fisheries. Trout that inhabit northwestern Mexico are the southernmost native salmonid populations in the world, and the least studied in North America. They are unfortunately also facing threats to their continued existence. Previous work has described one endemic species, the Mexican golden trout (O. chrysogaster), and one endemic subspecies, Nelson's trout (O. mykiss nelsoni), in Mexico, but previous work indicated that there is vastly more biodiversity in this group than formally described. Here we conducted a comprehensive genetic analysis of this important group of fishes using novel genetic markers and techniques to elucidate the biodiversity of trout inhabiting northwestern Mexico, examine genetic population structure of Mexican trout and their relationships to other species of Pacific trout, and measure introgression from non-native hatchery rainbow trout. We confirmed substantial genetic diversity and extremely strong genetic differentiation present in the Mexican trout complex, not only between basins but also between some locations within basins, with at least four species-level taxa present. We also revealed significant divergence between Mexican trout and other trout species and found that introgression from non-native rainbow trout is present but limited, and that the genetic integrity of native trout is still maintained in most locations. This information will help to guide effective conservation strategies for this important group of fishes. PMID:26509445

  6. Genetic Structure of Pacific Trout at the Extreme Southern End of Their Native Range

    PubMed Central

    Abadía-Cardoso, Alicia; Garza, John Carlos; Mayden, Richard L.; García de León, Francisco Javier

    2015-01-01

    Salmonid fishes are cold water piscivores with a native distribution spanning nearly the entire temperate and subarctic northern hemisphere. Trout in the genus Oncorhynchus are the most widespread salmonid fishes and are among the most important fish species in the world, due to their extensive use in aquaculture and valuable fisheries. Trout that inhabit northwestern Mexico are the southernmost native salmonid populations in the world, and the least studied in North America. They are unfortunately also facing threats to their continued existence. Previous work has described one endemic species, the Mexican golden trout (O. chrysogaster), and one endemic subspecies, Nelson’s trout (O. mykiss nelsoni), in Mexico, but previous work indicated that there is vastly more biodiversity in this group than formally described. Here we conducted a comprehensive genetic analysis of this important group of fishes using novel genetic markers and techniques to elucidate the biodiversity of trout inhabiting northwestern Mexico, examine genetic population structure of Mexican trout and their relationships to other species of Pacific trout, and measure introgression from non-native hatchery rainbow trout. We confirmed substantial genetic diversity and extremely strong genetic differentiation present in the Mexican trout complex, not only between basins but also between some locations within basins, with at least four species-level taxa present. We also revealed significant divergence between Mexican trout and other trout species and found that introgression from non-native rainbow trout is present but limited, and that the genetic integrity of native trout is still maintained in most locations. This information will help to guide effective conservation strategies for this important group of fishes. PMID:26509445

  7. Structure Prediction of Protein Complexes

    NASA Astrophysics Data System (ADS)

    Pierce, Brian; Weng, Zhiping

    Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.

  8. Protein structure, stability and folding in the cell -- in silico biophysical approaches

    NASA Astrophysics Data System (ADS)

    Cheung, Margaret

    2010-03-01

    How the crowded environment inside a cell affects the structural conformation of a protein with aspherical shape is a vital question because the geometry of proteins and protein-protein complexes are far from globules in vivo. Here we address this question by combining computational and experimental studies of a spherical protein (i.e. apoflavodoxin), a football-shaped protein (i.e., Borrelia burgdorferi VlsE) and a dumbbell-shaped protein (i.e. calmodulin) under crowded, cell-like conditions. The results show that macromolecular crowding affects protein folding dynamics as well as an overall protein shape associated with changes in secondary structures. Our work demonstrates the malleability of ``native'' proteins and implies that crowding-induced shape changes may be important for protein function and malfunction in vivo.

  9. Population Structure and Genetic Diversity of Native and Invasive Populations of Solanum rostratum (Solanaceae)

    PubMed Central

    Zhao, Jiali; Solís-Montero, Lislie; Lou, Anru; Vallejo-Marín, Mario

    2013-01-01

    Aims We investigate native and introduced populations of Solanum rostratum, an annual, self-compatible plant that has been introduced around the globe. This study is the first to compare the genetic diversity of Solanum rostratum between native and introduced populations. We aim to (1) determine the level of genetic diversity across the studied regions; (2) explore the likely origins of invasive populations in China; and (3) investigate whether there is the evidence of multiple introductions into China. Methods We genotyped 329 individuals at 10 microsatellite loci to determine the levels of genetic diversity and to investigate population structure of native and introduced populations of S. rostratum. We studied five populations in each of three regions across two continents: Mexico, the U.S.A. and China. Important Findings We found the highest genetic diversity among Mexican populations of S. rostratum. Genetic diversity was significantly lower in Chinese and U.S.A. populations, but we found no regional difference in inbreeding coefficients (FIS) or population differentiation (FST). Population structure analyses indicate that Chinese and U.S.A. populations are more closely related to each other than to sampled Mexican populations, revealing that introduced populations in China share an origin with the sampled U.S.A. populations. The distinctiveness between some introduced populations indicates multiple introductions of S. rostratum into China. PMID:24224008

  10. Protein structure prediction with local adjust tabu search algorithm

    PubMed Central

    2014-01-01

    Background Protein folding structure prediction is one of the most challenging problems in the bioinformatics domain. Because of the complexity of the realistic protein structure, the simplified structure model and the computational method should be adopted in the research. The AB off-lattice model is one of the simplification models, which only considers two classes of amino acids, hydrophobic (A) residues and hydrophilic (B) residues. Results The main work of this paper is to discuss how to optimize the lowest energy configurations in 2D off-lattice model and 3D off-lattice model by using Fibonacci sequences and real protein sequences. In order to avoid falling into local minimum and faster convergence to the global minimum, we introduce a novel method (SATS) to the protein structure problem, which combines simulated annealing algorithm and tabu search algorithm. Various strategies, such as the new encoding strategy, the adaptive neighborhood generation strategy and the local adjustment strategy, are adopted successfully for high-speed searching the optimal conformation corresponds to the lowest energy of the protein sequences. Experimental results show that some of the results obtained by the improved SATS are better than those reported in previous literatures, and we can sure that the lowest energy folding state for short Fibonacci sequences have been found. Conclusions Although the off-lattice models is not very realistic, they can reflect some important characteristics of the realistic protein. It can be found that 3D off-lattice model is more like native folding structure of the realistic protein than 2D off-lattice model. In addition, compared with some previous researches, the proposed hybrid algorithm can more effectively and more quickly search the spatial folding structure of a protein chain. PMID:25474708

  11. PDBFlex: exploring flexibility in protein structures.

    PubMed

    Hrabe, Thomas; Li, Zhanwen; Sedova, Mayya; Rotkiewicz, Piotr; Jaroszewski, Lukasz; Godzik, Adam

    2016-01-01

    The PDBFlex database, available freely and with no login requirements at http://pdbflex.org, provides information on flexibility of protein structures as revealed by the analysis of variations between depositions of different structural models of the same protein in the Protein Data Bank (PDB). PDBFlex collects information on all instances of such depositions, identifying them by a 95% sequence identity threshold, performs analysis of their structural differences and clusters them according to their structural similarities for easy analysis. The PDBFlex contains tools and viewers enabling in-depth examination of structural variability including: 2D-scaling visualization of RMSD distances between structures of the same protein, graphs of average local RMSD in the aligned structures of protein chains, graphical presentation of differences in secondary structure and observed structural disorder (unresolved residues), difference distance maps between all sets of coordinates and 3D views of individual structures and simulated transitions between different conformations, the latter displayed using JSMol visualization software. PMID:26615193

  12. PDBFlex: exploring flexibility in protein structures

    PubMed Central

    Hrabe, Thomas; Li, Zhanwen; Sedova, Mayya; Rotkiewicz, Piotr; Jaroszewski, Lukasz; Godzik, Adam

    2016-01-01

    The PDBFlex database, available freely and with no login requirements at http://pdbflex.org, provides information on flexibility of protein structures as revealed by the analysis of variations between depositions of different structural models of the same protein in the Protein Data Bank (PDB). PDBFlex collects information on all instances of such depositions, identifying them by a 95% sequence identity threshold, performs analysis of their structural differences and clusters them according to their structural similarities for easy analysis. The PDBFlex contains tools and viewers enabling in-depth examination of structural variability including: 2D-scaling visualization of RMSD distances between structures of the same protein, graphs of average local RMSD in the aligned structures of protein chains, graphical presentation of differences in secondary structure and observed structural disorder (unresolved residues), difference distance maps between all sets of coordinates and 3D views of individual structures and simulated transitions between different conformations, the latter displayed using JSMol visualization software. PMID:26615193

  13. Structure of the gene V protein of bacteriophage f1 determined by multiwavelength x-ray diffraction on the selenomethionyl protein.

    PubMed Central

    Skinner, M M; Zhang, H; Leschnitzer, D H; Guan, Y; Bellamy, H; Sweet, R M; Gray, C W; Konings, R N; Wang, A H; Terwilliger, T C

    1994-01-01

    The crystal structure of the dimeric gene V protein of bacteriophage f1 was determined using multiwavelength anomalous diffraction on the selenomethionine-containing wild-type and isoleucine-47-->methionine mutant proteins with x-ray diffraction data phased to 2.5 A resolution. The structure of the wild-type protein has been refined to an R factor of 19.2% using native data to 1.8 A resolution. The structure of the gene V protein was used to obtain a model for the protein portion of the gene V protein-single-stranded DNA complex. Images PMID:8134350

  14. [Protein structure: Folding and prions].

    PubMed

    Rey-Gayo, Antonio; Calbo Torrecilla, Francisco

    2002-04-01

    Transmissible spongiform encephalopathies have become a subject of prime social concern in recent years because of its relation to "mad cow disease" and their potential for transmission to humans. Among the most important scientific aspects of these diseases are the peculiar characteristics of the agent involved in their transmission. In this article we briefly describe the outstanding features of prions, the most widely accepted hypothesis for these diseases. We focus on the molecular characteristics of this protein, coded in the genome of the affected host, and describe the conformational alterations in the protein's tertiary structure that have been blamed for its pathologic activity. Our aim is to summarize the state-of-the-art knowledge on prions, the hypotheses proposed to explain mechanisms of disease transmission without agents containing genetic material, and some specific peculiarities of this new infectious agent. The links between this knowledge and possible therapeutic strategies to overcome the disease justify, once again, close interaction among chemistry, molecular biology, and medicine. PMID:11996702

  15. Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

    SciTech Connect

    Nakane, Takanori; Song, Changyong; Suzuki, Mamoru; Nango, Eriko; Kobayashi, Jun; Masuda, Tetsuya; Inoue, Shigeyuki; Mizohata, Eiichi; Nakatsu, Toru; Tanaka, Tomoyuki; Tanaka, Rie; Shimamura, Tatsuro; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Iwata, So; Sugahara, Michihiro

    2015-11-27

    Sulfur SAD phasing facilitates the structure determination of diverse native proteins using femtosecond X-rays from free-electron lasers via serial femtosecond crystallography. Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

  16. Dynamic insight into protein structure utilizing red edge excitation shift.

    PubMed

    Chattopadhyay, Amitabha; Haldar, Sourav

    2014-01-21

    Proteins are considered the workhorses in the cellular machinery. They are often organized in a highly ordered conformation in the crowded cellular environment. These conformations display characteristic dynamics over a range of time scales. An emerging consensus is that protein function is critically dependent on its dynamics. The subtle interplay between structure and dynamics is a hallmark of protein organization and is essential for its function. Depending on the environmental context, proteins can adopt a range of conformations such as native, molten globule, unfolded (denatured), and misfolded states. Although protein crystallography is a well established technique, it is not always possible to characterize various protein conformations by X-ray crystallography due to transient nature of these states. Even in cases where structural characterization is possible, the information obtained lacks dynamic component, which is needed to understand protein function. In this overall scenario, approaches that reveal information on protein dynamics are much appreciated. Dynamics of confined water has interesting implications in protein folding. Interfacial hydration combines the motion of water molecules with the slow moving protein molecules. The red edge excitation shift (REES) approach becomes relevant in this context. REES is defined as the shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption spectrum. REES arises due to slow rates (relative to fluorescence lifetime) of solvent relaxation (reorientation) around an excited state fluorophore in organized assemblies such as proteins. Consequently, REES depends on the environment-induced motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. In the case of a protein, the confined water in the protein creates a dipolar field that acts as the solvent for a fluorophore

  17. A Fourier analysis of symmetry in protein structure.

    PubMed

    Taylor, William R; Heringa, Jaap; Baud, Franck; Flores, Tomas P

    2002-02-01

    The score matrix from a structure comparison program (SAP) was used to search for repeated structures using a Fourier analysis. When tested with artificial data, a simple Fourier transform of the smoothed matrix provided a clear signal of the repeat periodicity that could be used to extract the repeating units with the SAP program. The strength of the Fourier signal was calibrated against the signal from model proteins. The most useful of these was the novel random-walk approach employed to generate realistic 'fake' structures. On the basis of these it was possible to conclude that only a small proportion of protein structures have an unexpected degree of symmetry. Artificially generated 'ideal' folds provided an upper limit on the strength of signal that could be expected from a 'perfectly' repeating compact structure. Unexpectedly, some of the very regular beta-propellor folds attained the same strength but the majority of symmetric structures lay below this region. When native proteins were ranked by the power of their spectrum a wide variety of fold types were seen to score highly. In the betaalpha class, these included the globular betaalpha proteins and the more repetitive leucine-rich betaalpha folds. In the all-beta class; beta-propellors, beta-prisms and beta-helices were found as well as the more globular gamma-crystalin domains. When this ranked list was filtered to remove proteins that contained detectable internal sequence similarity (using the program REPRO), the list became exclusively composed of just globular betaalpha class proteins and in the top 50 re-ranked proteins, only a single 4-fold propellor structure remained. PMID:11917144

  18. Water-protein interaction in native and partially unfolded equine cytochrome c

    NASA Astrophysics Data System (ADS)

    Banci, Lucia

    1998-12-01

    The problem of the interaction of water solvent with proteins has been addressed by investigating the water 1H nuclear magnetic relaxation dispersion (NMRD) profiles of cytochrome c solutions. It is shown that the 1H NMRD profiles are accounted for by 1, a sizeable contribution from exchangeable protein protons (mostly from lysine side chains) and 2, a modest contribution from long-lived water. It is also shown that the number of exchangeable protons is sizeably increased in the oxidized but not in the reduced protein in the presence of the unfolding agent guanidinium chloride at a 3M concentration. This additional contribution arises mostly from backbone protons, as evidenced by high resolution NMR data which provide significant and independent data on the structure and the dynamic behaviour of the partly unfolded oxidized protein. Higher accessibility to short lived water molecules is proposed also. For the analysis of the 1H NMRD data a complete relaxation matrix approach is presented that is analogous, but not identical, to one recently described. This approach permits the simultaneous incorporation of exchangeable protein protons and an unlimited number of water molecules in pre-defined protein binding sites.

  19. Constrained Peptides as Miniature Protein Structures

    PubMed Central

    Yin, Hang

    2012-01-01

    This paper discusses the recent developments of protein engineering using both covalent and noncovalent bonds to constrain peptides, forcing them into designed protein secondary structures. These constrained peptides subsequently can be used as peptidomimetics for biological functions such as regulations of protein-protein interactions. PMID:25969758

  20. Thermodynamics of native protein/foreign surface interactions. IV. Calorimetric and microelectrophoretic study of human fibrinogen sorption onto glass and LTI-carbon.

    PubMed

    Chiu, T H; Nyilas, E; Lederman, D M

    1976-01-01

    1. According to a working hypothesis put forward in the previous papers of this series2-4, the initial phases of native blood/foreign surface interactions have been considered within the framework of a physicochemical model of contact activation at the molecular level. The salient features of this hypothesis are: a) the arrival and adsorption of native plasma proteins on a contact surface overwhelmingly precedes that of the cellular blood components; b) the interaction energy that arises between a particular foreign surface and native plasma proteins settling on it, is a characteristic quantity depending upon the effective surface molecular structure as well as the nature of the proteins; c) the "intensity" of native protein/foreign surface interactions can be treated in terms of thermodynamic quantities since these energy terms are independent of the type of forces acting between protein and surface; and d) depending upon the degree to which the adsorption of a native protein is thermodynamically favored by enthalpy and/or entropy factors, the interaction energy can be utilized to induce conformational changes of varying degree in the sorbed protein. 2. Using glass and low temperature isotropic (LTI) carbon adsorbents, i.e., a known procoagulant and a relevant biomaterial, respectively, the adsorption properties and the potential surface-induced conformational changes of high-purity native human fibrinogen (clottability greater than or equal to 92%) were studied, at 25 degrees C, by 3 independent methods. In all of the experiments performed, a) both adsorbents were employed in the form of particles less than or equal to 1.0 mu representing specific surface areas of 9.85 M2/Gm and 27.7 (nominal) M2/Gm for the glass and LTI-carbon powders, respectively, and b) the fibrinogen was absorbed from a standaridized buffer (pH = 7.2, ionic strength 0.05) using the same fixed surface area/protein solution volume ratio with a given adsorbent. 3. The 25 degrees C adsorption

  1. Revealing Ligand Binding Sites and Quantifying Subunit Variants of Noncovalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

    NASA Astrophysics Data System (ADS)

    Li, Huilin; Wongkongkathep, Piriya; Van Orden, Steve L.; Ogorzalek Loo, Rachel R.; Loo, Joseph A.

    2014-12-01

    "Native" mass spectrometry (MS) has been proven to be increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode, and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD of the hADH dimer shows that each subunit (E and S chains) binds not only to two zinc atoms, but also the NAD/NADH ligand, with a higher NAD/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

  2. Molecular structure in peripheral dog breeds: Portuguese native breeds as a case study.

    PubMed

    Pires, A E; Amorim, I R; Ginja, C; Gomes, M; Godinho, I; Simões, F; Oom, M; Petrucci-Fonseca, F; Matos, J; Bruford, M W

    2009-08-01

    Genetic variability in purebred dogs is known to be highly structured, with differences among breeds accounting for approximately 30% of the genetic variation. However, analysis of the genetic structure in non-cosmopolitan breeds and local populations is still limited. Nine Portuguese native dog breeds, and other peripheral dog populations (five) with regional affinities, were characterized using 16 microsatellites and 225 amplified fragment length polymorphism (AFLP) markers, and the pattern of genetic differentiation was investigated. Although the level of breed differentiation detected is below that of other dog breeds, there is in most cases a correlation between breed affiliation and molecular structure. AFLP markers and Bayesian clustering methods allowed an average of 73.1% of individuals to be correctly assigned to source populations, providing robust genotypic assessment of breed affiliation. A geographical genetic structure was also detected, which suggests a limited influence of African dogs on the Iberian breeds. The sampling effect on the estimation of population structure was evaluated and there was a 2.2% decrease in genetic differentiation among breeds when working animals were included. Genetic diversity of stray dogs was also assessed and there is no evidence that they pose a threat to the preservation of the gene pool of native dog breeds. PMID:19298456

  3. Infrared Structural Biology: Detect Functionally Important Structural Motions of Proteins

    NASA Astrophysics Data System (ADS)

    Xie, Aihua

    Proteins are dynamic. Lack of dynamic structures of proteins hampers our understanding of protein functions. Infrared structural biology (IRSB) is an emerging technology. There are several advantages of IRSB for mechanistic studies of proteins: (1) its excellent dynamic range (detecting structural motions from picoseconds to >= seconds); (2) its high structural sensitivity (detect tiny but functionally important structural motions such as proton transfer and changes in hydrogen bonding interaction); (3) its ability to detect different structural motions simultaneously. Successful development of infrared structural biology demands not only new experimental techniques (from infrared technologies to chemical synthesis and cell biology), but also new data processing (how to translate infrared signals into quantitative structural information of proteins). These topics will be discussed as well as examples of how to use IRSB to study structure-function relationship of proteins. This work was supported by NSF DBI1338097 and OCAST HR10-078.

  4. Automated High Throughput Protein Crystallization Screening at Nanoliter Scale and Protein Structural Study on Lactate Dehydrogenase

    SciTech Connect

    Fenglei Li

    2006-08-09

    The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties. Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed in this system to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme has capability of eliminating common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening. Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. In this crystallization method, a gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. In addition

  5. Practical lessons from protein structure prediction

    PubMed Central

    Ginalski, Krzysztof; Grishin, Nick V.; Godzik, Adam; Rychlewski, Leszek

    2005-01-01

    Despite recent efforts to develop automated protein structure determination protocols, structural genomics projects are slow in generating fold assignments for complete proteomes, and spatial structures remain unknown for many protein families. Alternative cheap and fast methods to assign folds using prediction algorithms continue to provide valuable structural information for many proteins. The development of high-quality prediction methods has been boosted in the last years by objective community-wide assessment experiments. This paper gives an overview of the currently available practical approaches to protein structure prediction capable of generating accurate fold assignment. Recent advances in assessment of the prediction quality are also discussed. PMID:15805122

  6. Structural templates for comparative protein docking

    PubMed Central

    Anishchenko, Ivan; Kundrotas, Petras J.; Tuzikov, Alexander V.; Vakser, Ilya A.

    2014-01-01

    Structural characterization of protein-protein interactions is important for understanding life processes. Because of the inherent limitations of experimental techniques, such characterization requires computational approaches. Along with the traditional protein-protein docking (free search for a match between two proteins), comparative (template-based) modeling of protein-protein complexes has been gaining popularity. Its development puts an emphasis on full and partial structural similarity between the target protein monomers and the protein-protein complexes previously determined by experimental techniques (templates). The template-based docking relies on the quality and diversity of the template set. We present a carefully curated, non-redundant library of templates containing 4,950 full structures of binary complexes and 5,936 protein-protein interfaces extracted from the full structures at 12Å distance cut-off. Redundancy in the libraries was removed by clustering the PDB structures based on structural similarity. The value of the clustering threshold was determined from the analysis of the clusters and the docking performance on a benchmark set. High structural quality of the interfaces in the template and validation sets was achieved by automated procedures and manual curation. The library is included in the Dockground resource for molecular recognition studies at http://dockground.bioinformatics.ku.edu. PMID:25488330

  7. Formation of local native-like tertiary structures in the slow refolding reaction of human carbonic anhydrase II as monitored by circular dichroism on tryptophan mutants.

    PubMed

    Andersson, D; Freskgård, P O; Jonsson, B H; Carlsson, U

    1997-04-15

    In the present study, near-UV CD kinetic measurements on mutants, in which one Trp residue had been replaced, were performed to probe the development of asymmetric environments around specific Trp residues during the refolding of human carbonic anhydrase II (HCAII). In addition, the formation of the active site was probed by the binding of a fluorescent sulfonamide inhibitor. The development of the individual Trp CD spectra during refolding was obtained by subtracting the CD spectrum of the mutant lacking one Trp from that of HCAII at different time points. The same method was used for the particular Trp residues to obtain the kinetic CD traces monitored at a specific wavelength (270 nm). Trp residues 16, 97, and 245 were analyzed. Trp16 probes the N-terminal domain (amino acid residues 1-25), and this part is forming its tertiary structure slower than the major domain (amino acid residues 26-260) of the protein molecule, which contains the active site and a dominating beta-sheet. An essentially native structure of the major domain seems to act as a template for the correct folding of the N terminus. Trp97 is located in a hydrophobic cluster comprising beta-strands 3-5 in the protein core. Previously, we have shown that this region is remarkably stable and compact, and stopped-flow fluorescence data indicate that Trp97 is buried in an apolar compact cluster within a few milliseconds [Svensson, M., Jonasson, P., Freskgård, P.-O., Jonsson, B.-H., Lindgren, M., Martensson, L.-G., Gentile, M., Bóren, K., & Carlsson, U. (1995) Biochemistry 34, 8606-8620; Jonasson, P., Aronsson, G., Carlsson, U., & Jonsson, B.-H. (1997) Biochemistry 36 (in press)]. Here it is shown that the development of the native tertiary structure at Trp97 occurs in the minute time domain. Trp245 is located in a long loop between the N-terminal domain and the core structure. Although this Trp has attained native-like fluorescence properties within the dead time of the CD experiment, it assumes a

  8. A photon-free approach to transmembrane protein structure determination.

    PubMed

    Soto, Cinque S; Hannigan, Brett T; DeGrado, William F

    2011-12-01

    The structures of membrane proteins are generally solved using samples dissolved in micelles, bicelles, or occasionally phospholipid bilayers using X-ray diffraction or magnetic resonance. Because these are less than perfect mimics of true biological membranes, the structures are often confirmed by evaluating the effects of mutations on the properties of the protein in their native cellular environments. Low-resolution structures are also sometimes generated from the results of site-directed mutagenesis when other structural data are incomplete or not available. Here, we describe a rapid and automated approach to determine structures from data on site-directed mutants for the special case of homo-oligomeric helical bundles. The method uses as input an experimental profile of the effects of mutations on some property of the protein. This profile is then interpreted by assuming that positions that have large effects on structure/function when mutated project toward the center of the oligomeric bundle. Model bundles are generated, and correlation analysis is used to score which structures have inter-subunit C(β) distances between adjoining monomers that best correlate with the experimental profile. These structures are then clustered and refined using energy-based minimization methods. For a set of 10 homo-oligomeric TM protein structures ranging from dimers to pentamers, we show that our method predicts structures to within 1-2 Å backbone RMSD relative to X-ray and NMR structures. This level of agreement approaches the precision of NMR structures solved in different membrane mimetics. PMID:22024595

  9. Structural studies of human glioma pathogenesis-related protein 1

    PubMed Central

    Asojo, Oluwatoyin A.; Koski, Raymond A.; Bonafé, Nathalie

    2011-01-01

    Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85 Å resolution and that of a Zn2+ complex was refined to 2.2 Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn2+ similarly to snake-venom CRISPs, which are involved in Zn2+-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1. PMID:21931216

  10. Crystal structures of MBP fusion proteins.

    PubMed

    Waugh, David S

    2016-03-01

    Although chaperone-assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose-binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter-domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP-mediated structural distortions are very rare. PMID:26682969

  11. Population genetic structure of native versus naturalized sympatric shrub willows (Salix; Salicaceae).

    PubMed

    Lin, Juan; Gibbs, James P; Smart, Lawrence B

    2009-04-01

    Vegetative propagation of an introduced species can contribute significantly to its ability to spread and become naturalized, potentially in competition with native species. This study focused on the naturalization of a willow shrub, Salix purpurea, which was introduced to the United States from Europe and is commonly sympatric with the native shrub willow, S. eriocephala. Both species are capable of vegetative and sexual reproduction, but little is known about their relative frequency, nor the impact of clonal propagation on population-level genetic diversity. We analyzed genotypes at several microsatellite loci in 993 individuals belonging to 30 subpopulations of S. eriocephala and 28 subpopulations of S. purpurea in areas of sympatry across three watersheds to compare their genetic diversity and genetic structure. Our results revealed six subpopulations of S. purpurea containing plants with identical multilocus genotypes, while clonal individuals were rare among S. eriocephala populations. These species are dioecious with relatively high levels of heterozygosity, but S. eriocephala had much higher allelic diversity and genotypic diversity than did S. purpurea. These results strongly suggest that vegetative propagation has contributed to the naturalization of S. purpurea and has resulted in higher levels of genetic differentiation among S. purpurea populations than among native S. eriocephala populations. PMID:21628232

  12. Electrochemical atomic force microscopy imaging of redox-immunomarked proteins on native potyviruses: from subparticle to single-protein resolution.

    PubMed

    Nault, Laurent; Taofifenua, Cécilia; Anne, Agnès; Chovin, Arnaud; Demaille, Christophe; Besong-Ndika, Jane; Cardinale, Daniela; Carette, Noëlle; Michon, Thierry; Walter, Jocelyne

    2015-05-26

    We show herein that electrochemical atomic force microscopy (AFM-SECM), operated in molecule touching (Mt) mode and combined with redox immunomarking, enables the in situ mapping of the distribution of proteins on individual virus particles and makes localization of individual viral proteins possible. Acquisition of a topography image allows isolated virus particles to be identified and structurally characterized, while simultaneous acquisition of a current image allows the sought after protein, marked by redox antibodies, to be selectively located. We concomitantly show that Mt/AFM-SECM, due to its single-particle resolution, can also uniquely reveal the way redox functionalization endowed to viral particles is distributed both statistically among the viruses and spatially over individual virus particles. This possibility makes Mt/AFM-SECM a unique tool for viral nanotechnology. PMID:25905663

  13. The interface of protein structure, protein biophysics, and molecular evolution

    PubMed Central

    Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon

    2012-01-01

    Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

  14. Cryo-EM structure of a native, fully glycosylated, cleaved HIV-1 envelope trimer.

    PubMed

    Lee, Jeong Hyun; Ozorowski, Gabriel; Ward, Andrew B

    2016-03-01

    The envelope glycoprotein trimer (Env) on the surface of HIV-1 recognizes CD4(+) T cells and mediates viral entry. During this process, Env undergoes substantial conformational rearrangements, making it difficult to study in its native state. Soluble stabilized trimers have provided valuable insights into the Env structure, but they lack the hydrophobic membrane proximal external region (MPER, an important target of broadly neutralizing antibodies), the transmembrane domain, and the cytoplasmic tail. Here we present (i) a cryogenic electron microscopy (cryo-EM) structure of a clade B virus Env, which lacks only the cytoplasmic tail and is stabilized by the broadly neutralizing antibody PGT151, at a resolution of 4.2 angstroms and (ii) a reconstruction of this form of Env in complex with PGT151 and MPER-targeting antibody 10E8 at a resolution of 8.8 angstroms. These structures provide new insights into the wild-type Env structure. PMID:26941313

  15. NAPS: Network Analysis of Protein Structures.

    PubMed

    Chakrabarty, Broto; Parekh, Nita

    2016-07-01

    Traditionally, protein structures have been analysed by the secondary structure architecture and fold arrangement. An alternative approach that has shown promise is modelling proteins as a network of non-covalent interactions between amino acid residues. The network representation of proteins provide a systems approach to topological analysis of complex three-dimensional structures irrespective of secondary structure and fold type and provide insights into structure-function relationship. We have developed a web server for network based analysis of protein structures, NAPS, that facilitates quantitative and qualitative (visual) analysis of residue-residue interactions in: single chains, protein complex, modelled protein structures and trajectories (e.g. from molecular dynamics simulations). The user can specify atom type for network construction, distance range (in Å) and minimal amino acid separation along the sequence. NAPS provides users selection of node(s) and its neighbourhood based on centrality measures, physicochemical properties of amino acids or cluster of well-connected residues (k-cliques) for further analysis. Visual analysis of interacting domains and protein chains, and shortest path lengths between pair of residues are additional features that aid in functional analysis. NAPS support various analyses and visualization views for identifying functional residues, provide insight into mechanisms of protein folding, domain-domain and protein-protein interactions for understanding communication within and between proteins. URL:http://bioinf.iiit.ac.in/NAPS/. PMID:27151201

  16. Analysis of STR markers reveals high genetic structure in Portuguese native cattle.

    PubMed

    Ginja, Catarina; Telo Da Gama, Luís; Penedo, Maria Cecilia T

    2010-01-01

    Genetic structure and diversity of 13 Portuguese native and 3 imported cattle breeds were assessed with 39 microsatellites. Allelic richness per locus was high, with an overall average of 8.3 +/- 2.5. The mean observed and expected heterozygosities were 0.673 +/- 0.043 and 0.691 +/- 0.034, respectively. The mean number of alleles per breed ranged between 5.36 +/- 1.27 and 7.87 +/- 2.66. Brava de Lide and Mirandesa breeds had the lowest genetic diversity, whereas Minhota, Arouquesa, and Mertolenga had the highest. Significant (P < 0.05) heterozygote deficit was detected in all breeds except Garvonesa, Marinhoa, Minhota, and Limousin. Hardy-Weinberg deviations are most probably due to inbreeding, particularly in Alentejana, Brava de Lide, Mertolenga, and Ramo Grande (F(is) > 0, P < 0.0001). Based on the principal component and the Neighbor-Net analyses, Mirandesa was the most genetically distinct breed. Even though admixture was detected across all breeds (6.7%, q < 0.800), the molecular structure was consistent with original breed designations, with the exception of Cachena that had a clear influence of Barrosã (K = 15). Mertolenga showed substructure with independent clustering of red speckled animals. The percentage animals correctly assigned was >or=90 in all breeds except Cachena, Garvonesa, and Preta (q >or= 0.800). The results obtained here confirmed that high levels of genetic diversity exist within Portuguese native cattle and that the breeds are highly structured. Conservation measures should be implemented for all native breeds to minimize inbreeding. PMID:19965912

  17. Herbarium specimens reveal a historical shift in phylogeographic structure of common ragweed during native range disturbance.

    PubMed

    Martin, Michael D; Zimmer, Elizabeth A; Olsen, Morten T; Foote, Andrew D; Gilbert, M Thomas P; Brush, Grace S

    2014-04-01

    Invasive plants provide ample opportunity to study evolutionary shifts that occur after introduction to novel environments. However, although genetic characters pre-dating introduction can be important determinants of later success, large-scale investigations of historical genetic structure have not been feasible. Common ragweed (Ambrosia artemisiifolia L.) is an invasive weed native to North America that is known for its allergenic pollen. Palynological records from sediment cores indicate that this species was uncommon before European colonization of North America, and ragweed populations expanded rapidly as settlers deforested the landscape on a massive scale, later becoming an aggressive invasive with populations established globally. Towards a direct comparison of genetic structure now and during intense anthropogenic disturbance of the late 19th century, we sampled 45 natural populations of common ragweed across its native range as well as historical herbarium specimens collected up to 140 years ago. Bayesian clustering analyses of 453 modern and 473 historical samples genotyped at three chloroplast spacer regions and six nuclear microsatellite loci reveal that historical ragweed's spatial genetic structure mirrors both the palaeo-record of Ambrosia pollen deposition and the historical pattern of agricultural density across the landscape. Furthermore, for unknown reasons, this spatial genetic pattern has changed substantially in the intervening years. Following on previous work relating morphology and genetic expression between plants collected from eastern North America and Western Europe, we speculate that the cluster associated with humans' rapid transformation of the landscape is a likely source of these aggressive invasive populations. PMID:24450363

  18. Investigation of the structural invariance of the Ethnic Identity Scale with Native American youth.

    PubMed

    Yetter, Georgette; Foutch, Victoria

    2013-10-01

    This study examined the structural invariance of the 17-item Ethnic Identity Scale (EIS; Umaña-Taylor, Yazedjian, & Bámaca-Gomez, 2004) with a Native American sample. Participants were 144 students (55% female) enrolled in a Native American boarding school in the south-central U.S. Participants ranged in age from 13 to 19 years (M = 15.9, SD = 1.49). Confirmatory factor analysis was performed using maximum likelihood estimation. The original model was a poor fit for the data set. Removal of Items 1 and 2 and modeling the covariation between Items 6 and 11 resulted in an overall chi square value of Χ²(86, N = 144) = 152.21, p < .0001 (CFI = .936; RMSEA = .073; 90% CI on RMSEA = .054, .092), suggesting an acceptable fit. Substantial factor loadings were evident for the remaining 15 items. Analyses for the most part replicated Umaña-Taylor, Yazedjian, and Bámaca-Gomez's (2004) original 3-factor structure. We hypothesize that the minor differences between the present structure and that of Umaña-Taylor et al. (2004) may be an artifact of this particular sample. PMID:23731231

  19. The Structure of Clinical Consultation: A Case of Non-Native Speakers of English as Participants

    PubMed Central

    Bagheri, H.; Ibrahim, N. A.; Habil, H.

    2015-01-01

    Background: In many parts of the world, patients may find it difficult to visit doctors who share the same language and culture due to the intermingling of people and international recruitment of doctors among many other reasons. In these multilingual multicultural settings (MMSs), doctor-patient interactions face new communication challenges. This study aims to identify the structure of clinical consultation and its phases in an MMS where both doctors and patients are non-native speakers (NNSs) of English. Method: This study takes on a discourse analytic approach to examine the structure of clinical consultation as an activity type. 25 clinical consultation sessions between non-native speakers of English in a public healthcare centre in Malaysia were audio-recorded. Findings and Discussion: The results show that there are some deviations from the mainstream structure of clinical consultations although, in general, the pattern is compatible with previous studies. Deviations are particularly marked in the opening and closing phases of consultation. Conclusion: In almost all interactions, there is a straightforward manner of beginning medical consultations. The absence of greetings may have naturally reduced the length of talk. Hence, by directly entering medical talks, the doctors voice their concern on the curing aspects of the consultation rather than its caring facets. The preference of curing priority to caring is more goal-oriented and in alignment with the consultation as an activity type. PMID:25560336

  20. Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

    PubMed Central

    Nakane, Takanori; Song, Changyong; Suzuki, Mamoru; Nango, Eriko; Kobayashi, Jun; Masuda, Tetsuya; Inoue, Shigeyuki; Mizohata, Eiichi; Nakatsu, Toru; Tanaka, Tomoyuki; Tanaka, Rie; Shimamura, Tatsuro; Tono, Kensuke; Joti, Yasumasa; Kameshima, Takashi; Hatsui, Takaki; Yabashi, Makina; Nureki, Osamu; Iwata, So; Sugahara, Michihiro

    2015-01-01

    Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures. PMID:26627659

  1. Comparative Protein Structure Modeling Using Modeller

    PubMed Central

    Eswar, Narayanan; Marti-Renom, Marc A.; Madhusudhan, M.S.; Eramian, David; Shen, Min-yi; Pieper, Ursula

    2014-01-01

    Functional characterization of a protein sequence is one of the most frequent problems in biology. This task is usually facilitated by accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described. PMID:18428767

  2. Solid state NMR: The essential technology for helical membrane protein structural characterization

    PubMed Central

    Cross, Timothy A.; Ekanayake, Vindana; Paulino, Joana; Wright, Anna

    2014-01-01

    NMR spectroscopy of helical membrane proteins has been very challenging on multiple fronts. The expression and purification of these proteins while maintaining functionality has consumed countless graduate student hours. Sample preparations have depended on whether solution or solid-state NMR spectroscopy was to be performed – neither have been easy. In recent years it has become increasingly apparent that membrane mimic environments influence the structural result. Indeed, in these recent years we have rediscovered that Nobel laureate, Christian Anfinsen, did not say that protein structure was exclusively dictated by the amino acid sequence, but rather by the sequence in a given environment (Anfinsen, 1973) [106]. The environment matters, molecular interactions with the membrane environment are significant and many examples of distorted, non-native membrane protein structures have recently been documented in the literature. However, solid-state NMR structures of helical membrane proteins in proteoliposomes and bilayers are proving to be native structures that permit a high resolution characterization of their functional states. Indeed, solid-state NMR is uniquely able to characterize helical membrane protein structures in lipid environments without detergents. Recent progress in expression, purification, reconstitution, sample preparation and in the solid-state NMR spectroscopy of both oriented samples and magic angle spinning samples has demonstrated that helical membrane protein structures can be achieved in a timely fashion. Indeed, this is a spectacular opportunity for the NMR community to have a major impact on biomedical research through the solid-state NMR spectroscopy of these proteins. PMID:24412099

  3. Solid state NMR: The essential technology for helical membrane protein structural characterization

    NASA Astrophysics Data System (ADS)

    Cross, Timothy A.; Ekanayake, Vindana; Paulino, Joana; Wright, Anna

    2014-02-01

    NMR spectroscopy of helical membrane proteins has been very challenging on multiple fronts. The expression and purification of these proteins while maintaining functionality has consumed countless graduate student hours. Sample preparations have depended on whether solution or solid-state NMR spectroscopy was to be performed - neither have been easy. In recent years it has become increasingly apparent that membrane mimic environments influence the structural result. Indeed, in these recent years we have rediscovered that Nobel laureate, Christian Anfinsen, did not say that protein structure was exclusively dictated by the amino acid sequence, but rather by the sequence in a given environment (Anfinsen, 1973) [106]. The environment matters, molecular interactions with the membrane environment are significant and many examples of distorted, non-native membrane protein structures have recently been documented in the literature. However, solid-state NMR structures of helical membrane proteins in proteoliposomes and bilayers are proving to be native structures that permit a high resolution characterization of their functional states. Indeed, solid-state NMR is uniquely able to characterize helical membrane protein structures in lipid environments without detergents. Recent progress in expression, purification, reconstitution, sample preparation and in the solid-state NMR spectroscopy of both oriented samples and magic angle spinning samples has demonstrated that helical membrane protein structures can be achieved in a timely fashion. Indeed, this is a spectacular opportunity for the NMR community to have a major impact on biomedical research through the solid-state NMR spectroscopy of these proteins.

  4. Solid state NMR: The essential technology for helical membrane protein structural characterization.

    PubMed

    Cross, Timothy A; Ekanayake, Vindana; Paulino, Joana; Wright, Anna

    2014-02-01

    NMR spectroscopy of helical membrane proteins has been very challenging on multiple fronts. The expression and purification of these proteins while maintaining functionality has consumed countless graduate student hours. Sample preparations have depended on whether solution or solid-state NMR spectroscopy was to be performed - neither have been easy. In recent years it has become increasingly apparent that membrane mimic environments influence the structural result. Indeed, in these recent years we have rediscovered that Nobel laureate, Christian Anfinsen, did not say that protein structure was exclusively dictated by the amino acid sequence, but rather by the sequence in a given environment (Anfinsen, 1973) [106]. The environment matters, molecular interactions with the membrane environment are significant and many examples of distorted, non-native membrane protein structures have recently been documented in the literature. However, solid-state NMR structures of helical membrane proteins in proteoliposomes and bilayers are proving to be native structures that permit a high resolution characterization of their functional states. Indeed, solid-state NMR is uniquely able to characterize helical membrane protein structures in lipid environments without detergents. Recent progress in expression, purification, reconstitution, sample preparation and in the solid-state NMR spectroscopy of both oriented samples and magic angle spinning samples has demonstrated that helical membrane protein structures can be achieved in a timely fashion. Indeed, this is a spectacular opportunity for the NMR community to have a major impact on biomedical research through the solid-state NMR spectroscopy of these proteins. PMID:24412099

  5. DEAD-Box Helicase Proteins Disrupt RNA Tertiary Structure Through Helix Capture

    PubMed Central

    Pan, Cynthia; Potratz, Jeffrey P.; Cannon, Brian; Simpson, Zachary B.; Ziehr, Jessica L.; Tijerina, Pilar; Russell, Rick

    2014-01-01

    DEAD-box helicase proteins accelerate folding and rearrangements of highly structured RNAs and RNA–protein complexes (RNPs) in many essential cellular processes. Although DEAD-box proteins have been shown to use ATP to unwind short RNA helices, it is not known how they disrupt RNA tertiary structure. Here, we use single molecule fluorescence to show that the DEAD-box protein CYT-19 disrupts tertiary structure in a group I intron using a helix capture mechanism. CYT-19 binds to a helix within the structured RNA only after the helix spontaneously loses its tertiary contacts, and then CYT-19 uses ATP to unwind the helix, liberating the product strands. Ded1, a multifunctional yeast DEAD-box protein, gives analogous results with small but reproducible differences that may reflect its in vivo roles. The requirement for spontaneous dynamics likely targets DEAD-box proteins toward less stable RNA structures, which are likely to experience greater dynamic fluctuations, and provides a satisfying explanation for previous correlations between RNA stability and CYT-19 unfolding efficiency. Biologically, the ability to sense RNA stability probably biases DEAD-box proteins to act preferentially on less stable misfolded structures and thereby to promote native folding while minimizing spurious interactions with stable, natively folded RNAs. In addition, this straightforward mechanism for RNA remodeling does not require any specific structural environment of the helicase core and is likely to be relevant for DEAD-box proteins that promote RNA rearrangements of RNP complexes including the spliceosome and ribosome. PMID:25350280

  6. Functional role of TRPC proteins in native systems: implications from knockout and knock-down studies.

    PubMed

    Freichel, Marc; Vennekens, Rudi; Olausson, Jenny; Stolz, Susanne; Philipp, Stephan E; Weissgerber, Petra; Flockerzi, Veit

    2005-08-15

    Available data on transient receptor potential channel (TRPC) protein functions indicate that these proteins represent essential constituents of agonist-activated and phospholipase C-dependent cation entry pathways in primary cells which contribute to the elevation of cytosolic Ca2+. In addition, a striking number of biological functions have already been assigned to the various TRPC proteins, including mechanosensing activity (TRPC1), chemotropic axon guidance (TRPC1 and TRPC3), pheromone sensing and the regulation of sexual and social behaviour (TRPC2), endothelial-dependent regulation of vascular tone, endothelial permeability and neurotransmitter release (TRPC4), axonal growth (TRPC5), modulation of smooth muscle tone in blood vessels and lung and regulation of podocyte structure and function in the kidney (TRPC6). The lack of compounds which specifically block or activate TRPC proteins impairs the analysis of TRPC function in primary cells. We therefore concentrate in this contribution on (i) studies of TRPC-deficient mouse lines, (ii) data obtained by gene-silencing approaches using antisense oligonucleotides or RNA interference, (iii) expression experiments employing dominant negative TRPC constructs, and (iv) recent data correlating mutations of TRPC genes associated with human disease. PMID:15975974

  7. Effect of ionic liquid on the native and denatured state of a protein covalently attached to a probe: Solvation dynamics study

    NASA Astrophysics Data System (ADS)

    Chowdhury, Rajdeep; Mojumdar, Supratik Sen; Chattoraj, Shyamtanu; Bhattacharyya, Kankan

    2012-08-01

    Effect of a room temperature ionic liquid (RTIL, [pmim][Br]) on the solvation dynamics of a probe covalently attached to a protein (human serum albumin (HSA)) has been studied using femtosecond up-conversion. For this study, a solvation probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) has been covalently attached to the lone cysteine group (cys-34) of the protein HSA. Addition of 1.5 M RTIL or 6 M GdnHCl causes a red shift of the emission maxima of CPM bound to HSA by 3 nm and 12 nm, respectively. The average solvation time ⟨τs⟩ decreases from 650 ps (in native HSA) to 260 ps (˜2.5 times) in the presence of 1.5 M RTIL and to 60 ps (˜11 times) in the presence of 6 M GdnHCl. This is ascribed to unfolding of the protein by RTIL or GdnHCl and therefore making the probe CPM more exposed. When 1.5 M RTIL is added to the protein denatured by 6 M GdnHCl in advance, a further ˜5 nm red shift along with further ˜2 fold faster solvent relaxation (⟨τ⟩ ˜30 ps) is observed. Our previous fluorescence correlation spectroscopy study [D. K. Sasmal, T. Mondal, S. Sen Mojumdar, A. Choudhury, R. Banerjee, and K. Bhattacharyya, J. Phys. Chem. B 115, 13075 (2011), 10.1021/jp207829y] suggests that addition of RTIL to the protein denatured by 6 M GdnHCl causes a reduction in hydrodynamic radius (rh). It is demonstrated that in the presence of RTIL and GdnHCl, though the protein is structurally more compact, the local environment of CPM is very different from that in the native state.

  8. Template-based structure modeling of protein-protein interactions

    PubMed Central

    Szilagyi, Andras; Zhang, Yang

    2014-01-01

    The structure of protein-protein complexes can be constructed by using the known structure of other protein complexes as a template. The complex structure templates are generally detected either by homology-based sequence alignments or, given the structure of monomer components, by structure-based comparisons. Critical improvements have been made in recent years by utilizing interface recognition and by recombining monomer and complex template libraries. Encouraging progress has also been witnessed in genome-wide applications of template-based modeling, with modeling accuracy comparable to high-throughput experimental data. Nevertheless, bottlenecks exist due to the incompleteness of the proteinprotein complex structure library and the lack of methods for distant homologous template identification and full-length complex structure refinement. PMID:24721449

  9. A Reassessment of the Impact of European Contact on the Structure of Native American Genetic Diversity.

    PubMed

    Hunley, Keith; Gwin, Kiela; Liberman, Brendan

    2016-01-01

    Our current understanding of pre-Columbian history in the Americas rests in part on several trends identified in recent genetic studies. The goal of this study is to reexamine these trends in light of the impact of post-Columbian admixture and the methods used to study admixture. The previously-published data consist of 645 autosomal microsatellite genotypes from 1046 individuals in 63 populations. We used STRUCTURE to estimate ancestry proportions and tested the sensitivity of these estimates to the choice of the number of clusters, K. We used partial correlation analyses to examine the relationship between gene diversity and geographic distance from Beringia, controlling for non-Native American ancestry (from Africa, Europe and East Asia), and taking into account alternative paths of migration. Principal component analysis and multidimensional scaling were used to investigate the relationships between Andean and non-Andean populations and to explore gene-language correspondence. We found that 1) European and East Asian ancestry estimates decline as K increases, especially in Native Canadian populations, 2) a north-south decline in gene diversity is driven by low diversity in Amazonian and Paraguayan populations, not serial founder effects from Beringia, 3) controlling for non-Native American ancestry, populations in the Andes and Mesoamerica have higher gene diversity than populations in other regions, and 4) patterns of genetic and linguistic diversity are poorly correlated. We conclude that patterns of diversity previously attributed to pre-Columbian processes may in part reflect post-Columbian admixture and the choice of K in STRUCTURE analyses. Accounting for admixture, the pattern of diversity is inconsistent with a north-south founder effect process, though the genetic similarities between Mesoamerican and Andean populations are consistent with rapid dispersal along the western coast of the Americas. Further, even setting aside the disruptive effects of

  10. Native and thermally modified protein-polyphenol coassemblies: lactoferrin-based nanoparticles and submicrometer particles as protective vehicles for (-)-epigallocatechin-3-gallate.

    PubMed

    Yang, Wei; Xu, Chenqi; Liu, Fuguo; Yuan, Fang; Gao, Yanxiang

    2014-11-01

    The interactions between native, thermally modified lactoferrin (LF) and (-)-epigallocatechin-3-gallate (EGCG) at pH 3.5, 5.0, and 6.5 were investigated. Turbidity, particle size, and charge of LF-EGCG complexes were mainly dominated by pH value and secondary structure of protein. At pH 3.5 and 5.0, LF-EGCG complexes were nanoparticles which had high ζ-potential, small size, and soluble state. At pH 6.5, they were submicrometer particles which exhibited low ζ-potential, large size, and insoluble state. The infrared spectra of freeze-dried LF-EGCG complexes showed that they were different from LF and EGCG alone. Far-UV CD results indicated that heat denaturation might irreversibly alter the secondary structure of LF and EGCG induced a progressive increase in the proportion of α-helix structure at the cost of β-sheet and unordered coil structure of LF at pH 3.5, 5.0, and 6.5. EGCG exhibited a strong affinity for native LF but a weak affinity for thermally modified LF at pH 5.0 and 6.5. An inverse result was observed at pH 3.5. These results could have potential for the development of food formulations based on LF as a carrier of bioactive compounds. PMID:25310084

  11. A new protein structure representation for efficient protein function prediction.

    PubMed

    Maghawry, Huda A; Mostafa, Mostafa G M; Gharib, Tarek F

    2014-12-01

    One of the challenging problems in bioinformatics is the prediction of protein function. Protein function is the main key that can be used to classify different proteins. Protein function can be inferred experimentally with very small throughput or computationally with very high throughput. Computational methods are sequence based or structure based. Structure-based methods produce more accurate protein function prediction. In this article, we propose a new protein structure representation for efficient protein function prediction. The representation is based on three-dimensional patterns of protein residues. In the analysis, we used protein function based on enzyme activity through six mechanistically diverse enzyme superfamilies: amidohydrolase, crotonase, haloacid dehalogenase, isoprenoid synthase type I, and vicinal oxygen chelate. We applied three different classification methods, naïve Bayes, k-nearest neighbors, and random forest, to predict the enzyme superfamily of a given protein. The prediction accuracy using the proposed representation outperforms a recently introduced representation method that is based only on the distance patterns. The results show that the proposed representation achieved prediction accuracy up to 98%, with improvement of about 10% on average. PMID:25343279

  12. Protein Structures Revealed at Record Pace

    ScienceCinema

    Greg Hura

    2010-01-08

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  13. Protein Structures Revealed at Record Pace

    ScienceCinema

    Hura, Greg

    2013-05-29

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  14. Protein Structures Revealed at Record Pace

    SciTech Connect

    Hura, Greg

    2009-01-01

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  15. Protein Structures Revealed at Record Pace

    SciTech Connect

    Greg Hura

    2009-07-09

    The structure of a protein in days -- not months or years -- ushers in a new era in genomics research. Berkeley Lab scientists have developed a high-throughput protein pipeline that could expedite the development of biofuels and elucidate how proteins carry out lifes vital functions.

  16. Native fishes in the Truckee River: Are in-stream structures and patterns of population genetic structure related?

    PubMed

    Peacock, Mary M; Gustin, Mae S; Kirchoff, Veronica S; Robinson, Morgan L; Hekkala, Evon; Pizzarro-Barraza, Claudia; Loux, Tim

    2016-09-01

    In-stream structures are recognized as significant impediments to movement for freshwater fishes. Apex predators such as salmonids have been the focus of much research on the impacts of such barriers to population dynamics and population viability however much less research has focused on native fishes, where in-stream structures may have a greater impact on long term population viability of these smaller, less mobile species. Patterns of genetic structure on a riverscape can provide information on which structures represent real barriers to movement for fish species and under what specific flow conditions. Here we characterize the impact of 41 dam and diversion structures on movement dynamics under varying flow conditions for a suite of six native fishes found in the Truckee River of California and Nevada. Microsatellite loci were used to estimate total allelic diversity, effective population size and assess genetic population structure. Although there is spatial overlap among species within the river there are clear differences in species distributions within the watershed. Observed population genetic structure was associated with in-stream structures, but only under low flow conditions. High total discharge in 2006 allowed fish to move over potential barriers resulting in no observed population genetic structure for any species in 2007. The efficacy of in-stream structures to impede movement and isolate fish emerged only after multiple years of low flow conditions. Our results suggest that restricted movement of fish species, as a result of in-stream barriers, can be mitigated by flow management. However, as flow dynamics are likely to be altered under global climate change, fragmentation due to barriers could isolate stream fishes into small subpopulations susceptible to both demographic losses and losses of genetic variation. PMID:27135585

  17. Predicting protein dynamics from structural ensembles

    NASA Astrophysics Data System (ADS)

    Copperman, J.; Guenza, M. G.

    2015-12-01

    The biological properties of proteins are uniquely determined by their structure and dynamics. A protein in solution populates a structural ensemble of metastable configurations around the global fold. From overall rotation to local fluctuations, the dynamics of proteins can cover several orders of magnitude in time scales. We propose a simulation-free coarse-grained approach which utilizes knowledge of the important metastable folded states of the protein to predict the protein dynamics. This approach is based upon the Langevin Equation for Protein Dynamics (LE4PD), a Langevin formalism in the coordinates of the protein backbone. The linear modes of this Langevin formalism organize the fluctuations of the protein, so that more extended dynamical cooperativity relates to increasing energy barriers to mode diffusion. The accuracy of the LE4PD is verified by analyzing the predicted dynamics across a set of seven different proteins for which both relaxation data and NMR solution structures are available. Using experimental NMR conformers as the input structural ensembles, LE4PD predicts quantitatively accurate results, with correlation coefficient ρ = 0.93 to NMR backbone relaxation measurements for the seven proteins. The NMR solution structure derived ensemble and predicted dynamical relaxation is compared with molecular dynamics simulation-derived structural ensembles and LE4PD predictions and is consistent in the time scale of the simulations. The use of the experimental NMR conformers frees the approach from computationally demanding simulations.

  18. Impact of non-native terrestrial mammals on the structure of the terrestrial mammal food web of Newfoundland, Canada.

    PubMed

    Strong, Justin S; Leroux, Shawn J

    2014-01-01

    The island of Newfoundland is unique because it has as many non-native terrestrial mammals as native ones. The impacts of non-native species on native flora and fauna can be profound and invasive species have been identified as one of the primary drivers of species extinction. Few studies, however, have investigated the effects of a non-native species assemblage on community and ecosystem properties. We reviewed the literature to build the first terrestrial mammal food web for the island of Newfoundland and then used network analyses to investigate how the timing of introductions and trophic position of non-native species has affected the structure of the terrestrial mammal food web in Newfoundland. The first non-native mammals (house mouse and brown rat) became established in Newfoundland with human settlement in the late 15th and early 16th centuries. Coyotes and southern red-backed voles are the most recent mammals to establish themselves on the island in 1985 and 1998, respectively. The fraction of intermediate species increased with the addition of non-native mammals over time whereas the fraction of basal and top species declined over time. This increase in intermediate species mediated by non-native species arrivals led to an overall increase in the terrestrial mammal food web connectance and generality (i.e. mean number of prey per predator). This diverse prey base and sources of carrion may have facilitated the natural establishment of coyotes on the island. Also, there is some evidence that the introduction of non-native prey species such as the southern red-backed vole has contributed to the recovery of the threatened American marten. Long-term monitoring of the food web is required to understand and predict the impacts of the diverse novel interactions that are developing in the terrestrial mammal food web of Newfoundland. PMID:25170923

  19. Impact of Non-Native Terrestrial Mammals on the Structure of the Terrestrial Mammal Food Web of Newfoundland, Canada

    PubMed Central

    Strong, Justin S.; Leroux, Shawn J.

    2014-01-01

    The island of Newfoundland is unique because it has as many non-native terrestrial mammals as native ones. The impacts of non-native species on native flora and fauna can be profound and invasive species have been identified as one of the primary drivers of species extinction. Few studies, however, have investigated the effects of a non-native species assemblage on community and ecosystem properties. We reviewed the literature to build the first terrestrial mammal food web for the island of Newfoundland and then used network analyses to investigate how the timing of introductions and trophic position of non-native species has affected the structure of the terrestrial mammal food web in Newfoundland. The first non-native mammals (house mouse and brown rat) became established in Newfoundland with human settlement in the late 15th and early 16th centuries. Coyotes and southern red-backed voles are the most recent mammals to establish themselves on the island in 1985 and 1998, respectively. The fraction of intermediate species increased with the addition of non-native mammals over time whereas the fraction of basal and top species declined over time. This increase in intermediate species mediated by non-native species arrivals led to an overall increase in the terrestrial mammal food web connectance and generality (i.e. mean number of prey per predator). This diverse prey base and sources of carrion may have facilitated the natural establishment of coyotes on the island. Also, there is some evidence that the introduction of non-native prey species such as the southern red-backed vole has contributed to the recovery of the threatened American marten. Long-term monitoring of the food web is required to understand and predict the impacts of the diverse novel interactions that are developing in the terrestrial mammal food web of Newfoundland. PMID:25170923

  20. Assessment of the utility of contact-based restraints in accelerating the prediction of protein structure using molecular dynamics simulations.

    PubMed

    Raval, Alpan; Piana, Stefano; Eastwood, Michael P; Shaw, David E

    2016-01-01

    Molecular dynamics (MD) simulation is a well-established tool for the computational study of protein structure and dynamics, but its application to the important problem of protein structure prediction remains challenging, in part because extremely long timescales can be required to reach the native structure. Here, we examine the extent to which the use of low-resolution information in the form of residue-residue contacts, which can often be inferred from bioinformatics or experimental studies, can accelerate the determination of protein structure in simulation. We incorporated sets of 62, 31, or 15 contact-based restraints in MD simulations of ubiquitin, a benchmark system known to fold to the native state on the millisecond timescale in unrestrained simulations. One-third of the restrained simulations folded to the native state within a few tens of microseconds-a speedup of over an order of magnitude compared with unrestrained simulations and a demonstration of the potential for limited amounts of structural information to accelerate structure determination. Almost all of the remaining ubiquitin simulations reached near-native conformations within a few tens of microseconds, but remained trapped there, apparently due to the restraints. We discuss potential methodological improvements that would facilitate escape from these near-native traps and allow more simulations to quickly reach the native state. Finally, using a target from the Critical Assessment of protein Structure Prediction (CASP) experiment, we show that distance restraints can improve simulation accuracy: In our simulations, restraints stabilized the native state of the protein, enabling a reasonable structural model to be inferred. PMID:26266489

  1. Raman microprobe investigation of molecular structure and organization in the native state of woody tissue

    SciTech Connect

    Atalla, R.H.

    1989-08-01

    Although the primary emphasis of our program has remained with the application of Raman spectroscopy to the study of native tissue, the scope of the work has been expanded to include a number of complementary approaches. These have included Solid State 13C NMR, autoradiography of radiolabeled woody tissue sections, and the generation of biomimetic tertiary aggregates which simulate states of aggregation characteristic of cell walls. Our Raman spectroscopic studies have resulted in progress in the areas of interpretation of the spectral features, and confirmation of the variability of the patterns of orientation of lignin reported earlier. We have assembled and made operational our new microprobe and spectrometer systems acquired under the DOE-URIP program. We have also demonstrated that, operating with gated detection and pulsed laser excitation, we can discriminate against the laser-excited fluorescence characteristic of most woody tissue. Our studies of celluloses, which combine Raman spectroscopy and 13C NMR have shown that all native celluloses are composites of two forms which have the same secondary structure but different tertiary structures.

  2. Structure-approximating inverse protein folding problem in the 2D HP model.

    PubMed

    Gupta, Arvind; Manuch, Ján; Stacho, Ladislav

    2005-12-01

    The inverse protein folding problem is that of designing an amino acid sequence which has a particular native protein fold. This problem arises in drug design where a particular structure is necessary to ensure proper protein-protein interactions. In this paper, we show that in the 2D HP model of Dill it is possible to solve this problem for a broad class of structures. These structures can be used to closely approximate any given structure. One of the most important properties of a good protein (in drug design) is its stability--the aptitude not to fold simultaneously into other structures. We show that for a number of basic structures, our sequences have a unique fold. PMID:16379538

  3. Genome-wide Membrane Protein Structure Prediction

    PubMed Central

    Piccoli, Stefano; Suku, Eda; Garonzi, Marianna; Giorgetti, Alejandro

    2013-01-01

    Transmembrane proteins allow cells to extensively communicate with the external world in a very accurate and specific way. They form principal nodes in several signaling pathways and attract large interest in therapeutic intervention, as the majority pharmaceutical compounds target membrane proteins. Thus, according to the current genome annotation methods, a detailed structural/functional characterization at the protein level of each of the elements codified in the genome is also required. The extreme difficulty in obtaining high-resolution three-dimensional structures, calls for computational approaches. Here we review to which extent the efforts made in the last few years, combining the structural characterization of membrane proteins with protein bioinformatics techniques, could help describing membrane proteins at a genome-wide scale. In particular we analyze the use of comparative modeling techniques as a way of overcoming the lack of high-resolution three-dimensional structures in the human membrane proteome. PMID:24403851

  4. Early glycation products produce pentosidine cross-links on native proteins. novel mechanism of pentosidine formation and propagation of glycation.

    PubMed

    Chellan, P; Nagaraj, R H

    2001-02-01

    Bovine lens alpha-crystallin was immobilized on EAH-Sepharose gel and glycated using d-ribose. Incubation with 500 and 100 mm d-ribose for 2 and 15 days produced short-term glycated (STGP gel) and long-term glycated proteins (LTGP gel). Both STGP and LTGP gels produced oxygen free radicals. Hydroxyl radical production was twice that in STGP gel compared with the LTGP gel. Incubation with the glycated gels produced pentosidine in a mixture of N-alpha-acetylarginine + N-alpha-acetyllysine, bovine lens proteins (BLP), and lysozyme; the amounts measured with STGP gel were higher than those with LTGP gel. Reactive oxygen species scavengers decreased the formation of pentosidine. Pentosidine was also formed in BLP when incubated with water-insoluble proteins extracted from aged or brunescent human lenses. Early glycated proteins from aged or diabetic lenses were bound to a boronate affinity column, the protein-containing gel was incubated with BLP, and pentosidine was measured in the incubation mixtures. With this method we found that diabetic lens proteins produced more pentosidine on BLP than did aged lens proteins. Further investigation indicates that two and three carbon carbohydrates possibly formed from oxidative cleavage of early glycation products are involved in pentosidine formation. Based on our findings, we propose a novel pathway for pentosidine formation on native proteins from glycated proteins. PMID:11076948

  5. Website on Protein Interaction and Protein Structure Related Work

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj; Liang, Shoudan; Biegel, Bryan (Technical Monitor)

    2003-01-01

    In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2) Protein Structures; and 3) Current Transport through Small Molecules.

  6. Protein-protein interface prediction based on hexagon structure similarity.

    PubMed

    Guo, Fei; Ding, Yijie; Li, Shuai Cheng; Shen, Chao; Wang, Lusheng

    2016-08-01

    Studies on protein-protein interaction are important in proteome research. How to build more effective models based on sequence information, structure information and physicochemical characteristics, is the key technology in protein-protein interface prediction. In this paper, we study the protein-protein interface prediction problem. We propose a novel method for identifying residues on interfaces from an input protein with both sequence and 3D structure information, based on hexagon structure similarity. Experiments show that our method achieves better results than some state-of-the-art methods for identifying protein-protein interface. Comparing to existing methods, our approach improves F-measure value by at least 0.03. On a common dataset consisting of 41 complexes, our method has overall precision and recall values of 63% and 57%. On Benchmark v4.0, our method has overall precision and recall values of 55% and 56%. On CAPRI targets, our method has overall precision and recall values of 52% and 55%. PMID:26936323

  7. Revealing Ligand Binding Sites and Quantifying Subunit Variants of Non-Covalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

    PubMed Central

    Li, Huilin; Wongkongkathep, Piriya; Van Orden, Steve L.; Loo, Rachel R. Ogorzalek; Loo, Joseph A.

    2015-01-01

    “Native” mass spectrometry (MS) has been proven increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD MS hADH dimer shows that each subunit (E and S chain) binds not only to two zinc atoms, but also the NAD+/NADH ligand, with a higher NAD+/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains. PMID:24912433

  8. Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.

    PubMed Central

    Jurica, Melissa S; Licklider, Lawrence J; Gygi, Steven R; Grigorieff, Nikolaus; Moore, Melissa J

    2002-01-01

    We describe characterization of spliceosomes affinity purified under native conditions. These spliceosomes consist largely of C complex containing splicing intermediates. After C complex assembly on an MS2 affinity-tagged pre-mRNA substrate containing a 3' splice site mutation, followed by RNase H digestion of earlier complexes, spliceosomes were purified by size exclusion and affinity selection. This protocol yielded 40S C complexes in sufficient quantities to visualize in negative stain by electron microscopy. Complexes purified in this way contain U2, U5, and U6 snRNAs, but very little U1 or U4 snRNA. Analysis by tandem mass spectrometry confirmed the presence of core snRNP proteins (SM and LSM), U2 and U5 snRNP-specific proteins, and the second step factors Prp16, Prp17, Slu7, and Prp22. In contrast, proteins specific to earlier splicing complexes, such as U2AF and U1 snRNP components, were not detected in C complex, but were present in similarly purified H complex. Images of these spliceosomes revealed single particles with dimensions of approximately 270 x 240 A that assort into well-defined classes. These images represent an important first step toward attaining a comprehensive three-dimensional understanding of pre-mRNA splicing. PMID:11991638

  9. From Ramachandran Maps to Tertiary Structures of Proteins.

    PubMed

    DasGupta, Debarati; Kaushik, Rahul; Jayaram, B

    2015-08-27

    Sequence to structure of proteins is an unsolved problem. A possible coarse grained resolution to this entails specification of all the torsional (Φ, Ψ) angles along the backbone of the polypeptide chain. The Ramachandran map quite elegantly depicts the allowed conformational (Φ, Ψ) space of proteins which is still very large for the purposes of accurate structure generation. We have divided the allowed (Φ, Ψ) space in Ramachandran maps into 27 distinct conformations sufficient to regenerate a structure to within 5 Å from the native, at least for small proteins, thus reducing the structure prediction problem to a specification of an alphanumeric string, i.e., the amino acid sequence together with one of the 27 conformations preferred by each amino acid residue. This still theoretically results in 27(n) conformations for a protein comprising "n" amino acids. We then investigated the spatial correlations at the two-residue (dipeptide) and three-residue (tripeptide) levels in what may be described as higher order Ramachandran maps, with the premise that the allowed conformational space starts to shrink as we introduce neighborhood effects. We found, for instance, for a tripeptide which potentially can exist in any of the 27(3) "allowed" conformations, three-fourths of these conformations are redundant to the 95% confidence level, suggesting sequence context dependent preferred conformations. We then created a look-up table of preferred conformations at the tripeptide level and correlated them with energetically favorable conformations. We found in particular that Boltzmann probabilities calculated from van der Waals energies for each conformation of tripeptides correlate well with the observed populations in the structural database (the average correlation coefficient is ∼0.8). An alpha-numeric string and hence the tertiary structure can be generated for any sequence from the look-up table within minutes on a single processor and to a higher level of accuracy

  10. Conformational flexibility and protein folding: rigid structural fragments connected by flexible joints in subtilisin BPN.

    PubMed Central

    Honig, B; Ray, A; Levinthal, C

    1976-01-01

    Conformational energy calculations are used to analyze the interactions of structural substructures in subtilisin BPN. These substructures are kept fixed or "rigid" so that the only variables in the calculations are the backbone segments that separate them. The flexible segments are assumed to be free turns. Using this representation of the protein it is possible to predict both a likely order of events along a folding pathway and preferred modes of conformational changes of the native protein. Moreover, when the native structure has been perturbed by moving the substructures apart, it is possible to assess the range of interactions that return the protein, upon energy minimization, to its original conformation. These results suggest an approach to the folding problem based on the piecemeal formation of tertiary structure from smaller prefolded fragments. PMID:1064867

  11. Local backbone structure prediction of proteins.

    PubMed

    de Brevern, Alexandre G; Benros, Cristina; Gautier, Romain; Valadié, Héléne; Hazout, Serge; Etchebest, Catherine

    2004-01-01

    A statistical analysis of the PDB structures has led us to define a new set of small 3D structural prototypes called Protein Blocks (PBs). This structural alphabet includes 16 PBs, each one is defined by the (phi, psi) dihedral angles of 5 consecutive residues. The amino acid distributions observed in sequence windows encompassing these PBs are used to predict by a Bayesian approach the local 3D structure of proteins from the sole knowledge of their sequences. LocPred is a software which allows the users to submit a protein sequence and performs a prediction in terms of PBs. The prediction results are given both textually and graphically. PMID:15724288

  12. A minimal sequence code for switching protein structure and function.

    PubMed

    Alexander, Patrick A; He, Yanan; Chen, Yihong; Orban, John; Bryan, Philip N

    2009-12-15

    We present here a structural and mechanistic description of how a protein changes its fold and function, mutation by mutation. Our approach was to create 2 proteins that (i) are stably folded into 2 different folds, (ii) have 2 different functions, and (iii) are very similar in sequence. In this simplified sequence space we explore the mutational path from one fold to another. We show that an IgG-binding, 4beta+alpha fold can be transformed into an albumin-binding, 3-alpha fold via a mutational pathway in which neither function nor native structure is completely lost. The stabilities of all mutants along the pathway are evaluated, key high-resolution structures are determined by NMR, and an explanation of the switching mechanism is provided. We show that the conformational switch from 4beta+alpha to 3-alpha structure can occur via a single amino acid substitution. On one side of the switch point, the 4beta+alpha fold is >90% populated (pH 7.2, 20 degrees C). A single mutation switches the conformation to the 3-alpha fold, which is >90% populated (pH 7.2, 20 degrees C). We further show that a bifunctional protein exists at the switch point with affinity for both IgG and albumin. PMID:19923431

  13. Optimized Null Model for Protein Structure Networks

    PubMed Central

    Lappe, Michael; Pržulj, Nataša

    2009-01-01

    Much attention has recently been given to the statistical significance of topological features observed in biological networks. Here, we consider residue interaction graphs (RIGs) as network representations of protein structures with residues as nodes and inter-residue interactions as edges. Degree-preserving randomized models have been widely used for this purpose in biomolecular networks. However, such a single summary statistic of a network may not be detailed enough to capture the complex topological characteristics of protein structures and their network counterparts. Here, we investigate a variety of topological properties of RIGs to find a well fitting network null model for them. The RIGs are derived from a structurally diverse protein data set at various distance cut-offs and for different groups of interacting atoms. We compare the network structure of RIGs to several random graph models. We show that 3-dimensional geometric random graphs, that model spatial relationships between objects, provide the best fit to RIGs. We investigate the relationship between the strength of the fit and various protein structural features. We show that the fit depends on protein size, structural class, and thermostability, but not on quaternary structure. We apply our model to the identification of significantly over-represented structural building blocks, i.e., network motifs, in protein structure networks. As expected, choosing geometric graphs as a null model results in the most specific identification of motifs. Our geometric random graph model may facilitate further graph-based studies of protein conformation space and have important implications for protein structure comparison and prediction. The choice of a well-fitting null model is crucial for finding structural motifs that play an important role in protein folding, stability and function. To our knowledge, this is the first study that addresses the challenge of finding an optimized null model for RIGs, by

  14. Soluble Non-ammonia Nitrogen in Ruminal and Omasal Digesta of Korean Native Steers Supplemented with Soluble Proteins

    PubMed Central

    Choi, C. W.; Kim, K. H.; Chang, S. S.; Choi, N. J.

    2012-01-01

    An experiment was conducted to study the effect of soluble protein supplements on concentration of soluble non-ammonia nitrogen (SNAN) in the liquid phase of ruminal (RD) and omasal digesta (OD) of Korean native steers, and to investigate diurnal pattern in SNAN concentration in RD and OD. Three ruminally cannulated Korean native steers in a 3×3 Latin square design consumed a basal diet of rice straw and corn-based concentrate (control), and that supplemented (kg/d DM basis) with intact casein (0.24; IC) or acid hydrolyzed casein (0.46; AHC). Ruminal digesta was sampled using a vacuum pump, whereas OD was collected using an omasal sampling system at 2.0 h intervals after a morning feeding. The SNAN fractions (free amino acid (AA), peptide and soluble protein) in RD and OD were assessed using the ninhydrin assay. Concentrations of free AA and total SNAN in RD were significantly (p<0.05) lower than those in OD. Although free AA concentration was relatively high, mean peptide was quantitatively the most important fraction of total SNAN in both RD and OD, indicating that degradation of peptide to AA rather than hydrolysis of soluble protein to peptide or deamination may be the most limiting step in rumen proteolysis of Korean native steers. Diurnal variation in peptide concentration in OD for the soluble protein supplemented diets during the feeding cycle peaked 2 h post-feeding and decreased thereafter whereas that for the control was relatively constant during the entire feeding cycle. Diurnal variation in peptide concentration was rather similar between RD and OD. PMID:25049690

  15. Contrasting Role of Temperature in Structuring Regional Patterns of Invasive and Native Pestilential Stink Bugs

    PubMed Central

    Venugopal, P. Dilip; Dively, Galen P.; Herbert, Ames; Malone, Sean; Whalen, Joanne; Lamp, William O.

    2016-01-01

    Objectives Assessment and identification of spatial structures in the distribution and abundance of invasive species is important for unraveling the underlying ecological processes. The invasive agricultural insect pest Halyomorpha halys that causes severe economic losses in the United States is currently expanding both within United States and across Europe. We examined the drivers of H. halys invasion by characterizing the distribution and abundance patterns of H. halys and native stink bugs (Chinavia hilaris and Euschistus servus) across eight different spatial scales. We then quantified the interactive and individual influences of temperature, and measures of resource availability and distance from source populations, and their relevant spatial scales. We used Moran’s Eigenvector Maps based on Gabriel graph framework to quantify spatial relationships among the soybean fields in mid-Atlantic Unites States surveyed for stink bugs. Findings Results from the multi-spatial scale, multivariate analyses showed that temperature and its interaction with resource availability and distance from source populations structures the patterns in H. halys at very broad spatial scale. H. halys abundance decreased with increasing average June temperature and distance from source population. H. halys were not recorded at fields with average June temperature higher than 23.5°C. In parts with suitable climate, high H. halys abundance was positively associated with percentage developed open area and percentage deciduous forests at 250m scale. Broad scale patterns in native stink bugs were positively associated with increasing forest cover and, in contrast to the invasive H. halys, increasing mean July temperature. Our results identify the contrasting role of temperature in structuring regional patterns in H. halys and native stink bugs, while demonstrating its interaction with resource availability and distance from source populations for structuring H. halys patterns. Conclusion

  16. Protein-associated water and secondary structure effect removal of blood proteins from metallic substrates.

    PubMed

    Anand, Gaurav; Zhang, Fuming; Linhardt, Robert J; Belfort, Georges

    2011-03-01

    Removing adsorbed protein from metals has significant health and industrial consequences. There are numerous protein-adsorption studies using model self-assembled monolayers or polymeric substrates but hardly any high-resolution measurements of adsorption and removal of proteins on industrially relevant transition metals. Surgeons and ship owners desire clean metal surfaces to reduce transmission of disease via surgical instruments and minimize surface fouling (to reduce friction and corrosion), respectively. A major finding of this work is that, besides hydrophobic interaction adhesion energy, water content in an adsorbed protein layer and secondary structure of proteins determined the access and hence ability to remove adsorbed proteins from metal surfaces with a strong alkaline-surfactant solution (NaOH and 5 mg/mL SDS in PBS at pH 11). This is demonstrated with three blood proteins (bovine serum albumin, immunoglobulin, and fibrinogen) and four transition metal substrates and stainless steel (platinum (Pt), gold (Au), tungsten (W), titanium (Ti), and 316 grade stainless steel (SS)). All the metallic substrates were checked for chemical contaminations like carbon and sulfur and were characterized using X-ray photoelectron spectroscopy (XPS). While Pt and Au surfaces were oxide-free (fairly inert elements), W, Ti, and SS substrates were associated with native oxide. Difference measurements between a quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance spectroscopy (SPR) provided a measure of the water content in the protein-adsorbed layers. Hydrophobic adhesion forces, obtained with atomic force microscopy, between the proteins and the metals correlated with the amount of the adsorbed protein-water complex. Thus, the amount of protein adsorbed decreased with Pt, Au, W, Ti and SS, in this order. Neither sessile contact angle nor surface roughness of the metal substrates was useful as predictors here. All three globular proteins

  17. PSSweb: protein structural statistics web server.

    PubMed

    Gaillard, Thomas; Stote, Roland H; Dejaegere, Annick

    2016-07-01

    With the increasing number of protein structures available, there is a need for tools capable of automating the comparison of ensembles of structures, a common requirement in structural biology and bioinformatics. PSSweb is a web server for protein structural statistics. It takes as input an ensemble of PDB files of protein structures, performs a multiple sequence alignment and computes structural statistics for each position of the alignment. Different optional functionalities are proposed: structure superposition, Cartesian coordinate statistics, dihedral angle calculation and statistics, and a cluster analysis based on dihedral angles. An interactive report is generated, containing a summary of the results, tables, figures and 3D visualization of superposed structures. The server is available at http://pssweb.org. PMID:27174930

  18. PSSweb: protein structural statistics web server

    PubMed Central

    Gaillard, Thomas; Stote, Roland H.; Dejaegere, Annick

    2016-01-01

    With the increasing number of protein structures available, there is a need for tools capable of automating the comparison of ensembles of structures, a common requirement in structural biology and bioinformatics. PSSweb is a web server for protein structural statistics. It takes as input an ensemble of PDB files of protein structures, performs a multiple sequence alignment and computes structural statistics for each position of the alignment. Different optional functionalities are proposed: structure superposition, Cartesian coordinate statistics, dihedral angle calculation and statistics, and a cluster analysis based on dihedral angles. An interactive report is generated, containing a summary of the results, tables, figures and 3D visualization of superposed structures. The server is available at http://pssweb.org. PMID:27174930

  19. Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics.

    PubMed

    Melani, Rafael D; Skinner, Owen S; Fornelli, Luca; Domont, Gilberto B; Compton, Philip D; Kelleher, Neil L

    2016-07-01

    Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric l-amino acid oxidase (∼130 kDa) and the multichain toxin cobra venom factor (∼147 kDa). The l-amino acid oxidase complex exhibited two clusters of multiproteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new antivenoms or other biotherapeutics. PMID:27178327

  20. Protein Structure Determination Using Protein Threading and Sparse NMR Data

    SciTech Connect

    Crawford, O.H.; Einstein, J.R.; Xu, D.; Xu, Y.

    1999-11-14

    It is well known that the NMR method for protein structure determination applies to small proteins and that its effectiveness decreases very rapidly as the molecular weight increases beyond about 30 kD. We have recently developed a method for protein structure determination that can fully utilize partial NMR data as calculation constraints. The core of the method is a threading algorithm that guarantees to find a globally optimal alignment between a query sequence and a template structure, under distance constraints specified by NMR/NOE data. Our preliminary tests have demonstrated that a small number of NMR/NOE distance restraints can significantly improve threading performance in both fold recognition and threading-alignment accuracy, and can possibly extend threading's scope of applicability from structural homologs to structural analogs. An accurate backbone structure generated by NMR-constrained threading can then provide a significant amount of structural information, equivalent to that provided by the NMR method with many NMR/NOE restraints; and hence can greatly reduce the amount of NMR data typically required for accurate structure determination. Our preliminary study suggests that a small number of NMR/NOE restraints may suffice to determine adequately the all-atom structure when those restraints are incorporated in a procedure combining threading, modeling of loops and sidechains, and molecular dynamics simulation. Potentially, this new technique can expand NMR's capability to larger proteins.

  1. Mapping membrane protein structure with fluorescence

    PubMed Central

    Taraska, Justin W.

    2012-01-01

    Membrane proteins regulate many cellular processes including signaling cascades, ion transport, membrane fusion, and cell-to-cell communications. Understanding the architecture and conformational fluctuations of these proteins is critical to understanding their regulation and functions. Fluorescence methods including intensity mapping, fluorescence resonance energy transfer, and photo-induced electron transfer, allow for targeted measurements of domains within membrane proteins. These methods can reveal how a protein is structured and how it transitions between different conformational states. Here, I will review recent work done using fluorescence to map the structures of membrane proteins, focusing on how each of these methods can be applied to understanding the dynamic nature of individual membrane proteins and protein complexes. PMID:22445227

  2. Profiles of structural heterogeneity in native lipooligosaccharides of Neisseria and cytokine induction.

    PubMed

    John, Constance M; Liu, Mingfeng; Jarvis, Gary A

    2009-03-01

    Fine differences in the phosphorylation and acylation of lipooligosaccharide (LOS) from Neisseria species are thought to profoundly influence the virulence of the organisms and the innate immune responses of the host, such as signaling through toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells (TREM). MALDI time-of-flight (TOF) mass spectrometry was used to characterize heterogeneity in the native LOS from Neisseria gonorrheae and N. meningitidis. A sample preparation methodology previously reported for Escherichia coli lipopolysaccharide (LPS) employing deposition of untreated LOS on a thin layer of a film composed of 2,4,6-trihydroxyacetophenone and nitrocellulose was used. Prominent peaks were observed corresponding to molecular ions and to fragment ions primarily formed by cleavage between the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and the lipid A (LA). Analyses of these data and comparison with spectra of the corresponding O-deacylated or hydrogen fluoride-treated LOS enabled the detection of novel species that apparently differed by the expression of up to three phosphates with one or more phosphoethanolamine (PEA) groups on the LA. We found that the heterogeneity profile of acylation and phosphorylation correlates with the induction of proinflammatory cytokines in THP-1 monocytic cells. This methodology enabled us to rapidly profile components of structural variants of native LOS that are of importance biologically. PMID:18832773

  3. Homology-Based Modeling of Protein Structure

    NASA Astrophysics Data System (ADS)

    Xiang, Zhexin

    The human genome project has already discovered millions of proteins (http://www.swissprot.com). The potential of the genome project can only be fully realized once we can assign, understand, manipulate, and predict the function of these new proteins (Sanchez and Sali, 1997; Frishman et al., 2000; Domingues et al., 2000). Predicting protein function generally requires knowledge of protein three-dimensional structure (Blundell et al., 1978;Weber, 1990), which is ultimately determined by protein sequence (Anfinsen, 1973). Protein structure determination using experimental methods such as X-ray crystallography or NMR spectroscopy is very time consuming (Johnson et al. 1994). To date, fewer than 2% of the known proteins have had their structures solved experimentally. In 2004, more than half a million new proteins were sequenced that almost doubled the efforts in the previous year, but only 5300 structures were solved. Although the rate of experimental structure determination will continue to increase, the number of newly discovered sequences grows much faster than the number of structures solved (see Fig. 10.1).

  4. Protein structure prediction from sequence variation

    PubMed Central

    Marks, Debora S; Hopf, Thomas A; Sander, Chris

    2015-01-01

    Genomic sequences contain rich evolutionary information about functional constraints on macromolecules such as proteins. This information can be efficiently mined to detect evolutionary couplings between residues in proteins and address the long-standing challenge to compute protein three-dimensional structures from amino acid sequences. Substantial progress has recently been made on this problem owing to the explosive growth in available sequences and the application of global statistical methods. In addition to three-dimensional structure, the improved understanding of covariation may help identify functional residues involved in ligand binding, protein-complex formation and conformational changes. We expect computation of covariation patterns to complement experimental structural biology in elucidating the full spectrum of protein structures, their functional interactions and evolutionary dynamics. PMID:23138306

  5. Lessons from making the Structural Classification of Proteins (SCOP) and their implications for protein structure modelling

    PubMed Central

    Andreeva, Antonina

    2016-01-01

    The Structural Classification of Proteins (SCOP) database has facilitated the development of many tools and algorithms and it has been successfully used in protein structure prediction and large-scale genome annotations. During the development of SCOP, numerous exceptions were found to topological rules, along with complex evolutionary scenarios and peculiarities in proteins including the ability to fold into alternative structures. This article reviews cases of structural variations observed for individual proteins and among groups of homologues, knowledge of which is essential for protein structure modelling. PMID:27284063

  6. Lessons from making the Structural Classification of Proteins (SCOP) and their implications for protein structure modelling.

    PubMed

    Andreeva, Antonina

    2016-06-15

    The Structural Classification of Proteins (SCOP) database has facilitated the development of many tools and algorithms and it has been successfully used in protein structure prediction and large-scale genome annotations. During the development of SCOP, numerous exceptions were found to topological rules, along with complex evolutionary scenarios and peculiarities in proteins including the ability to fold into alternative structures. This article reviews cases of structural variations observed for individual proteins and among groups of homologues, knowledge of which is essential for protein structure modelling. PMID:27284063

  7. Criteria for folding in structure-based models of proteins

    NASA Astrophysics Data System (ADS)

    Wołek, Karol; Cieplak, Marek

    2016-05-01

    In structure-based models of proteins, one often assumes that folding is accomplished when all contacts are established. This assumption may frequently lead to a conceptual problem that folding takes place in a temperature region of very low thermodynamic stability, especially when the contact map used is too sparse. We consider six different structure-based models and show that allowing for a small, but model-dependent, percentage of the native contacts not being established boosts the folding temperature substantially while affecting the time scales of folding only in a minor way. We also compare other properties of the six models. We show that the choice of the description of the backbone stiffness has a substantial effect on the values of characteristic temperatures that relate both to equilibrium and kinetic properties. Models without any backbone stiffness (like the self-organized polymer) are found to perform similar to those with the stiffness, including in the studies of stretching.

  8. Criteria for folding in structure-based models of proteins.

    PubMed

    Wołek, Karol; Cieplak, Marek

    2016-05-14

    In structure-based models of proteins, one often assumes that folding is accomplished when all contacts are established. This assumption may frequently lead to a conceptual problem that folding takes place in a temperature region of very low thermodynamic stability, especially when the contact map used is too sparse. We consider six different structure-based models and show that allowing for a small, but model-dependent, percentage of the native contacts not being established boosts the folding temperature substantially while affecting the time scales of folding only in a minor way. We also compare other properties of the six models. We show that the choice of the description of the backbone stiffness has a substantial effect on the values of characteristic temperatures that relate both to equilibrium and kinetic properties. Models without any backbone stiffness (like the self-organized polymer) are found to perform similar to those with the stiffness, including in the studies of stretching. PMID:27179507

  9. Improvement of protein structure comparison using a structural alphabet.

    PubMed

    Joseph, Agnel Praveen; Srinivasan, N; de Brevern, Alexandre G

    2011-09-01

    The three dimensional structure of a protein provides major insights into its function. Protein structure comparison has implications in functional and evolutionary studies. A structural alphabet (SA) is a library of local protein structure prototypes that can abstract every part of protein main chain conformation. Protein Blocks (PBs) is a widely used SA, composed of 16 prototypes, each representing a pentapeptide backbone conformation defined in terms of dihedral angles. Through this description, the 3D structural information can be translated into a 1D sequence of PBs. In a previous study, we have used this approach to compare protein structures encoded in terms of PBs. A classical sequence alignment procedure based on dynamic programming was used, with a dedicated PB Substitution Matrix (SM). PB-based pairwise structural alignment method gave an excellent performance, when compared to other established methods for mining. In this study, we have (i) refined the SMs and (ii) improved the Protein Block Alignment methodology (named as iPBA). The SM was normalized in regards to sequence and structural similarity. Alignment of protein structures often involves similar structural regions separated by dissimilar stretches. A dynamic programming algorithm that weighs these local similar stretches has been designed. Amino acid substitutions scores were also coupled linearly with the PB substitutions. iPBA improves (i) the mining efficiency rate by 6.8% and (ii) more than 82% of the alignments have a better quality. A higher efficiency in aligning multi-domain proteins could be also demonstrated. The quality of alignment is better than DALI and MUSTANG in 81.3% of the cases. Thus our study has resulted in an impressive improvement in the quality of protein structural alignment. PMID:21569819

  10. Predictive recognition of native proteins by cucurbit[7]uril in a complex mixture.

    PubMed

    Li, Wei; Bockus, Andrew T; Vinciguerra, Brittany; Isaacs, Lyle; Urbach, Adam R

    2016-06-30

    The recognition of human growth hormone (hGH) by the synthetic host molecule cucurbit[7]uril (Q7) was predicted on the basis of its N-terminal phenylalanine. An aqueous-compatible resin with covalently immobilized Q7 groups was prepared and shown to recognize native insulin and hGH in simple and complex mixtures. PMID:27311878

  11. Expression of a foreign Rubisco small subunit in tobacco with reduced levels of the native protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cDNA, ArRbcS3, for the small subunit of Rubisco from Amaranthus retroflexus (pigweed) was expressed in tobacco (Nicotiana tabacum) under the control of a strong leaf-specific Lhcb promoter. The coding region of the ArRbcS3 was fused to the plastid targeting sequence of the native tobacco rbcS to...

  12. Protein thermal denaturation is modulated by central residues in the protein structure network.

    PubMed

    Souza, Valquiria P; Ikegami, Cecília M; Arantes, Guilherme M; Marana, Sandro R

    2016-03-01

    Network structural analysis, known as residue interaction networks or graphs (RIN or RIG, respectively) or protein structural networks or graphs (PSN or PSG, respectively), comprises a useful tool for detecting important residues for protein function, stability, folding and allostery. In RIN, the tertiary structure is represented by a network in which residues (nodes) are connected by interactions (edges). Such structural networks have consistently presented a few central residues that are important for shortening the pathways linking any two residues in a protein structure. To experimentally demonstrate that central residues effectively participate in protein properties, mutations were directed to seven central residues of the β-glucosidase Sfβgly (β-d-glucoside glucohydrolase; EC 3.2.1.21). These mutations reduced the thermal stability of the enzyme, as evaluated by changes in transition temperature (Tm ) and the denaturation rate at 45 °C. Moreover, mutations directed to the vicinity of a central residue also caused significant decreases in the Tm of Sfβgly and clearly increased the unfolding rate constant at 45 °C. However, mutations at noncentral residues or at surrounding residues did not affect the thermal stability of Sfβgly. Therefore, the data reported in the present study suggest that the perturbation of the central residues reduced the stability of the native structure of Sfβgly. These results are in agreement with previous findings showing that networks are robust, whereas attacks on central nodes cause network failure. Finally, the present study demonstrates that central residues underlie the functional properties of proteins. PMID:26785700

  13. Solubilization of native integral membrane proteins in aqueous buffer by non-covalent chelation with monomethoxy polyethylene glycol (mPEG) polymers

    PubMed Central

    Janaratne, Thamara K.; Okach, Linda; Brock, Ansgar; Lesley, Scott A.

    2011-01-01

    Highly hydrophobic integral membrane proteins (IMPs) are typically purified in excess detergent media, often resulting in rapid inactivation and denaturation of the protein. One promising approach to solve this problem is to couple hydrophilic polymers, such as monomethoxypolyethylene glycol (mPEG) to IMPs under mild conditions in place of detergents. However, the broad application of this approach is hampered by poor reaction efficiencies, low tolerance of detergent stabilized membrane proteins to reaction conditions and a lack of proper site-specific reversible approaches. Here we have developed a straightforward, efficient and mild approach to site-specific non-covalent binding of long-chain polymers to recombinant IMPs. This method uses the hexa-histidine tag (His-Tag) often used for purification of recombinant proteins as an attachment site for mPEGs. Solubility studies performed using five different IMPs confirmed that all tested mPEG-bound IMPs were completely soluble and stable in detergent free aqueous buffer compared to their precipitated native proteins under the identical circumstances. Activity assays and circular dichroism (CD) spectroscopy confirmed the structural integrity of modified IMPs. PMID:21740061

  14. Pair potentials for protein folding: choice of reference states and sensitivity of predicted native states to variations in the interaction schemes.

    PubMed Central

    Betancourt, M. R.; Thirumalai, D.

    1999-01-01

    We examine the similarities and differences between two widely used knowledge-based potentials, which are expressed as contact matrices (consisting of 210 elements) that gives a scale for interaction energies between the naturally occurring amino acid residues. These are the Miyazawa-Jernigan contact interaction matrix M and the potential matrix S derived by Skolnick J et al., 1997, Protein Sci 6:676-688. Although the correlation between the two matrices is good, there is a relatively large dispersion between the elements. We show that when Thr is chosen as a reference solvent within the Miyazawa and Jernigan scheme, the dispersion between the M and S matrices is reduced. The resulting interaction matrix B gives hydrophobicities that are in very good agreement with experiment. The small dispersion between the S and B matrices, which arises due to differing reference states, is shown to have dramatic effect on the predicted native states of lattice models of proteins. These findings and other arguments are used to suggest that for reliable predictions of protein structures, pairwise additive potentials are not sufficient. We also establish that optimized protein sequences can tolerate relatively large random errors in the pair potentials. We conjecture that three body interaction may be needed to predict the folds of proteins in a reliable manner. PMID:10048329

  15. Taking advantage of local structure descriptors to analyze interresidue contacts in protein structures and protein complexes.

    PubMed

    Martin, Juliette; Regad, Leslie; Etchebest, Catherine; Camproux, Anne-Claude

    2008-11-15

    Interresidue protein contacts in proteins structures and at protein-protein interface are classically described by the amino acid types of interacting residues and the local structural context of the contact, if any, is described using secondary structures. In this study, we present an alternate analysis of interresidue contact using local structures defined by the structural alphabet introduced by Camproux et al. This structural alphabet allows to describe a 3D structure as a sequence of prototype fragments called structural letters, of 27 different types. Each residue can then be assigned to a particular local structure, even in loop regions. The analysis of interresidue contacts within protein structures defined using Voronoï tessellations reveals that pairwise contact specificity is greater in terms of structural letters than amino acids. Using a simple heuristic based on specificity score comparison, we find that 74% of the long-range contacts within protein structures are better described using structural letters than amino acid types. The investigation is extended to a set of protein-protein complexes, showing that the similar global rules apply as for intraprotein contacts, with 64% of the interprotein contacts best described by local structures. We then present an evaluation of pairing functions integrating structural letters to decoy scoring and show that some complexes could benefit from the use of structural letter-based pairing functions. PMID:18491388

  16. Embracing proteins: structural themes in aptamer-protein complexes.

    PubMed

    Gelinas, Amy D; Davies, Douglas R; Janjic, Nebojsa

    2016-02-01

    Understanding the structural rules that govern specific, high-affinity binding characteristic of aptamer-protein interactions is important in view of the increasing use of aptamers across many applications. From the modest number of 16 aptamer-protein structures currently available, trends are emerging. The flexible phosphodiester backbone allows folding into precise three-dimensional structures using known nucleic acid motifs as scaffolds that orient specific functional groups for target recognition. Still, completely novel motifs essential for structure and function are found in modified aptamers with diversity-enhancing side chains. Aptamers and antibodies, two classes of macromolecules used as affinity reagents with entirely different backbones and composition, recognize protein epitopes of similar size and with comparably high shape complementarity. PMID:26919170

  17. Principles for designing ideal protein structures

    PubMed Central

    Koga, Nobuyasu; Tatsumi-Koga, Rie; Liu, Gaohua; Xiao, Rong; Acton, Thomas B.; Montelione, Gaetano T.; Baker, David

    2013-01-01

    Unlike random heteropolymers, natural proteins fold into unique ordered structures. Understanding how these are encoded in amino-acid sequences is complicated by energetically unfavourable non-ideal features—for example kinked α-helices, bulged β-strands, strained loops and buried polar groups—that arise in proteins from evolutionary selection for biological function or from neutral drift. Here we describe an approach to designing ideal protein structures stabilized by completely consistent local and non-local interactions. The approach is based on a set of rules relating secondary structure patterns to protein tertiary motifs, which make possible the design of funnel-shaped protein folding energy landscapes leading into the target folded state. Guided by these rules, we designed sequences predicted to fold into ideal protein structures consisting of α-helices, β-strands and minimal loops. Designs for five different topologies were found to be monomeric and very stable and to adopt structures in solution nearly identical to the computational models. These results illuminate how the folding funnels of natural proteins arise and provide the foundation for engineering a new generation of functional proteins free from natural evolution. PMID:23135467

  18. Protein Structure Is Related to RNA Structural Reactivity In Vivo.

    PubMed

    Tang, Yin; Assmann, Sarah M; Bevilacqua, Philip C

    2016-02-27

    We assessed whether in vivo mRNA structural reactivity and the structure of the encoded protein are related. This is the first investigation of such a relationship that utilizes information on RNA structure obtained in living cells. Based on our recent genome-wide Structure-seq analysis in Arabidopsis thaliana, we report that, as a meta property, regions of individual mRNAs that code for protein domains generally have higher reactivity to DMS (dimethyl sulfate), a chemical that covalently modifies accessible As and Cs, than regions that encode protein domain junctions. This relationship is prominent for proteins annotated for catalytic activity and reversed in proteins annotated for binding and transcription regulatory activity. Upon analyzing intrinsically disordered proteins, we found a similar pattern for disordered regions as compared to ordered regions: regions of individual mRNAs that code for ordered regions have significantly higher DMS reactivity than regions that code for intrinsically disordered regions. Based on these effects, we hypothesize that the decreased DMS reactivity of RNA regions that encode protein domain junctions or intrinsically disordered regions may reflect increased RNA structure that may slow translation, allowing time for the nascent protein domain or ordered region of the protein to fold, thereby reducing protein misfolding. In addition, a drop in DMS reactivity was observed on portions of mRNA sequences that correspond to the C-termini of protein domains, suggesting ribosome protection at these mRNA regions. Structural relationships between mRNAs and their encoded proteins may have evolved to allow efficient and accurate protein folding. PMID:26598238

  19. Structural proteins of Herpesvirus saimiri.

    PubMed Central

    Keil, G; Fleckenstein, B; Bodemer, W

    1983-01-01

    Herpesvirus saimiri particles were purified from productively infected owl monkey kidney cell cultures, and the virion polypeptides were analyzed by polyacrylamide gel electrophoresis. A total of 21 predominant proteins were found in lysates of H. saimiri 11 particles by Coomassie blue staining or by [35S]methionine labeling and autoradiography; all proteins were between 160,000 and 12,000 daltons in size. They are most probably virion constituents, as most of them were precipitated by immune sera, and no dominant proteins of equivalent sizes were found in mock-infected cultures. Four glycoproteins (gp 155/160, gp 128, gp 84/90, gp 55) and three polypeptides that appeared not to be glycosylated (p71, p35, p28) were assigned to the envelope or matrix of virions, whereas at least four phosphoproteins (pp132, pp118, pp55, pp13) and ten polypeptides without apparent secondary modification (p155/160, p106, p96, p67, p53, p36, p32, p15, p14, p12) were found in the nucleocapsid fraction. Analysis of virion proteins from different H. saimiri strains did not reveal appreciable differences in the migration behavior of most polypeptides, including all glycoproteins; however, determination of a strain-specific size pattern was possible for three of four phosphoproteins. The overall similarity in protein architecture of H. saimiri strains obviously does not reflect the variability in biology, such as oncogenic properties. In comparison, DNA sequence divergences appear to remain a better taxonomic criterion for strain distinction. Images PMID:6312078

  20. Datamining protein structure databanks for crystallization patterns of proteins.

    PubMed

    Valafar, Homayoun; Prestegard, James H; Valafar, Faramarz

    2002-12-01

    A study of 345 protein structures selected among 1,500 structures determined by nuclear magnetic resonance (NMR) methods, revealed useful correlations between crystallization properties and several parameters for the studied proteins. NMR methods of structure determination do not require the growth of protein crystals, and hence allow comparison of properties of proteins that have or have not been the subject of crystallographic approaches. One- and two-dimensional statistical analyses of the data confirmed a hypothesized relation between the size of the molecule and its crystallization potential. Furthermore, two-dimensional Bayesian analysis revealed a significant relationship between relative ratio of different secondary structures and the likelihood of success for crystallization trials. The most immediate result is an apparent correlation of crystallization potential with protein size. Further analysis of the data revealed a relationship between the unstructured fraction of proteins and the success of its crystallization. Utilization of Bayesian analysis on the latter correlation resulted in a prediction performance of about 64%, whereas a two-dimensional Bayesian analysis succeeded with a performance of about 75%. PMID:12594078

  1. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    SciTech Connect

    Yu,P.

    2007-01-01

    The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  2. Fast loop modeling for protein structures

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  3. Investigation of the low frequency dynamics of heme proteins: native and mutant cytochrome P450(cam) and redox partner complexes.

    PubMed

    Karunakaran, Venugopal; Denisov, Ilia; Sligar, Stephen G; Champion, Paul M

    2011-05-12

    Vibrational coherence spectroscopy (VCS) is used to investigate the low-frequency dynamics of camphor-free and camphor-bound cytochrome P450(cam) (CYP 101) and its L358P mutant. The low-frequency heme vibrations are found to be perturbed upon binding to the electron transfer partner putidaredoxin (Pdx). A strong correlation between the "detuned" vibrational coherence spectrum, which monitors frequencies between 100 and 400 cm(-1), and the lower frequency part of the Raman spectrum is also demonstrated. The very low frequency region ≤200 cm(-1), uniquely accessed by open-band VCS measurements, reveals a mode near 103 cm(-1) in P450(cam) when camphor is not present in the distal pocket. This reflects the presence of a specific heme distortion, such as saddling or ruffling, in the substrate-free state where water is coordinated to the low-spin iron atom. Such distortions are likely to retard the rate of electron transfer to the substrate-free protein. The presence of strong mode near ∼33 cm(-1) in the camphor-bound form suggests a significant heme-doming distortion, which is supported by analysis using normal coordinate structural decomposition. Pdx also displays a strong coherent vibration near 30 cm(-1) that in principle could be involved in vibrational resonance with its electron transfer target. A splitting of the 33 cm(-1) feature and intensification of a mode near 78 cm(-1) appear when the P450(cam)/Pdx complex is formed. These observations are consistent with vibrational mixing and heme geometric distortions upon Pdx binding that are coincident with the increased thiolate electron donation to the heme. The appearance of a mode near 65 cm(-1) in the coherence spectra of the L358P mutant is comparable to the mode at 78 cm(-1) seen in the P450(cam)/Pdx complex and is consistent with the view that the heme and its environment in the L358P mutant are similar to the Pdx-bound native protein. Resonance Raman spectra are presented for both P450(cam) and the L358P

  4. In vivo deuteration of a native bacterial biopolymer for structural elucidation using SANS

    NASA Astrophysics Data System (ADS)

    Holden, P. J.; Russell, R. A.; Stone, D. J. M.; Garvey, C. J.; Foster, L. J. R.

    2004-07-01

    In order to facilitate future structural studies, biodeuteration of bacterial polyhydroxyalkanoates (PHAs) was investigated. We report here the in vivo deuteration of poly 3-hydroxyoctanoate (PHO) produced by its native host, the bacterium Pseudomonas oleovorans. Bacterial biomass was produced in bioreactor studies by growth on hydrogenated substrates and PHO was subsequently produced intracellularly (10-20% w/w) during batch fed growth on deuterated octanoic acid under oxygen limitation. GC-MS analyses of the PHO demonstrated that 13 of the 15 hydrogen atoms had been replaced with deuterium (except in position 3), the remaining two hydrogen presumably being derived from water. A SANS contrast variation study was conducted on whole cells and the results indicate the potential to discriminate inclusion bodies formed from deuterated precursor from an otherwise hydrogenated background.

  5. Unlocking the eukaryotic membrane protein structural proteome

    PubMed Central

    Lee, John Kyongwon; Stroud, Robert Michael

    2012-01-01

    Summary Most of the 231 unique membrane protein structures (as of 3/2010) are of bacterial membrane proteins (MPs) expressed in bacteria, or eukaryotic MPs from natural sources. However eukaryotic membrane proteins, especially those with more than three membrane crossings rarely succumb to any suitable expression in bacterial cells. They typically require expression in eukaryotic cells that can provide appropriate endoplasmic reticulum, chaperones, targeting and post-translational processing. In evidence, only ~20 eukaryotic MP structures have resulted from heterologous expression. This is required for a general approach to target particular human or pathogen membrane proteins of importance to human health. The first of these appeared in 2005. Our review addresses the special issues that pertain to the expression of eukaryotic and human membrane proteins, and recent advances in the tool kit for crystallization and structure determination. PMID:20739007

  6. AMASS: a database for investigating protein structures

    PubMed Central

    Mielke, Clinton J.; Mandarino, Lawrence J.; Dinu, Valentin

    2014-01-01

    Motivation: Modern techniques have produced many sequence annotation databases and protein structure portals, but these Web resources are rarely integrated in ways that permit straightforward exploration of protein functional residues and their co-localization. Results: We have created the AMASS database, which maps 1D sequence annotation databases to 3D protein structures with an intuitive visualization interface. Our platform also provides an analysis service that screens mass spectrometry sequence data for post-translational modifications that reside in functionally relevant locations within protein structures. The system is built on the premise that functional residues such as active sites, cancer mutations and post-translational modifications within proteins may co-localize and share common functions. Availability and implementation: AMASS database is implemented with Biopython and Apache as a freely available Web server at amass-db.org. Contact: clinton.mielke@gmail.com PMID:24497503

  7. Protein structure designability: The consequences of amino-acid alphabet and the underlying energy model

    NASA Astrophysics Data System (ADS)

    Buchler, Nicolas Emile G.

    2001-11-01

    It has been noted by scientists that certain native, protein structures occur more frequently than others in the Protein Data Bank. A variety of models have been developed to explain this phenomenon by considering protein structure "designability". A protein structure is more designable if a larger fraction of all possible sequences can fold into it. Such highly-designable structures are more likely to have been found and maintained through the process of evolution hence they are likely to be over-represented. Proteins are biopolymers and the key to designability lies with understanding the thermodynamic constraints of how a biologically relevant protein sequence folds into a native protein structure. We begin by discussing protein folding in the context of free energy landscapes and phase transitions. The role of protein evolution, critical transitions, such as "freezing" into the native state ( Tf), collapse into molten globules (Ttheta), and glassy phase transitions (Tg), folding-funnels, and free energy landscape measures is a well studied subject of protein folding both computationally and theoretically. In particular, the relationship between folding funnels and relevant phase transition temperatures (T f, Tg) can be related to free energy landscape measures, such as foldability F and energy gap Delta. Using the Random Energy Model, we analytically demonstrate the statistical relationship between F , Delta. Using simplified models of protein structure and interactions, we have explored the relationship between protein structure designability, foldability, interaction parameters, and amino-acid alphabet. We shed new light on which structures are expected to be highly designable, for which types of energy models (solvation or pair-contact), and why these structures are designable. Moreover, we can also understand why free energy landscape measures, such as foldability F and energy gap Delta, must be correlated to protein structure designability. We further

  8. Protein structure database search and evolutionary classification.

    PubMed

    Yang, Jinn-Moon; Tung, Chi-Hua

    2006-01-01

    As more protein structures become available and structural genomics efforts provide structural models in a genome-wide strategy, there is a growing need for fast and accurate methods for discovering homologous proteins and evolutionary classifications of newly determined structures. We have developed 3D-BLAST, in part, to address these issues. 3D-BLAST is as fast as BLAST and calculates the statistical significance (E-value) of an alignment to indicate the reliability of the prediction. Using this method, we first identified 23 states of the structural alphabet that represent pattern profiles of the backbone fragments and then used them to represent protein structure databases as structural alphabet sequence databases (SADB). Our method enhanced BLAST as a search method, using a new structural alphabet substitution matrix (SASM) to find the longest common substructures with high-scoring structured segment pairs from an SADB database. Using personal computers with Intel Pentium4 (2.8 GHz) processors, our method searched more than 10 000 protein structures in 1.3 s and achieved a good agreement with search results from detailed structure alignment methods. [3D-BLAST is available at http://3d-blast.life.nctu.edu.tw]. PMID:16885238

  9. Stand Structural Controls on Evapotranspiration in Native and Invaded Tropical Montane Cloud Forest in Hawai'i

    NASA Astrophysics Data System (ADS)

    Giambelluca, T. W.; Delay, J. K.; Asner, G. P.; Martin, R. E.; Nullet, M. A.; Huang, M.; Mudd, R. G.; Takahashi, M.

    2008-12-01

    Tropical montane cloud forests (TMCFs) in Hawai'i are important zones of water input and stores of critically important native plant and animal species. Invasion by alien tree species threatens these forests and may alter the hydrological services they provide. At two TMCF sites in Hawai'i, one within native Metrosideros polymorpha forest and the other at a site heavily invaded by Psidium cattleianum, we are conducting measurements of stand-level evapotranspiration (ET), transpiration (using sapflow techniques), energy balance, and related processes. Previously presented results showed that ET as a function of available energy was 27% higher at the invaded site than the native site, with the difference rising to 53% during dry- canopy periods. In this presentation, mechanisms for the observed higher ET rate at the invaded site are explored. The difference in measured xylem flow velocities of native and alien trees cannot explain the observed stand level ET difference. Tree basal area is lower at the invaded site than the native site, again contrary to the ET difference. However, the alien trees have much smaller stem diameters, on average, than the native trees, with little or no heartwood. Hence, the cross-sectional xylem area is much greater in the invaded stand, facilitating higher transpiration rates. These results demonstrate the importance of stand structural controls on ET and raise questions about whether higher ET is a transient feature of the succession or a persistent characteristic of invasive trees.

  10. Proteins with Novel Structure, Function and Dynamics

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew

    2014-01-01

    Recently, a small enzyme that ligates two RNA fragments with the rate of 10(exp 6) above background was evolved in vitro (Seelig and Szostak, Nature 448:828-831, 2007). This enzyme does not resemble any contemporary protein (Chao et al., Nature Chem. Biol. 9:81-83, 2013). It consists of a dynamic, catalytic loop, a small, rigid core containing two zinc ions coordinated by neighboring amino acids, and two highly flexible tails that might be unimportant for protein function. In contrast to other proteins, this enzyme does not contain ordered secondary structure elements, such as alpha-helix or beta-sheet. The loop is kept together by just two interactions of a charged residue and a histidine with a zinc ion, which they coordinate on the opposite side of the loop. Such structure appears to be very fragile. Surprisingly, computer simulations indicate otherwise. As the coordinating, charged residue is mutated to alanine, another, nearby charged residue takes its place, thus keeping the structure nearly intact. If this residue is also substituted by alanine a salt bridge involving two other, charged residues on the opposite sides of the loop keeps the loop in place. These adjustments are facilitated by high flexibility of the protein. Computational predictions have been confirmed experimentally, as both mutants retain full activity and overall structure. These results challenge our notions about what is required for protein activity and about the relationship between protein dynamics, stability and robustness. We hypothesize that small, highly dynamic proteins could be both active and fault tolerant in ways that many other proteins are not, i.e. they can adjust to retain their structure and activity even if subjected to mutations in structurally critical regions. This opens the doors for designing proteins with novel functions, structures and dynamics that have not been yet considered.

  11. Microfluidic Diffusion Analysis of the Sizes and Interactions of Proteins under Native Solution Conditions.

    PubMed

    Arosio, Paolo; Müller, Thomas; Rajah, Luke; Yates, Emma V; Aprile, Francesco A; Zhang, Yingbo; Cohen, Samuel I A; White, Duncan A; Herling, Therese W; De Genst, Erwin J; Linse, Sara; Vendruscolo, Michele; Dobson, Christopher M; Knowles, Tuomas P J

    2016-01-26

    Characterizing the sizes and interactions of macromolecules under native conditions is a challenging problem in many areas of molecular sciences, which fundamentally arises from the polydisperse nature of biomolecular mixtures. Here, we describe a microfluidic platform for diffusional sizing based on monitoring micron-scale mass transport simultaneously in space and time. We show that the global analysis of such combined space-time data enables the hydrodynamic radii of individual species within mixtures to be determined directly by deconvoluting average signals into the contributions from the individual species. We demonstrate that the ability to perform rapid noninvasive sizing allows this method to be used to characterize interactions between biomolecules under native conditions. We illustrate the potential of the technique by implementing a single-step quantitative immunoassay that operates on a time scale of seconds and detects specific interactions between biomolecules within complex mixtures. PMID:26678709

  12. Contemporary Methodology for Protein Structure Determination.

    ERIC Educational Resources Information Center

    Hunkapiller, Michael W.; And Others

    1984-01-01

    Describes the nature and capabilities of methods used to characterize protein and peptide structure, indicating that they have undergone changes which have improved the speed, reliability, and applicability of the process. Also indicates that high-performance liquid chromatography and gel electrophoresis have made purifying proteins and peptides a…

  13. PEGylated nanoparticles: protein corona and secondary structure

    NASA Astrophysics Data System (ADS)

    Runa, Sabiha; Hill, Alexandra; Cochran, Victoria L.; Payne, Christine K.

    2014-09-01

    Nanoparticles have important biological and biomedical applications ranging from drug and gene delivery to biosensing. In the presence of extracellular proteins, a "corona" of proteins adsorbs on the surface of the nanoparticles, altering their interaction with cells, including immune cells. Nanoparticles are often functionalized with polyethylene glycol (PEG) to reduce this non-specific adsorption of proteins. To understand the change in protein corona that occurs following PEGylation, we first quantified the adsorption of blood serum proteins on bare and PEGylated gold nanoparticles using gel electrophoresis. We find a threefold decrease in the amount of protein adsorbed on PEGylated gold nanoparticles compared to the bare gold nanoparticles, showing that PEG reduces, but does not prevent, corona formation. To determine if the secondary structure of corona proteins was altered upon adsorption onto the bare and PEGylated gold nanoparticles, we use CD spectroscopy to characterize the secondary structure of bovine serum albumin following incubation with the nanoparticles. Our results show no significant change in protein secondary structure following incubation with bare or PEGylated nanoparticles. Further examination of the secondary structure of bovine serum albumin, α2-macroglobulin, and transferrin in the presence of free PEG showed similar results. These findings provide important insights for the use of PEGylated gold nanoparticles under physiological conditions.

  14. Information-driven structural modelling of protein-protein interactions.

    PubMed

    Rodrigues, João P G L M; Karaca, Ezgi; Bonvin, Alexandre M J J

    2015-01-01

    Protein-protein docking aims at predicting the three-dimensional structure of a protein complex starting from the free forms of the individual partners. As assessed in the CAPRI community-wide experiment, the most successful docking algorithms combine pure laws of physics with information derived from various experimental or bioinformatics sources. Of these so-called "information-driven" approaches, HADDOCK stands out as one of the most successful representatives. In this chapter, we briefly summarize which experimental information can be used to drive the docking prediction in HADDOCK, and then focus on the docking protocol itself. We discuss and illustrate with a tutorial example a "classical" protein-protein docking prediction, as well as more recent developments for modelling multi-body systems and large conformational changes. PMID:25330973

  15. Human complement protein C9 is a calcium binding protein. Structural and functional implications.

    PubMed

    Thielens, N M; Lohner, K; Esser, A F

    1988-05-15

    Human complement protein C9 is shown to be a metalloprotein that binds 1 mol of Ca2+/mol of C9 with a dissociation constant of 3 micron as measured by equilibrium dialysis. Incubation with EDTA removes the bound calcium, resulting in a apoprotein with decreased thermal stability. This loss in stability leads to aggregation and, therefore, to loss of hemolytic activity upon heating to a few degrees above the physiological temperature. Heat-induced aggregation of apoC9 can be prevented by salts that stabilize proteins according to the Hofmeister series of lyotropic ions, suggesting that the ion in native C9 may ligand with more than one structural element or domain of the protein. Ligand blotting indicates that the calcium binding site is located in the amino-terminal half of the protein. Removal of calcium by inclusion of EDTA in assay mixtures has no effect on the hemolytic activity of C9, and its capacity to bind to C8 in solution, or to small unilamellar lipid vesicles at temperatures at or below the physiological range. Although we do not know yet the precise structural and functional role of the bound calcium, it is clear that it provides thermal stability to C9 and it may have a function in regulation of membrane insertion. PMID:3360798

  16. Detection of native protein ions in aqueous solution under ambient conditions by electrospray laser desorption/ionization mass spectrometry.

    PubMed

    Shiea, Jentaie; Yuan, Cheng-Hui; Huang, Min-Zong; Cheng, Sy-Chyi; Ma, Ya-Lin; Tseng, Wei-Lung; Chang, Hui-Chiu; Hung, Wen-Chun

    2008-07-01

    Liquid electrospray laser desorption/ionization (ELDI) mass spectrometry allows desorption and ionization of proteins directly from aqueous solutions and biological fluids under ambient conditions. Native protein ions such as those of myoglobin, cytochrome c, and hemoglobin were obtained. A droplet (ca. 5 microL) containing the protein molecules and micrometer-sized particles (e.g., carbon graphite powder) is irradiated with a pulsed UV laser. The laser energy adsorbed by the inert particles is transferred to the surrounding solvent and protein molecules, leading to their desorption; the desorbed gaseous molecules are then postionized within an electrospray (ESI) plume to generate the ESI-like protein ions. With the use of this technique, we detected only the protonated protein ions in various biological fluids (including human tears, cow milk, serum, and bacterial extracts) without interference from their corresponding sodiated or potassiated adduct ions. In addition, we rapidly quantified the levels of glycosylated hemoglobin present in drops of whole blood obtained from diabetic patients without the need of sample pretreatment. PMID:18510347

  17. Exploring representations of protein structure for automated remote homology detection and mapping of protein structure space

    PubMed Central

    2014-01-01

    Background Due to rapid sequencing of genomes, there are now millions of deposited protein sequences with no known function. Fast sequence-based comparisons allow detecting close homologs for a protein of interest to transfer functional information from the homologs to the given protein. Sequence-based comparison cannot detect remote homologs, in which evolution has adjusted the sequence while largely preserving structure. Structure-based comparisons can detect remote homologs but most methods for doing so are too expensive to apply at a large scale over structural databases of proteins. Recently, fragment-based structural representations have been proposed that allow fast detection of remote homologs with reasonable accuracy. These representations have also been used to obtain linearly-reducible maps of protein structure space. It has been shown, as additionally supported from analysis in this paper that such maps preserve functional co-localization of the protein structure space. Methods Inspired by a recent application of the Latent Dirichlet Allocation (LDA) model for conducting structural comparisons of proteins, we propose higher-order LDA-obtained topic-based representations of protein structures to provide an alternative route for remote homology detection and organization of the protein structure space in few dimensions. Various techniques based on natural language processing are proposed and employed to aid the analysis of topics in the protein structure domain. Results We show that a topic-based representation is just as effective as a fragment-based one at automated detection of remote homologs and organization of protein structure space. We conduct a detailed analysis of the information content in the topic-based representation, showing that topics have semantic meaning. The fragment-based and topic-based representations are also shown to allow prediction of superfamily membership. Conclusions This work opens exciting venues in designing novel

  18. Structural Characteristics of Novel Protein Folds

    PubMed Central

    Fernandez-Fuentes, Narcis; Dybas, Joseph M.; Fiser, Andras

    2010-01-01

    Folds are the basic building blocks of protein structures. Understanding the emergence of novel protein folds is an important step towards understanding the rules governing the evolution of protein structure and function and for developing tools for protein structure modeling and design. We explored the frequency of occurrences of an exhaustively classified library of supersecondary structural elements (Smotifs), in protein structures, in order to identify features that would define a fold as novel compared to previously known structures. We found that a surprisingly small set of Smotifs is sufficient to describe all known folds. Furthermore, novel folds do not require novel Smotifs, but rather are a new combination of existing ones. Novel folds can be typified by the inclusion of a relatively higher number of rarely occurring Smotifs in their structures and, to a lesser extent, by a novel topological combination of commonly occurring Smotifs. When investigating the structural features of Smotifs, we found that the top 10% of most frequent ones have a higher fraction of internal contacts, while some of the most rare motifs are larger, and contain a longer loop region. PMID:20421995

  19. 3D protein structure prediction using Imperialist Competitive algorithm and half sphere exposure prediction.

    PubMed

    Khaji, Erfan; Karami, Masoumeh; Garkani-Nejad, Zahra

    2016-02-21

    Predicting the native structure of proteins based on half-sphere exposure and contact numbers has been studied deeply within recent years. Online predictors of these vectors and secondary structures of amino acids sequences have made it possible to design a function for the folding process. By choosing variant structures and directs for each secondary structure, a random conformation can be generated, and a potential function can then be assigned. Minimizing the potential function utilizing meta-heuristic algorithms is the final step of finding the native structure of a given amino acid sequence. In this work, Imperialist Competitive algorithm was used in order to accelerate the process of minimization. Moreover, we applied an adaptive procedure to apply revolutionary changes. Finally, we considered a more accurate tool for prediction of secondary structure. The results of the computational experiments on standard benchmark show the superiority of the new algorithm over the previous methods with similar potential function. PMID:26718864

  20. Protein Block Expert (PBE): a web-based protein structure analysis server using a structural alphabet.

    PubMed

    Tyagi, M; Sharma, P; Swamy, C S; Cadet, F; Srinivasan, N; de Brevern, A G; Offmann, B

    2006-07-01

    Encoding protein 3D structures into 1D string using short structural prototypes or structural alphabets opens a new front for structure comparison and analysis. Using the well-documented 16 motifs of Protein Blocks (PBs) as structural alphabet, we have developed a methodology to compare protein structures that are encoded as sequences of PBs by aligning them using dynamic programming which uses a substitution matrix for PBs. This methodology is implemented in the applications available in Protein Block Expert (PBE) server. PBE addresses common issues in the field of protein structure analysis such as comparison of proteins structures and identification of protein structures in structural databanks that resemble a given structure. PBE-T provides facility to transform any PDB file into sequences of PBs. PBE-ALIGNc performs comparison of two protein structures based on the alignment of their corresponding PB sequences. PBE-ALIGNm is a facility for mining SCOP database for similar structures based on the alignment of PBs. Besides, PBE provides an interface to a database (PBE-SAdb) of preprocessed PB sequences from SCOP culled at 95% and of all-against-all pairwise PB alignments at family and superfamily levels. PBE server is freely available at http://bioinformatics.univ-reunion.fr/PBE/. PMID:16844973

  1. Artefacts and biases affecting the evaluation of scoring functions on decoy sets for protein structure prediction

    PubMed Central

    Handl, Julia; Knowles, Joshua; Lovell, Simon C.

    2009-01-01

    Motivation: Decoy datasets, consisting of a solved protein structure and numerous alternative native-like structures, are in common use for the evaluation of scoring functions in protein structure prediction. Several pitfalls with the use of these datasets have been identified in the literature, as well as useful guidelines for generating more effective decoy datasets. We contribute to this ongoing discussion an empirical assessment of several decoy datasets commonly used in experimental studies. Results: We find that artefacts and sampling issues in the large majority of these data make it trivial to discriminate the native structure. This underlines that evaluation based on the rank/z-score of the native is a weak test of scoring function performance. Moreover, sampling biases present in the way decoy sets are generated or used can strongly affect other types of evaluation measures such as the correlation between score and root mean squared deviation (RMSD) to the native. We demonstrate how, depending on type of bias and evaluation context, sampling biases may lead to both over- or under-estimation of the quality of scoring terms, functions or methods. Availability: Links to the software and data used in this study are available at http://dbkgroup.org/handl/decoy_sets. Contact: simon.lovell@manchester.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19297350

  2. Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection

    PubMed Central

    Alegre, Ana Claudia Paiva; Oliveira, Aline Ferreira; Dos Reis Almeida, Fausto Bruno; Roque-Barreira, Maria Cristina; Hanna, Ebert Seixas

    2014-01-01

    Background Paracoccin is a dual-function protein of the yeast Paracoccidioides brasiliensis that has lectin properties and N-acetylglucosaminidase activities. Proteomic analysis of a paracoccin preparation from P. brasiliensis revealed that the sequence matched that of the hypothetical protein encoded by PADG-3347 of isolate Pb-18, with a polypeptide sequence similar to the family 18 endochitinases. These endochitinases are multi-functional proteins, with distinct lectin and enzymatic domains. Methodology/principal findings The multi-exon assembly and the largest exon of the predicted ORF (PADG-3347), was cloned and expressed in Escherichia coli cells, and the features of the recombinant proteins were compared to those of the native paracoccin. The multi-exon protein was also used for protection assays in a mouse model of paracoccidioidomycosis. Conclusions/Significance Our results showed that the recombinant protein reproduced the biological properties described for the native protein—including binding to laminin in a manner that is dependent on carbohydrate recognition—showed N-acetylglucosaminidase activity, and stimulated murine peritoneal macrophages to produce high levels of TNF-α and nitric oxide. Considering the immunomodulatory potential of glycan-binding proteins, we also investigated whether prophylactic administration of recombinant paracoccin affected the course of experimental paracoccidioidomycosis in mice. In comparison to animals injected with vehicle (controls), mice treated with recombinant paracoccin displayed lower pulmonary fungal burdens and reduced pulmonary granulomas. These protective effects were associated with augmented pulmonary levels of IL-12 and IFN-γ. We also observed that injection of paracoccin three days before challenge was the most efficient administration protocol, as the induced Th1 immunity was balanced by high levels of pulmonary IL-10, which may prevent the tissue damage caused by exacerbated inflammation. The

  3. Structure and protein-protein interactions of methanol dehydrogenase from Methylococcus capsulatus (Bath).

    PubMed

    Culpepper, Megen A; Rosenzweig, Amy C

    2014-10-01

    In the initial steps of their metabolic pathway, methanotrophic bacteria oxidize methane to methanol with methane monooxygenases (MMOs) and methanol to formaldehyde with methanol dehydrogenases (MDHs). Several lines of evidence suggest that the membrane-bound or particulate MMO (pMMO) and MDH interact to form a metabolic supercomplex. To further investigate the possible existence of such a supercomplex, native MDH from Methylococcus capsulatus (Bath) has been purified and characterized by size exclusion chromatography with multi-angle light scattering and X-ray crystallography. M. capsulatus (Bath) MDH is primarily a dimer in solution, although an oligomeric species with a molecular mass of ∼450-560 kDa forms at higher protein concentrations. The 2.57 Å resolution crystal structure reveals an overall fold and α2β2 dimeric architecture similar to those of other MDH structures. In addition, biolayer interferometry studies demonstrate specific protein-protein interactions between MDH and M. capsulatus (Bath) pMMO as well as between MDH and the truncated recombinant periplasmic domains of M. capsulatus (Bath) pMMO (spmoB). These interactions exhibit KD values of 833 ± 409 nM and 9.0 ± 7.7 μM, respectively. The biochemical data combined with analysis of the crystal lattice interactions observed in the MDH structure suggest a model in which MDH and pMMO associate not as a discrete, stoichiometric complex but as a larger assembly scaffolded by the intracytoplasmic membranes. PMID:25185034

  4. Protein GB1 Folding and Assembly from Structural Elements

    PubMed Central

    Bauer, Mikael C.; Xue, Wei-Feng; Linse, Sara

    2009-01-01

    Folding of the Protein G B1 domain (PGB1) shifts with increasing salt concentration from a cooperative assembly of inherently unstructured subdomains to an assembly of partly pre-folded structures. The salt-dependence of pre-folding contributes to the stability minimum observed at physiological salt conditions. Our conclusions are based on a study in which the reconstitution of PGB1 from two fragments was studied as a function of salt concentrations and temperature using circular dichroism spectroscopy. Salt was found to induce an increase in β-hairpin structure for the C-terminal fragment (residues 41 – 56), whereas no major salt effect on structure was observed for the isolated N-terminal fragment (residues 1 – 41). In line with the increasing evidence on the interrelation between fragment complementation and stability of the corresponding intact protein, we also find that salt effects on reconstitution can be predicted from salt dependence of the stability of the intact protein. Our data show that our variant (which has the mutations T2Q, N8D, N37D and reconstitutes in a manner similar to the wild type) displays the lowest equilibrium association constant around physiological salt concentration, with higher affinity observed both at lower and higher salt concentration. This corroborates the salt effects on the stability towards denaturation of the intact protein, for which the stability at physiological salt is lower compared to both lower and higher salt concentrations. Hence we conclude that reconstitution reports on molecular factors that govern the native states of proteins. PMID:19468325

  5. The activity of protein phosphatase 5 towards native clients is modulated by the middle- and C-terminal domains of Hsp90

    PubMed Central

    Haslbeck, Veronika; Eckl, Julia M.; Drazic, Adrian; Rutz, Daniel A.; Lorenz, Oliver R.; Zimmermann, Kerstin; Kriehuber, Thomas; Lindemann, Claudia; Madl, Tobias; Richter, Klaus

    2015-01-01

    Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex. PMID:26593036

  6. Quantitative proteomic dissection of a native 14-3-3ε interacting protein complex associated with hepatocellular carcinoma.

    PubMed

    Bai, Chen; Tang, Siwei; Bai, Chen; Chen, Xian

    2014-04-01

    The 14-3-3 proteins regulate diverse biological processes that are implicated in cancer development, and seven 14-3-3 isoforms were identified with isoform-specific roles in different human tumors. In our previous work, we dissected the interactome of 14-3-3ε formed during the DNA damage response in a hepatocellular carcinoma (HCC) cell using an AACT/SILAC-based quantitative proteomic approach. In this study, we used a similar proteomic approach to profile/identify the 14-3-3ε interactome formed in native HCC cells. Functional categorization and data-dependent network analysis of the native HCC-specific 14-3-3ε interactome revealed that 14-3-3ε is involved in the regulation of multiple biological processes (BPs)/pathways, including cell cycle control, apoptosis, signal transduction, transport, cell adhesion, carbohydrate metabolism, and nucleic acid metabolism. Biological validation further supports that 14-3-3ε, via association with multiple BP/pathway-specific proteins, coordinates the regulation of proliferation, survival, and metastasis of HCC. The findings in this study, together with those of our previous study, provide an extensive profile of the 14-3-3ε interaction network in HCC cells, which should be valuable for understanding the pathology of HCC and HCC therapy. PMID:24363202

  7. Synthetic RNA–protein modules integrated with native translation mechanisms to control gene expression in malaria parasites

    PubMed Central

    Ganesan, Suresh M.; Falla, Alejandra; Goldfless, Stephen J.; Nasamu, Armiyaw S.; Niles, Jacquin C.

    2016-01-01

    Synthetic posttranscriptional regulation of gene expression is important for understanding fundamental biology and programming new cellular processes in synthetic biology. Previous strategies for regulating translation in eukaryotes have focused on disrupting individual steps in translation, including initiation and mRNA cleavage. In emphasizing modularity and cross-organism functionality, these systems are designed to operate orthogonally to native control mechanisms. Here we introduce a broadly applicable strategy for robustly controlling protein translation by integrating synthetic translational control via a small-molecule-regulated RNA–protein module with native mechanisms that simultaneously regulate multiple facets of cellular RNA fate. We demonstrate that this strategy reduces ‘leakiness' to improve overall expression dynamic range, and can be implemented without sacrificing modularity and cross-organism functionality. We illustrate this in Saccharomyces cerevisae and the non-model human malarial parasite, Plasmodium falciparum. Given the limited functional genetics toolkit available for P. falciparum, we establish the utility of this strategy for defining essential genes. PMID:26925876

  8. Resolution of 16 to 20 chlorophyll-protein complexes using a low ionic strength native green gel system.

    PubMed

    Allen, K D; Staehelin, L A

    1991-04-01

    Conventional native "green gel" systems resolve at most 10 chlorophyll-protein complexes from thylakoid membranes of higher plants and green algae. Such analyses suggest a simplicity of the thylakoid membrane that is not supported by a growing body of evidence on the heterogeneity of photosystems I and II (PSI and PSII) and their associated antennae (LHCI and LHCII). We report here the development and characterization of a low ionic strength native "green gel" system that resolves from 16 to 20, mostly large chlorophyll-protein complexes from a variety of higher plant and green algal species with very little release of free pigment. In Chlamydomonas, this system resolves multiple PSI-LHCI complexes, multiple PSII-LHCII complexes, four oligomeric LHCII complexes, as well as several low electrophoretic mobility reaction center complexes, and a number of small complexes. We have obtained similar resolution with a large number of higher plant and green algal species. We also demonstrate how this system can be used as a sort of "fingerprinting" technique to distinguish thylakoids of different species, and for the analysis of photosynthetic mutants, using the chlorophyll b-less chlorina f2 mutant of barley as an example. PMID:1867380

  9. Resolution-enhanced native acidic gel electrophoresis: a method for resolving, sizing, and quantifying prion protein oligomers.

    PubMed

    Ladner, Carol L; Wishart, David S

    2012-07-01

    The formation of β-sheet-rich prion protein (PrP(β)) oligomers from native or cellular PrP(c) is thought to be a key step in the development of prion diseases. To assist in this characterization process we have developed a rapid and remarkably high resolution gel electrophoresis technique called RENAGE (resolution-enhanced native acidic gel electrophoresis) for separating, sizing, and quantifying oligomeric PrP(β) complexes. PrP(β) oligomers formed via either urea/salt or acid conversion can be resolved by RENAGE into a clear set of oligomeric bands differing by just one subunit. Calibration of the size of the PrP(β) oligomer bands was made possible with a cross-linked mouse PrP(90-232) ladder (1- to 11-mer) generated using ruthenium bipyridyl-based photoinduced cross-linking of unmodified proteins (PICUP). This PrP PICUP ladder allowed the size and abundance of PrP(β) oligomers formed from urea/salt and acid conversion to be determined. This distribution consists of 7-, 8-, 9-, 10-, and 11-mers, with the most abundant species being the 8-mer. The high-resolution separation afforded by RENAGE has allowed us to investigate distinctive size and population changes in PrP(β) oligomers formed under various conversion conditions, with various construct lengths, from various species or in the presence of anti-prion compounds. PMID:22490465

  10. Quantitative determination of native proteins in individual human erythrocytes by capillary zone electrophoresis with laser-induced fluorescence detection

    SciTech Connect

    Lee, T.T.; Yeung, E.S. Iowa State Univ., Ames )

    1992-12-01

    Intracellular fluid within single human erythrocytes is analyzed by capillary electrophoresis with laser-excited native protein fluorescence. Good signal-to-noise is achieved, allowing even minor components to be quantified. Non-Gaussian distributions were found for total protein, fraction carbonic anhydrase, fraction hemoglobin A[sub 0], and an unidentified component. Variations among a group of 29 cells for each quantity are as much as 1 order of magnitude, even though erythrocytes are known to be fairly homogeneous in size distribution. Variations in fraction hemoglobin A[sub 0] reflect differences in in vitro oxidation rates to methemoglobin. A positive correlation was observed between carbonic anhydrase and hemoglobin A[sub 0] for individual cells. This is consistent with the presence of erythrocytes of different ages within the population, with the older cells being less capable of maintaining enzyme activity and preventing oxidative damage. 35 refs., 10 figs., 1 tab.

  11. Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.

    SciTech Connect

    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.

  12. 3Drefine: Consistent Protein Structure Refinement by Optimizing Hydrogen Bonding Network and Atomic-Level Energy Minimization

    PubMed Central

    Bhattacharya, Debswapna; Cheng, Jianlin

    2013-01-01

    One of the major limitations of computational protein structure prediction is the deviation of predicted models from their experimentally derived true, native structures. The limitations often hinder the possibility of applying computational protein structure prediction methods in biochemical assignment and drug design that are very sensitive to structural details. Refinement of these low-resolution predicted models to high-resolution structures close to the native state, however, has proven to be extremely challenging. Thus, protein structure refinement remains a largely unsolved problem. Critical assessment of techniques for protein structure prediction (CASP) specifically indicated that most predictors participating in the refinement category still did not consistently improve model quality. Here, we propose a two-step refinement protocol, called 3Drefine, to consistently bring the initial model closer to the native structure. The first step is based on optimization of hydrogen bonding (HB) network and the second step applies atomic-level energy minimization on the optimized model using a composite physics and knowledge-based force fields. The approach has been evaluated on the CASP benchmark data and it exhibits consistent improvement over the initial structure in both global and local structural quality measures. 3Drefine method is also computationally inexpensive, consuming only few minutes of CPU time to refine a protein of typical length (300 residues). PMID:22927229

  13. Purification and in vitro activities of the native nitrogen fixation control proteins NifA and NifL.

    PubMed

    Austin, S; Buck, M; Cannon, W; Eydmann, T; Dixon, R

    1994-06-01

    The prokaryotic enhancer-binding protein NifA stimulates transcription at a distance by binding to sequences upstream of nitrogen fixation (nif) promoters and catalyzing the formation of open promoter complexes by RNA polymerase containing the alternative sigma factor, sigma 54. The activity of NifA in vivo is modulated by the negative regulatory protein NifL in response to environmental oxygen and fixed nitrogen. To date, a detailed biochemical analysis of these proteins from the model diazotroph Klebsiella pneumoniae has been hindered by their insolubility. We have now purified NifA and NifL from Azotobacter vinelandii in their native form. NifA is competent in specific DNA binding, transcriptional activation, and response to negative regulation by NifL in vitro. In contrast to the conserved mechanism of phosphotransfer demonstrated by other two-component regulatory systems, our results support a model in which NifL regulates the activity of NifA via a protein-protein steric block interaction rather than a catalytic modification of NifA. PMID:8206822

  14. Rapid search for tertiary fragments reveals protein sequence-structure relationships.

    PubMed

    Zhou, Jianfu; Grigoryan, Gevorg

    2015-04-01

    Finding backbone substructures from the Protein Data Bank that match an arbitrary query structural motif, composed of multiple disjoint segments, is a problem of growing relevance in structure prediction and protein design. Although numerous protein structure search approaches have been proposed, methods that address this specific task without additional restrictions and on practical time scales are generally lacking. Here, we propose a solution, dubbed MASTER, that is both rapid, enabling searches over the Protein Data Bank in a matter of seconds, and provably correct, finding all matches below a user-specified root-mean-square deviation cutoff. We show that despite the potentially exponential time complexity of the problem, running times in practice are modest even for queries with many segments. The ability to explore naturally plausible structural and sequence variations around a given motif has the potential to synthesize its design principles in an automated manner; so we go on to illustrate the utility of MASTER to protein structural biology. We demonstrate its capacity to rapidly establish structure-sequence relationships, uncover the native designability landscapes of tertiary structural motifs, identify structural signatures of binding, and automatically rewire protein topologies. Given the broad utility of protein tertiary fragment searches, we hope that providing MASTER in an open-source format will enable novel advances in understanding, predicting, and designing protein structure. PMID:25420575

  15. Temperature induced structural transitions from native to unfolded aggregated states of tobacco etch virus protease

    NASA Astrophysics Data System (ADS)

    Zhu, Guo-Fei; Ren, Si-Yan; Xi, Lei; Du, Lin-Fang; Zhu, Xiao-Feng

    2015-02-01

    Tobacco etch virus protease (TEVp) is widely used to remove fusion tags from recombinant proteins because of its high and unique specificity. This work describes the conformational and the thermodynamic properties in the unfolding/refolding process of TEVp3M (three-point mutant: L56V/S135G/S219V) induced by temperature. With temperature increasing from 20 to 100 °C, the CD spectra showed a transition trend from α-helix to β-sheet, and the fluorescence emission, synchronous fluorescence, ANS and RLS spectroscopy consistently revealed that the temperature-induced unfolding process behaved in a three-state manner, for there was a relatively stable intermediate state observed around 50 °C. The reversibility of thermal unfolding of TEVp3M further showed that the transition from the native to the intermediate state was reversible (below 50 °C), however the transition from the intermediate to the unfolded state was irreversible (above 60 °C). Moreover, aggregates were observed above 60 °C as revealed by SDS-PAGE, Thioflavin-T fluorescence and Congo red absorbance.

  16. Simplified protein models can rival all atom simulations in predicting folding pathways and structure

    PubMed Central

    Adhikari, Aashish N.; Freed, Karl F.; Sosnick, Tobin R.

    2014-01-01

    We demonstrate the ability of simultaneously determining a protein’s folding pathway and structure using a properly formulated model without prior knowledge of the native structure. Our model employs a natural coordinate system for describing proteins and a search strategy inspired by the observation that real proteins fold in a sequential fashion by incrementally stabilizing native-like substructures or "foldons". Comparable folding pathways and structures are obtained for the twelve proteins recently studied using atomistic molecular dynamics simulations [K. Lindorff-Larsen, S. Piana, R.O. Dror, D. E. Shaw, Science 334, 517 (2011)], with our calculations running several orders of magnitude faster. We find that native-like propensities in the unfolded state do not necessarily determine the order of structure formation, a departure from a major conclusion of the MD study. Instead, our results support a more expansive view wherein intrinsic local structural propensities may be enhanced or overridden in the folding process by environmental context. The success of our search strategy validates it as an expedient mechanism for folding both in silico and in vivo. PMID:23889448

  17. Protein folding: When ribosomes pick the structure

    NASA Astrophysics Data System (ADS)

    Sivertsson, Elin M.; Itzhaki, Laura S.

    2014-05-01

    Anfinsen's principle tells us that the folded structure of a protein is determined solely by its sequence. Now, it has been shown that the rate at which a polypeptide chain is synthesized in the cell can affect which of two alternative folded structures it adopts.

  18. Automated protein motif generation in the structure-based protein function prediction tool ProMOL.

    PubMed

    Osipovitch, Mikhail; Lambrecht, Mitchell; Baker, Cameron; Madha, Shariq; Mills, Jeffrey L; Craig, Paul A; Bernstein, Herbert J

    2015-12-01

    ProMOL, a plugin for the PyMOL molecular graphics system, is a structure-based protein function prediction tool. ProMOL includes a set of routines for building motif templates that are used for screening query structures for enzyme active sites. Previously, each motif template was generated manually and required supervision in the optimization of parameters for sensitivity and selectivity. We developed an algorithm and workflow for the automation of motif building and testing routines in ProMOL. The algorithm uses a set of empirically derived parameters for optimization and requires little user intervention. The automated motif generation algorithm was first tested in a performance comparison with a set of manually generated motifs based on identical active sites from the same 112 PDB entries. The two sets of motifs were equally effective in identifying alignments with homologs and in rejecting alignments with unrelated structures. A second set of 296 active site motifs were generated automatically, based on Catalytic Site Atlas entries with literature citations, as an expansion of the library of existing manually generated motif templates. The new motif templates exhibited comparable performance to the existing ones in terms of hit rates against native structures, homologs with the same EC and Pfam designations, and randomly selected unrelated structures with a different EC designation at the first EC digit, as well as in terms of RMSD values obtained from local structural alignments of motifs and query structures. This research is supported by NIH grant GM078077. PMID:26573864

  19. An All-Atom Force Field for Tertiary Structure Prediction of Helical Proteins

    PubMed Central

    Herges, T.; Wenzel, W.

    2004-01-01

    We have developed an all-atom free-energy force field (PFF01) for protein tertiary structure prediction. PFF01 is based on physical interactions and was parameterized using experimental structures of a family of proteins believed to span a wide variety of possible folds. It contains empirical, although sequence-independent terms for hydrogen bonding. Its solvent-accessible surface area solvent model was first fit to transfer energies of small peptides. The parameters of the solvent model were then further optimized to stabilize the native structure of a single protein, the autonomously folding villin headpiece, against competing low-energy decoys. Here we validate the force field for five nonhomologous helical proteins with 20–60 amino acids. For each protein, decoys with 2–3 Å backbone root mean-square deviation and correct experimental Cβ–Cβ distance constraints emerge as those with the lowest energy. PMID:15507688

  20. Conformational flexibility and structural dynamics in GPCR-mediated G protein activation: a perspective

    PubMed Central

    Preininger, Anita M.; Meiler, Jens; Hamm, Heidi

    2013-01-01

    Structure and dynamics of G proteins and their cognate receptors, both alone and in complex, are becoming increasingly accessible to experimental techniques. Understanding the conformational changes and timelines which govern these changes can lead to new insights into the processes of ligand binding and associated G protein activation. Experimental systems may involve the use of, or otherwise stabilize, non-native environments. This can complicate our understanding of structural and dynamical features of processes such as the ionic lock, Tryptophan toggle, and G protein flexibility. While elements in the receptor’s transmembrane helices and the C-terminal α5 helix of Gα undergo well defined structural changes, regions subject to conformational flexibility may be important in fine-tuning the interactions between activated receptors and G proteins. The pairing of computational and experimental approaches will continue to provide powerful tools to probe the conformation and dynamics of receptor-mediated G protein activation. PMID:23602809

  1. Structural characterization of the photoswitchable fluorescent protein Dronpa-C62S

    SciTech Connect

    Nam, Ki-Hyun; Kwon, Oh Yeun; Sugiyama, Kanako; Lee, Won-Ho; Kim, Young Kwan; Song, Hyun Kyu; Kim, Eunice Eunkyung; Park, Sam-Yong; Jeon, Hyesung . E-mail: hjeon@kist.re.kr; Hwang, Kwang Yeon . E-mail: chahong@korea.ac.kr

    2007-03-23

    The photoswitching behavior of green fluorescent proteins (GFPs) or GFP-like proteins is increasingly recognized as a new technique for optical marking. Recently, Ando and his colleagues developed a new green fluorescent protein Dronpa, which possesses the unique photochromic property of being photoswitchable in a non-destructive manner. To better understand this mechanism, we determined the crystal structures of a new GFP Dronpa and its mutant C62S, at 1.9 A and 1.8 A, respectively. Determination of the structures demonstrates that a unique hydrogen-bonding network and the sulfur atom of the chromophore are critical to the photoswitching property of Dronpa. Reversible photoswitching was lost in cells expressing the Dronpa-C62S upon repetitive irradiation compared to the native protein. Structural and mutational analyses reveal the chemical basis for the functional properties of photoswitchable fluorescent proteins and provide the basis for subsequent coherent engineering of this subfamily of Dronpa homolog's.

  2. Protein structural domains: definition and prediction.

    PubMed

    Ezkurdia, Iakes; Tress, Michael L

    2011-11-01

    Recognition and prediction of structural domains in proteins is an important part of structure and function prediction. This unit lists the range of tools available for domain prediction, and describes sequence and structural analysis tools that complement domain prediction methods. Also detailed are the basic domain prediction steps, along with suggested strategies for different protein sequences and potential pitfalls in domain boundary prediction. The difficult problem of domain orientation prediction is also discussed. All the resources necessary for domain boundary prediction are accessible via publicly available Web servers and databases and do not require computational expertise. PMID:22045561

  3. Factor Structure of the Wechsler Intelligence Scales for Children-Fourth Edition among Referred Native American Students

    ERIC Educational Resources Information Center

    Nakano, Selena; Watkins, Marley W.

    2013-01-01

    The Native American population is severely underrepresented in empirical test validity research despite being overrepresented in special education programs and at increased risk for psychoeducational evaluation. The structural validity of the Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV) was investigated with a sample of 176,…

  4. Protein structure, spectral properties, and photobiological function of lumazine protein

    NASA Astrophysics Data System (ADS)

    Lee, John W.; Bradley, Elizabeth A.; O'Kane, Dennis J.

    1992-04-01

    Protein sequence analysis, nuclear magnetic resonance, and fluorescence dynamics have been applied in a determination of the interactions of the lumazine derivative with the amino acid residues in the proposed ligand binding site of lumazine protein. It is these interactions that `tune' the excited state properties of the bound lumazine so that it can perform its photobiological function as the emitter of bioluminescence in Photobacterium species. A three- way sequence alignment shows that lumazine protein is homologous with the yellow- fluorescent protein of Vibrio fischeri and the riboflavin synthase from Bacillus subtilis. This last enzyme is ubiquitous in procaryotes, and utilizes two of these same lumazines as substrates for the production of riboflavin. By analogy with riboflavin synthase, a short sequence in the lumazine protein has been suggested as the ligand binding site. In riboflavin synthase there is a second binding site, but this is absent in lumazine protein, thus negating any synthase activity for this protein. Hydrogen bonds to the residues in this binding domain and `freeze' the lumazine structure into the highly polar tautomer deduced from NMR evidence. This also accounts for the rigidity of binding shown by the 23 ns (2 degree(s)C) rotational correlation time of the bound ligand as well as the strong blue shift of the fluorescence maximum, from 490 nm free to 475 nm when bound.

  5. Benchmarking Inverse Statistical Approaches for Protein Structure and Design with Exactly Solvable Models

    PubMed Central

    Jacquin, Hugo; Shakhnovich, Eugene; Cocco, Simona; Monasson, Rémi

    2016-01-01

    Inverse statistical approaches to determine protein structure and function from Multiple Sequence Alignments (MSA) are emerging as powerful tools in computational biology. However the underlying assumptions of the relationship between the inferred effective Potts Hamiltonian and real protein structure and energetics remain untested so far. Here we use lattice protein model (LP) to benchmark those inverse statistical approaches. We build MSA of highly stable sequences in target LP structures, and infer the effective pairwise Potts Hamiltonians from those MSA. We find that inferred Potts Hamiltonians reproduce many important aspects of ‘true’ LP structures and energetics. Careful analysis reveals that effective pairwise couplings in inferred Potts Hamiltonians depend not only on the energetics of the native structure but also on competing folds; in particular, the coupling values reflect both positive design (stabilization of native conformation) and negative design (destabilization of competing folds). In addition to providing detailed structural information, the inferred Potts models used as protein Hamiltonian for design of new sequences are able to generate with high probability completely new sequences with the desired folds, which is not possible using independent-site models. Those are remarkable results as the effective LP Hamiltonians used to generate MSA are not simple pairwise models due to the competition between the folds. Our findings elucidate the reasons for the success of inverse approaches to the modelling of proteins from sequence data, and their limitations. PMID:27177270

  6. SCOP: a structural classification of proteins database.

    PubMed Central

    Hubbard, T J; Murzin, A G; Brenner, S E; Chothia, C

    1997-01-01

    The Structural Classification of Proteins (SCOP) database provides a detailed and comprehensive description of the relationships of all known proteins structures. The classification is on hierarchical levels: the first two levels, family and superfamily, describe near and far evolutionary relationships; the third, fold, describes geometrical relationships. The distinction between evolutionary relationships and those that arise from the physics and chemistry of proteins is a feature that is unique to this database, so far. SCOP also provides for each structure links to atomic co-ordinates, images of the structures, interactive viewers, sequence data, data on any conformational changes related to function and literature references. The database is freely accessible on the World Wide Web (WWW) with an entry point at URL http://scop.mrc-lmb.cam.ac.uk/scop/ PMID:9016544

  7. Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations.

    PubMed

    Barnett, Gregory V; Qi, Wei; Amin, Samiul; Neil Lewis, E; Roberts, Christopher J

    2015-12-01

    Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity. This report investigates non-native aggregation of a model protein, α-chymotrypsinogen, under accelerated conditions at elevated protein concentrations. Far-UV circular dichroism and Raman scattering show structural changes during aggregation. Size exclusion chromatography and laser light scattering are used to monitor the progression of aggregate growth and monomer loss. Monomer loss is concomitant with increased β-sheet structures as monomers are added to aggregates, which illustrate a transition from a native monomeric state to an aggregate state. Aggregates grow predominantly through monomer-addition, resulting in a semi-flexible polymer morphology. Analysis of aggregation growth kinetics shows that pH strongly affects the characteristic timescales for nucleation (τn) and growth (τg), while the initial protein concentration has only minor effects on τn or τg. Low-shear viscosity measurements follow a common scaling relationship between average aggregate molecular weight (Mw(agg)) and concentration (σ), which is consistent with semi-dilute polymer-solution theory. The results establish a link between aggregate growth mechanisms, which couple Mw(agg) and σ, to increases in solution viscosity even at these intermediate protein concentrations (less than 3w/v %). PMID:26284891

  8. Recurrent Structural Motifs in Non-Homologous Protein Structures

    PubMed Central

    Johansson, Maria U.; Zoete, Vincent; Guex, Nicolas

    2013-01-01

    We have extracted an extensive collection of recurrent structural motifs (RSMs), which consist of sequentially non-contiguous structural motifs (4–6 residues), each of which appears with very similar conformation in three or more mutually unrelated protein structures. We find that the proteins in our set are covered to a substantial extent by the recurrent non-contiguous structural motifs, especially the helix and strand regions. Computational alanine scanning calculations indicate that the average folding free energy changes upon alanine mutation for most types of non-alanine residues are higher for amino acids that are present in recurrent structural motifs than for amino acids that are not. The non-alanine amino acids that are most common in the recurrent structural motifs, i.e., phenylalanine, isoleucine, leucine, valine and tyrosine and the less abundant methionine and tryptophan, have the largest folding free energy changes. This indicates that the recurrent structural motifs, as we define them, describe recurrent structural patterns that are important for protein stability. In view of their properties, such structural motifs are potentially useful for inter-residue contact prediction and protein structure refinement. PMID:23574940

  9. Structure and Non-Structure of Centrosomal Proteins

    PubMed Central

    Bertero, Michela G.; Boutin, Maïlys; Guarín, Nayibe; Méndez-Giraldez, Raúl; Nuñez, Alfonso; Pedrero, Juan G.; Redondo, Pilar; Sanz, María; Speroni, Silvia; Teichert, Florian; Bruix, Marta; Carazo, José M.; Gonzalez, Cayetano; Reina, José; Valpuesta, José M.; Vernos, Isabelle; Zabala, Juan C.; Montoya, Guillermo; Coll, Miquel; Bastolla, Ugo; Serrano, Luis

    2013-01-01

    Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php. PMID:23671615

  10. Human usage in the native range may determine future genetic structure of an invasion: insights from Acacia pycnantha

    PubMed Central

    2013-01-01

    Background The influence of introduction history and post-introduction dynamics on genetic diversity and structure has been a major research focus in invasion biology. However, genetic diversity and structure in the invasive range can also be affected by human-mediated processes in the native range prior to species introductions, an aspect often neglected in invasion biology. Here we aim to trace the native provenance of the invasive tree Acacia pycnantha by comparing the genetic diversity and structure between populations in the native Australian range and the invasive range in South Africa. This approach also allowed us to explore how human actions altered genetic structure before and after the introduction of A. pycnantha into South Africa. We hypothesized that extensive movement and replanting in A. pycnantha’s Australian range prior to its introduction to South Africa might result in highly admixed genotypes in the introduced range, comparable genetic diversity in both ranges, and therefore preclude an accurate determination of native provenance(s) of invasive populations. Results In the native range Bayesian assignment tests identified three genetic clusters with substantial admixture and could not clearly differentiate previously identified genetic entities, corroborating admixture as a result of replantings within Australia. Assignment tests that included invasive populations from South Africa indicated similar levels of admixture compared to Australian populations and a lack of genetic structure. Invasive populations of A. pycnantha in South Africa are as genetically diverse as native populations, and could not be assigned to particular native range regions. Conclusions Our results indicate that the genetic structure of A. pycnantha in Australia has been greatly altered through various planting initiatives. Specifically, there is little geographic structure and high levels of admixture. While numerous introduction history scenarios may explain the levels

  11. Rapid characterization of biotherapeutic proteins by size-exclusion chromatography coupled to native mass spectrometry

    PubMed Central

    Haberger, Markus; Leiss, Michael; Heidenreich, Anna-Katharina; Pester, Oxana; Hafenmair, Georg; Hook, Michaela; Bonnington, Lea; Wegele, Harald; Haindl, Markus; Reusch, Dietmar; Bulau, Patrick

    2016-01-01

    ABSTRACT High-molecular weight aggregates such as antibody dimers and other side products derived from incorrect light or heavy chain association typically represent critical product-related impurities for bispecific antibody formats. In this study, an approach employing ultra-pressure liquid chromatography size-exclusion separation combined with native electrospray ionization mass spectrometry for the simultaneous formation, identification and quantification of size variants in recombinant antibodies was developed. Samples exposed to storage and elevated temperature(s) enabled the identification of various bispecific antibody size variants. This test system hence allowed us to study the variants formed during formulation and bio-process development, and can thus be transferred to quality control units for routine in-process control and release analytics. In addition, native SEC-UV/MS not only facilitates the detailed analysis of low-abundant and non-covalent size variants during process characterization/validation studies, but is also essential for the SEC-UV method validation prior to admission to the market. PMID:26655595

  12. Structure of the photoactive yellow protein reconstituted with caffeic acid at 1.16 A resolution.

    PubMed

    van Aalten, Daan M F; Crielaard, Wim; Hellingwerf, Klaas J; Joshua-Tor, Leemor

    2002-04-01

    A structural study is described of the photoactive yellow protein (PYP) reconstituted with the chromophore derivative 3,4-dihydroxycinnamic acid. The crystal structure of PYP reconstituted with this chromophore at 1.16 A resolution is reported in space group P6(5). This is the first high-resolution structure of a photoreceptor containing a modified chromophore. The introduction of an extra hydroxyl group in the native chromophore (i.e. p-coumaric acid) appears to perturb the structure of the hybrid yellow protein only slightly. The chromophore is bound by the protein in two different conformations, separated by a rotation of 180 degrees of the catechol ring. In combination with available spectroscopic data, it is concluded that the caffeic acid chromophore binds to the protein in a strained conformation, which leads to a faster ejection from the chromophore-binding pocket upon pB formation. PMID:11914481

  13. Beta-turn propensities as paradigms for the analysis of structural motifs to engineer protein stability.

    PubMed Central

    Ohage, E. C.; Graml, W.; Walter, M. M.; Steinbacher, S.; Steipe, B.

    1997-01-01

    The thermodynamic stability of a protein provides an experimental metric for the relationship of protein sequence and native structure. We have investigated an approach based on an analysis of the structural database for stability engineering of an immunoglobulin variable domain. The most frequently occurring residues in specific positions of beta-turn motifs were predicted to increase the folding stability of mutants that were constructed by site-directed mutagenesis. Even in positions in which different residues are conserved in immunoglobulin sequences, the predictions were confirmed. Frequently, mutants with increased beta-turn propensities display increased folding cooperativities, suggesting pronounced effects on the unfolded state independent of the expected effect on conformational entropy. We conclude that structural motifs with predominantly local interactions can serve as templates with which patterns of sequence preferences can be extracted from the database of protein structures. Such preferences can predict the stability effects of mutations for protein engineering and design. PMID:9007995

  14. Rapid search for tertiary fragments reveals protein sequence–structure relationships

    PubMed Central

    Zhou, Jianfu; Grigoryan, Gevorg

    2015-01-01

    Finding backbone substructures from the Protein Data Bank that match an arbitrary query structural motif, composed of multiple disjoint segments, is a problem of growing relevance in structure prediction and protein design. Although numerous protein structure search approaches have been proposed, methods that address this specific task without additional restrictions and on practical time scales are generally lacking. Here, we propose a solution, dubbed MASTER, that is both rapid, enabling searches over the Protein Data Bank in a matter of seconds, and provably correct, finding all matches below a user-specified root-mean-square deviation cutoff. We show that despite the potentially exponential time complexity of the problem, running times in practice are modest even for queries with many segments. The ability to explore naturally plausible structural and sequence variations around a given motif has the potential to synthesize its design principles in an automated manner; so we go on to illustrate the utility of MASTER to protein structural biology. We demonstrate its capacity to rapidly establish structure–sequence relationships, uncover the native designability landscapes of tertiary structural motifs, identify structural signatures of binding, and automatically rewire protein topologies. Given the broad utility of protein tertiary fragment searches, we hope that providing MASTER in an open-source format will enable novel advances in understanding, predicting, and designing protein structure. PMID:25420575

  15. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    PubMed Central

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  16. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  17. Deciphering Supramolecular Structures with Protein-Protein Interaction Network Modeling

    PubMed Central

    Tsuji, Toshiyuki; Yoda, Takao; Shirai, Tsuyoshi

    2015-01-01

    Many biological molecules are assembled into supramolecules that are essential to perform complicated functions in the cell. However, experimental information about the structures of supramolecules is not sufficient at this point. We developed a method of predicting and modeling the structures of supramolecules in a biological network by combining structural data of the Protein Data Bank (PDB) and interaction data in IntAct databases. Templates for binary complexes in IntAct were extracted from PDB. Modeling was attempted by assembling binary complexes with superposed shared subunits. A total of 3,197 models were constructed, and 1,306 (41% of the total) contained at least one subunit absent from experimental structures. The models also suggested 970 (25% of the total) experimentally undetected subunit interfaces, and 41 human disease-related amino acid variants were mapped onto these model-suggested interfaces. The models demonstrated that protein-protein interaction network modeling is useful to fill the information gap between biological networks and structures. PMID:26549015

  18. Faster protein splicing with the Nostoc punctiforme DnaE intein using non-native extein residues.

    PubMed

    Cheriyan, Manoj; Pedamallu, Chandra Sekhar; Tori, Kazuo; Perler, Francine

    2013-03-01

    Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing. PMID:23306197

  19. A scoring function based on solvation thermodynamics for protein structure prediction

    PubMed Central

    Du, Shiqiao; Harano, Yuichi; Kinoshita, Masahiro; Sakurai, Minoru

    2012-01-01

    We predict protein structure using our recently developed free energy function for describing protein stability, which is focused on solvation thermodynamics. The function is combined with the current most reliable sampling methods, i.e., fragment assembly (FA) and comparative modeling (CM). The prediction is tested using 11 small proteins for which high-resolution crystal structures are available. For 8 of these proteins, sequence similarities are found in the database, and the prediction is performed with CM. Fairly accurate models with average Cα root mean square deviation (RMSD) ∼ 2.0 Å are successfully obtained for all cases. For the rest of the target proteins, we perform the prediction following FA protocols. For 2 cases, we obtain predicted models with an RMSD ∼ 3.0 Å as the best-scored structures. For the other case, the RMSD remains larger than 7 Å. For all the 11 target proteins, our scoring function identifies the experimentally determined native structure as the best structure. Starting from the predicted structure, replica exchange molecular dynamics is performed to further refine the structures. However, we are unable to improve its RMSD toward the experimental structure. The exhaustive sampling by coarse-grained normal mode analysis around the native structures reveals that our function has a linear correlation with RMSDs < 3.0 Å. These results suggest that the function is quite reliable for the protein structure prediction while the sampling method remains one of the major limiting factors in it. The aspects through which the methodology could further be improved are discussed.

  20. The TFE-induced transient native-like structure of the intrinsically disordered σ₄⁷⁰ domain of Escherichia coli RNA polymerase.

    PubMed

    Kaczka, Piotr; Winiewska, Maria; Zhukov, Igor; Rempoła, Bożenna; Bolewska, Krystyna; Łoziński, Tomasz; Ejchart, Andrzej; Poznańska, Anna; Wierzchowski, Kazimierz L; Poznański, Jarosław

    2014-12-01

    The transient folding of domain 4 of an E. coli RNA polymerase σ⁷⁰ subunit (rECσ₄⁷⁰) induced by an increasing concentration of 2,2,2-trifluoroethanol (TFE) in an aqueous solution was monitored by means of CD and heteronuclear NMR spectroscopy. NMR data, collected at a 30% TFE, allowed the estimation of the population of a locally folded rECσ₄⁷⁰ structure (CSI descriptors) and of local backbone dynamics ((15)N relaxation). The spontaneous organization of the helical regions of the initially unfolded protein into a TFE-induced 3D structure was revealed from structural constraints deduced from (15)N- to (13)C-edited NOESY spectra. In accordance with all the applied criteria, three highly populated α-helical regions, separated by much more flexible fragments, form a transient HLHTH motif resembling those found in PDB structures resolved for homologous proteins. All the data taken together demonstrate that TFE induces a transient native-like structure in the intrinsically disordered protein. PMID:25261014

  1. Predictions of Tertiary Structures of α-Helical Membrane Proteins by Replica-Exchange Method with Consideration of Helix Deformations

    NASA Astrophysics Data System (ADS)

    Urano, Ryo; Kokubo, Hironori; Okamoto, Yuko

    2015-08-01

    We propose an improved prediction method of the tertiary structures of α-helical membrane proteins based on the replica-exchange method by taking into account helix deformations. Our method has wide applications because transmembrane helices of native membrane proteins are often distorted. In order to test the effectiveness of the present method, we applied it to the structure predictions of glycophorin A and phospholamban. The results were in good agreement with experiments.

  2. Sequence, structure, and cooperativity in folding of elementary protein structural motifs.

    PubMed

    Lai, Jason K; Kubelka, Ginka S; Kubelka, Jan

    2015-08-11

    Residue-level unfolding of two helix-turn-helix proteins--one naturally occurring and one de novo designed--is reconstructed from multiple sets of site-specific (13)C isotopically edited infrared (IR) and circular dichroism (CD) data using Ising-like statistical-mechanical models. Several model variants are parameterized to test the importance of sequence-specific interactions (approximated by Miyazawa-Jernigan statistical potentials), local structural flexibility (derived from the ensemble of NMR structures), interhelical hydrogen bonds, and native contacts separated by intervening disordered regions (through the Wako-Saitô-Muñoz-Eaton scheme, which disallows such configurations). The models are optimized by directly simulating experimental observables: CD ellipticity at 222 nm for model proteins and their fragments and (13)C-amide I' bands for multiple isotopologues of each protein. We find that data can be quantitatively reproduced by the model that allows two interacting segments flanking a disordered loop (double sequence approximation) and incorporates flexibility in the native contact maps, but neither sequence-specific interactions nor hydrogen bonds are required. The near-identical free energy profiles as a function of the global order parameter are consistent with expected similar folding kinetics for nearly identical structures. However, the predicted folding mechanism for the two motifs is different, reflecting the order of local stability. We introduce free energy profiles for "experimental" reaction coordinates--namely, the degree of local folding as sensed by site-specific (13)C-edited IR, which highlight folding heterogeneity and contrast its overall, average description with the detailed, local picture. PMID:26216963

  3. Mathematics, thermodynamics, and modeling to address ten common misconceptions about protein structure, folding, and stability.

    PubMed

    Robic, Srebrenka

    2010-01-01

    To fully understand the roles proteins play in cellular processes, students need to grasp complex ideas about protein structure, folding, and stability. Our current understanding of these topics is based on mathematical models and experimental data. However, protein structure, folding, and stability are often introduced as descriptive, qualitative phenomena in undergraduate classes. In the process of learning about these topics, students often form incorrect ideas. For example, by learning about protein folding in the context of protein synthesis, students may come to an incorrect conclusion that once synthesized on the ribosome, a protein spends its entire cellular life time in its fully folded native confirmation. This is clearly not true; proteins are dynamic structures that undergo both local fluctuations and global unfolding events. To prevent and address such misconceptions, basic concepts of protein science can be introduced in the context of simple mathematical models and hands-on explorations of publicly available data sets. Ten common misconceptions about proteins are presented, along with suggestions for using equations, models, sequence, structure, and thermodynamic data to help students gain a deeper understanding of basic concepts relating to protein structure, folding, and stability. PMID:20810950

  4. Protein structures by spallation neutron crystallography

    PubMed Central

    Langan, Paul; Fisher, Zoë; Kovalevsky, Andrii; Mustyakimov, Marat; Sutcliffe Valone, Amanda; Unkefer, Cliff; Waltman, Mary Jo; Coates, Leighton; Adams, Paul D.; Afonine, Pavel V.; Bennett, Brad; Dealwis, Chris; Schoenborn, Benno P.

    2008-01-01

    The Protein Crystallography Station at Los Alamos Neutron Science Center is a high-performance beamline that forms the core of a capability for neutron macromolecular structure and function determination. This capability also includes the Macromolecular Neutron Crystallography (MNC) consortium between Los Alamos (LANL) and Lawrence Berkeley National Laboratories for developing computational tools for neutron protein crystallography, a biological deuteration laboratory, the National Stable Isotope Production Facility, and an MNC drug design consortium between LANL and Case Western Reserve University. PMID:18421142

  5. Structure and Function of Lipopolysaccharide Binding Protein

    NASA Astrophysics Data System (ADS)

    Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

    1990-09-01

    The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

  6. Trehalulose synthase native and carbohydrate complexed structures provide insights into sucrose isomerization.

    PubMed

    Ravaud, Stéphanie; Robert, Xavier; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2007-09-21

    Various diseases related to the overconsumption of sugar make a growing need for sugar substitutes. Because sucrose is an inexpensive and readily available d-glucose donor, the industrial potential for enzymatic synthesis o