Pre-calculated protein structure alignments at the RCSB PDB website.

PubMed

Prlic, Andreas; Bliven, Spencer; Rose, Peter W; Bluhm, Wolfgang F; Bizon, Chris; Godzik, Adam; Bourne, Philip E

2010-12-01

With the continuous growth of the RCSB Protein Data Bank (PDB), providing an up-to-date systematic structure comparison of all protein structures poses an ever growing challenge. Here, we present a comparison tool for calculating both 1D protein sequence and 3D protein structure alignments. This tool supports various applications at the RCSB PDB website. First, a structure alignment web service calculates pairwise alignments. Second, a stand-alone application runs alignments locally and visualizes the results. Third, pre-calculated 3D structure comparisons for the whole PDB are provided and updated on a weekly basis. These three applications allow users to discover novel relationships between proteins available either at the RCSB PDB or provided by the user. A web user interface is available at http://www.rcsb.org/pdb/workbench/workbench.do. The source code is available under the LGPL license from http://www.biojava.org. A source bundle, prepared for local execution, is available from http://source.rcsb.org andreas@sdsc.edu; pbourne@ucsd.edu.

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

PubMed Central

Zook, James D.; Molugu, Trivikram R.; Jacobsen, Neil E.; Lin, Guangxin; Soll, Jürgen; Cherry, Brian R.; Brown, Michael F.; Fromme, Petra

2013-01-01

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the 13C, 15N, 2H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, HN, CO, Cα, and Cβ chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein. PMID:24205117

Future directions of electron crystallography.

PubMed

Fujiyoshi, Yoshinori

2013-01-01

In biological science, there are still many interesting and fundamental yet difficult questions, such as those in neuroscience, remaining to be answered. Structural and functional studies of membrane proteins, which are key molecules of signal transduction in neural and other cells, are essential for understanding the molecular mechanisms of many fundamental biological processes. Technological and instrumental advancements of electron microscopy have facilitated comprehension of structural studies of biological components, such as membrane proteins. While X-ray crystallography has been the main method of structure analysis of proteins including membrane proteins, electron crystallography is now an established technique to analyze structures of membrane proteins in the lipid bilayer, which is close to their natural biological environment. By utilizing cryo-electron microscopes with helium-cooled specimen stages, structures of membrane proteins were analyzed at a resolution better than 3 Å. Such high-resolution structural analysis of membrane proteins by electron crystallography opens up the new research field of structural physiology. Considering the fact that the structures of integral membrane proteins in their native membrane environment without artifacts from crystal contacts are critical in understanding their physiological functions, electron crystallography will continue to be an important technology for structural analysis. In this chapter, I will present several examples to highlight important advantages and to suggest future directions of this technique.

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

PubMed

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

2008-10-23

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

Detergent-associated Solution Conformations of Helical and Beta-barrel Membrane Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mo, Yiming; Lee, Byung-Kwon; Ankner, John Francis

2008-01-01

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

Inorganic pyrophosphatases: structural diversity serving the function

NASA Astrophysics Data System (ADS)

Samygina, V. R.

2016-05-01

The review is devoted to ubiquitous enzymes, inorganic pyrophosphatases, which are essential in all living organisms. Despite the long history of investigations, these enzymes continue to attract interest. The review focuses on the three-dimensional structures of various representatives of this class of proteins. The structural diversity, the relationship between the structure and some properties of pyrophosphatases and various mechanisms of enzyme action related to the structural diversity of these enzymes are discussed. Interactions of pyrophosphatase with other proteins and possible practical applications are considered. The bibliography includes 56 references.

A limited universe of membrane protein families and folds

PubMed Central

Oberai, Amit; Ihm, Yungok; Kim, Sanguk; Bowie, James U.

2006-01-01

One of the goals of structural genomics is to obtain a structural representative of almost every fold in nature. A recent estimate suggests that 70%–80% of soluble protein domains identified in the first 1000 genome sequences should be covered by about 25,000 structures—a reasonably achievable goal. As no current estimates exist for the number of membrane protein families, however, it is not possible to know whether family coverage is a realistic goal for membrane proteins. Here we find that virtually all polytopic helical membrane protein families are present in the already known sequences so we can make an estimate of the total number of families. We find that only ∼700 polytopic membrane protein families account for 80% of structured residues and ∼1700 cover 90% of structured residues. While apparently a finite and reachable goal, we estimate that it will likely take more than three decades to obtain the structures needed for 90% residue coverage, if current trends continue. PMID:16815920

Tertiary structural propensities reveal fundamental sequence/structure relationships.

PubMed

Zheng, Fan; Zhang, Jian; Grigoryan, Gevorg

2015-05-05

Extracting useful generalizations from the continually growing Protein Data Bank (PDB) is of central importance. We hypothesize that the PDB contains valuable quantitative information on the level of local tertiary structural motifs (TERMs). We show that by breaking a protein structure into its constituent TERMs, and querying the PDB to characterize the natural ensemble matching each, we can estimate the compatibility of the structure with a given amino acid sequence through a metric we term "structure score." Considering submissions from recent Critical Assessment of Structure Prediction (CASP) experiments, we found a strong correlation (R = 0.69) between structure score and model accuracy, with poorly predicted regions readily identifiable. This performance exceeds that of leading atomistic statistical energy functions. Furthermore, TERM-based analysis of two prototypical multi-state proteins rapidly produced structural insights fully consistent with prior extensive experimental studies. We thus find that TERM-based analysis should have considerable utility for protein structural biology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Local-global alignment for finding 3D similarities in protein structures

DOEpatents

Zemla, Adam T [Brentwood, CA

2011-09-20

A method of finding 3D similarities in protein structures of a first molecule and a second molecule. The method comprises providing preselected information regarding the first molecule and the second molecule. Comparing the first molecule and the second molecule using Longest Continuous Segments (LCS) analysis. Comparing the first molecule and the second molecule using Global Distance Test (GDT) analysis. Comparing the first molecule and the second molecule using Local Global Alignment Scoring function (LGA_S) analysis. Verifying constructed alignment and repeating the steps to find the regions of 3D similarities in protein structures.

Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

PubMed Central

Hyung, Suk-Joon; Ruotolo, Brandon T.

2013-01-01

Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

The Role of Hexon Protein as a Molecular Mold in Patterning the Protein IX Organization in Human Adenoviruses.

PubMed

Reddy, Vijay S

2017-09-01

Adenoviruses are respiratory, ocular and enteric pathogens that form complex capsids, which are assembled from seven different structural proteins and composed of several core proteins that closely interact with the packaged dsDNA genome. The recent near-atomic resolution structures revealed that the interlacing continuous hexagonal network formed by the protein IX molecules is conserved among different human adenoviruses (HAdVs), but not in non-HAdVs. In this report, we propose a distinct role for the hexon protein as a "molecular mold" in enabling the formation of such hexagonal protein IX network that has been shown to preserve the stability and infectivity of HAdVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Adaptation of model proteins from cold to hot environments involves continuous and small adjustments of average parameters related to amino acid composition.

PubMed

De Vendittis, Emmanuele; Castellano, Immacolata; Cotugno, Roberta; Ruocco, Maria Rosaria; Raimo, Gennaro; Masullo, Mariorosario

2008-01-07

The growth temperature adaptation of six model proteins has been studied in 42 microorganisms belonging to eubacterial and archaeal kingdoms, covering optimum growth temperatures from 7 to 103 degrees C. The selected proteins include three elongation factors involved in translation, the enzymes glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, the cell division protein FtsZ. The common strategy of protein adaptation from cold to hot environments implies the occurrence of small changes in the amino acid composition, without altering the overall structure of the macromolecule. These continuous adjustments were investigated through parameters related to the amino acid composition of each protein. The average value per residue of mass, volume and accessible surface area allowed an evaluation of the usage of bulky residues, whereas the average hydrophobicity reflected that of hydrophobic residues. The specific proportion of bulky and hydrophobic residues in each protein almost linearly increased with the temperature of the host microorganism. This finding agrees with the structural and functional properties exhibited by proteins in differently adapted sources, thus explaining the great compactness or the high flexibility exhibited by (hyper)thermophilic or psychrophilic proteins, respectively. Indeed, heat-adapted proteins incline toward the usage of heavier-size and more hydrophobic residues with respect to mesophiles, whereas the cold-adapted macromolecules show the opposite behavior with a certain preference for smaller-size and less hydrophobic residues. An investigation on the different increase of bulky residues along with the growth temperature observed in the six model proteins suggests the relevance of the possible different role and/or structure organization played by protein domains. The significance of the linear correlations between growth temperature and parameters related to the amino acid composition improved when the analysis was collectively carried out on all model proteins.

Protein Structure Classification and Loop Modeling Using Multiple Ramachandran Distributions.

PubMed

Najibi, Seyed Morteza; Maadooliat, Mehdi; Zhou, Lan; Huang, Jianhua Z; Gao, Xin

2017-01-01

Recently, the study of protein structures using angular representations has attracted much attention among structural biologists. The main challenge is how to efficiently model the continuous conformational space of the protein structures based on the differences and similarities between different Ramachandran plots. Despite the presence of statistical methods for modeling angular data of proteins, there is still a substantial need for more sophisticated and faster statistical tools to model the large-scale circular datasets. To address this need, we have developed a nonparametric method for collective estimation of multiple bivariate density functions for a collection of populations of protein backbone angles. The proposed method takes into account the circular nature of the angular data using trigonometric spline which is more efficient compared to existing methods. This collective density estimation approach is widely applicable when there is a need to estimate multiple density functions from different populations with common features. Moreover, the coefficients of adaptive basis expansion for the fitted densities provide a low-dimensional representation that is useful for visualization, clustering, and classification of the densities. The proposed method provides a novel and unique perspective to two important and challenging problems in protein structure research: structure-based protein classification and angular-sampling-based protein loop structure prediction.

ECOD: An Evolutionary Classification of Protein Domains

PubMed Central

Kinch, Lisa N.; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V.

2014-01-01

Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or “fold”). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies. PMID:25474468

ECOD: an evolutionary classification of protein domains.

PubMed

Cheng, Hua; Schaeffer, R Dustin; Liao, Yuxing; Kinch, Lisa N; Pei, Jimin; Shi, Shuoyong; Kim, Bong-Hyun; Grishin, Nick V

2014-12-01

Understanding the evolution of a protein, including both close and distant relationships, often reveals insight into its structure and function. Fast and easy access to such up-to-date information facilitates research. We have developed a hierarchical evolutionary classification of all proteins with experimentally determined spatial structures, and presented it as an interactive and updatable online database. ECOD (Evolutionary Classification of protein Domains) is distinct from other structural classifications in that it groups domains primarily by evolutionary relationships (homology), rather than topology (or "fold"). This distinction highlights cases of homology between domains of differing topology to aid in understanding of protein structure evolution. ECOD uniquely emphasizes distantly related homologs that are difficult to detect, and thus catalogs the largest number of evolutionary links among structural domain classifications. Placing distant homologs together underscores the ancestral similarities of these proteins and draws attention to the most important regions of sequence and structure, as well as conserved functional sites. ECOD also recognizes closer sequence-based relationships between protein domains. Currently, approximately 100,000 protein structures are classified in ECOD into 9,000 sequence families clustered into close to 2,000 evolutionary groups. The classification is assisted by an automated pipeline that quickly and consistently classifies weekly releases of PDB structures and allows for continual updates. This synchronization with PDB uniquely distinguishes ECOD among all protein classifications. Finally, we present several case studies of homologous proteins not recorded in other classifications, illustrating the potential of how ECOD can be used to further biological and evolutionary studies.

WeFold: A Coopetition for Protein Structure Prediction

PubMed Central

Khoury, George A.; Liwo, Adam; Khatib, Firas; Zhou, Hongyi; Chopra, Gaurav; Bacardit, Jaume; Bortot, Leandro O.; Faccioli, Rodrigo A.; Deng, Xin; He, Yi; Krupa, Pawel; Li, Jilong; Mozolewska, Magdalena A.; Sieradzan, Adam K.; Smadbeck, James; Wirecki, Tomasz; Cooper, Seth; Flatten, Jeff; Xu, Kefan; Baker, David; Cheng, Jianlin; Delbem, Alexandre C. B.; Floudas, Christodoulos A.; Keasar, Chen; Levitt, Michael; Popović, Zoran; Scheraga, Harold A.; Skolnick, Jeffrey; Crivelli, Silvia N.; Players, Foldit

2014-01-01

The protein structure prediction problem continues to elude scientists. Despite the introduction of many methods, only modest gains were made over the last decade for certain classes of prediction targets. To address this challenge, a social-media based worldwide collaborative effort, named WeFold, was undertaken by thirteen labs. During the collaboration, the labs were simultaneously competing with each other. Here, we present the first attempt at “coopetition” in scientific research applied to the protein structure prediction and refinement problems. The coopetition was possible by allowing the participating labs to contribute different components of their protein structure prediction pipelines and create new hybrid pipelines that they tested during CASP10. This manuscript describes both successes and areas needing improvement as identified throughout the first WeFold experiment and discusses the efforts that are underway to advance this initiative. A footprint of all contributions and structures are publicly accessible at http://www.wefold.org. PMID:24677212

SOV_refine: A further refined definition of segment overlap score and its significance for protein structure similarity.

PubMed

Liu, Tong; Wang, Zheng

2018-01-01

The segment overlap score (SOV) has been used to evaluate the predicted protein secondary structures, a sequence composed of helix (H), strand (E), and coil (C), by comparing it with the native or reference secondary structures, another sequence of H, E, and C. SOV's advantage is that it can consider the size of continuous overlapping segments and assign extra allowance to longer continuous overlapping segments instead of only judging from the percentage of overlapping individual positions as Q3 score does. However, we have found a drawback from its previous definition, that is, it cannot ensure increasing allowance assignment when more residues in a segment are further predicted accurately. A new way of assigning allowance has been designed, which keeps all the advantages of the previous SOV score definitions and ensures that the amount of allowance assigned is incremental when more elements in a segment are predicted accurately. Furthermore, our improved SOV has achieved a higher correlation with the quality of protein models measured by GDT-TS score and TM-score, indicating its better abilities to evaluate tertiary structure quality at the secondary structure level. We analyzed the statistical significance of SOV scores and found the threshold values for distinguishing two protein structures (SOV_refine  > 0.19) and indicating whether two proteins are under the same CATH fold (SOV_refine > 0.94 and > 0.90 for three- and eight-state secondary structures respectively). We provided another two example applications, which are when used as a machine learning feature for protein model quality assessment and comparing different definitions of topologically associating domains. We proved that our newly defined SOV score resulted in better performance. The SOV score can be widely used in bioinformatics research and other fields that need to compare two sequences of letters in which continuous segments have important meanings. We also generalized the previous SOV definitions so that it can work for sequences composed of more than three states (e.g., it can work for the eight-state definition of protein secondary structures). A standalone software package has been implemented in Perl with source code released. The software can be downloaded from http://dna.cs.miami.edu/SOV/.

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors*

PubMed Central

Smith, Jeffrey S.; Rajagopal, Sudarshan

2016-01-01

The β-arrestins (βarrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, βarrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that βarrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for βarrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. PMID:26984408

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

PubMed

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

2011-08-01

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

Meeting Report: Structural Determination of Environmentally Responsive Proteins

PubMed Central

Reinlib, Leslie

2005-01-01

The three-dimensional structure of gene products continues to be a missing lynchpin between linear genome sequences and our understanding of the normal and abnormal function of proteins and pathways. Enhanced activity in this area is likely to lead to better understanding of how discrete changes in molecular patterns and conformation underlie functional changes in protein complexes and, with it, sensitivity of an individual to an exposure. The National Institute of Environmental Health Sciences convened a workshop of experts in structural determination and environmental health to solicit advice for future research in structural resolution relative to environmentally responsive proteins and pathways. The highest priorities recommended by the workshop were to support studies of structure, analysis, control, and design of conformational and functional states at molecular resolution for environmentally responsive molecules and complexes; promote understanding of dynamics, kinetics, and ligand responses; investigate the mechanisms and steps in posttranslational modifications, protein partnering, impact of genetic polymorphisms on structure/function, and ligand interactions; and encourage integrated experimental and computational approaches. The workshop participants also saw value in improving the throughput and purity of protein samples and macromolecular assemblies; developing optimal processes for design, production, and assembly of macromolecular complexes; encouraging studies on protein–protein and macromolecular interactions; and examining assemblies of individual proteins and their functions in pathways of interest for environmental health. PMID:16263521

[A structural protein study of the influenza A (H1N1) virus by polyacrylamide gel electrophoresis].

PubMed

Pérez Guevara, M T; Savón Valdés, C; Rivas Arjona, M; Goyenechea Hernández, A

1992-01-01

Influenza is an acute respiratory disease typically appearing as an epidemic. Three immunological types of the influenza virus are known: A, B and C. Continually, antigen changes occur, especially in type A. Therefore, a comparative study was carried out on 4 influenza A(H1N1) virus strains in relation to protein structure (surface antigens), by using polyacrylamide gel electrophoresis by the modified Laemmli method. The objective was to compare the structural proteins of the A/Havana/1292/78 (H1N1) national strain with the proteins of 3 international pattern strains. In all the cases, 6 bands were detected by densitometry. In the 4 strains studied the most abundant protein was M. Great differences between the Cuban strain and the 3 international patterns were not seen.

An estimated 5% of new protein structures solved today represent a new Pfam family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mistry, Jaina; Kloppmann, Edda; Rost, Burkhard

2013-11-01

This study uses the Pfam database to show that the sequence redundancy of protein structures deposited in the PDB is increasing. The possible reasons behind this trend are discussed. High-resolution structural knowledge is key to understanding how proteins function at the molecular level. The number of entries in the Protein Data Bank (PDB), the repository of all publicly available protein structures, continues to increase, with more than 8000 structures released in 2012 alone. The authors of this article have studied how structural coverage of the protein-sequence space has changed over time by monitoring the number of Pfam families that acquiredmore » their first representative structure each year from 1976 to 2012. Twenty years ago, for every 100 new PDB entries released, an estimated 20 Pfam families acquired their first structure. By 2012, this decreased to only about five families per 100 structures. The reasons behind the slower pace at which previously uncharacterized families are being structurally covered were investigated. It was found that although more than 50% of current Pfam families are still without a structural representative, this set is enriched in families that are small, functionally uncharacterized or rich in problem features such as intrinsically disordered and transmembrane regions. While these are important constraints, the reasons why it may not yet be time to give up the pursuit of a targeted but more comprehensive structural coverage of the protein-sequence space are discussed.« less

The value of protein structure classification information—Surveying the scientific literature

PubMed Central

Fox, Naomi K.; Brenner, Steven E.

2015-01-01

ABSTRACT The Structural Classification of Proteins (SCOP) and Class, Architecture, Topology, Homology (CATH) databases have been valuable resources for protein structure classification for over 20 years. Development of SCOP (version 1) concluded in June 2009 with SCOP 1.75. The SCOPe (SCOP–extended) database offers continued development of the classic SCOP hierarchy, adding over 33,000 structures. We have attempted to assess the impact of these two decade old resources and guide future development. To this end, we surveyed recent articles to learn how structure classification data are used. Of 571 articles published in 2012–2013 that cite SCOP, 439 actually use data from the resource. We found that the type of use was fairly evenly distributed among four top categories: A) study protein structure or evolution (27% of articles), B) train and/or benchmark algorithms (28% of articles), C) augment non‐SCOP datasets with SCOP classification (21% of articles), and D) examine the classification of one protein/a small set of proteins (22% of articles). Most articles described computational research, although 11% described purely experimental research, and a further 9% included both. We examined how CATH and SCOP were used in 158 articles that cited both databases: while some studies used only one dataset, the majority used data from both resources. Protein structure classification remains highly relevant for a diverse range of problems and settings. Proteins 2015; 83:2025–2038. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc. PMID:26313554

Which strategy for a protein crystallization project?

NASA Technical Reports Server (NTRS)

Kundrot, C. E.

2004-01-01

The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryocrystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

Which Strategy for a Protein Crystallization Project?

NASA Technical Reports Server (NTRS)

Kundrot, Craig E.

2003-01-01

The three-dimensional, atomic-resolution protein structures produced by X-ray crystallography over the past 50+ years have led to tremendous chemical understanding of fundamental biochemical processes. The pace of discovery in protein crystallography has increased greatly with advances in molecular biology, crystallization techniques, cryo-crystallography, area detectors, synchrotrons and computing. While the methods used to produce single, well-ordered crystals have also evolved over the years in response to increased understanding and advancing technology, crystallization strategies continue to be rooted in trial-and-error approaches. This review summarizes the current approaches in protein crystallization and surveys the first results to emerge from the structural genomics efforts.

LiveBench-1: continuous benchmarking of protein structure prediction servers.

PubMed

Bujnicki, J M; Elofsson, A; Fischer, D; Rychlewski, L

2001-02-01

We present a novel, continuous approach aimed at the large-scale assessment of the performance of available fold-recognition servers. Six popular servers were investigated: PDB-Blast, FFAS, T98-lib, GenTHREADER, 3D-PSSM, and INBGU. The assessment was conducted using as prediction targets a large number of selected protein structures released from October 1999 to April 2000. A target was selected if its sequence showed no significant similarity to any of the proteins previously available in the structural database. Overall, the servers were able to produce structurally similar models for one-half of the targets, but significantly accurate sequence-structure alignments were produced for only one-third of the targets. We further classified the targets into two sets: easy and hard. We found that all servers were able to find the correct answer for the vast majority of the easy targets if a structurally similar fold was present in the server's fold libraries. However, among the hard targets--where standard methods such as PSI-BLAST fail--the most sensitive fold-recognition servers were able to produce similar models for only 40% of the cases, half of which had a significantly accurate sequence-structure alignment. Among the hard targets, the presence of updated libraries appeared to be less critical for the ranking. An "ideally combined consensus" prediction, where the results of all servers are considered, would increase the percentage of correct assignments by 50%. Each server had a number of cases with a correct assignment, where the assignments of all the other servers were wrong. This emphasizes the benefits of considering more than one server in difficult prediction tasks. The LiveBench program (http://BioInfo.PL/LiveBench) is being continued, and all interested developers are cordially invited to join.

Directed molecular evolution to design advanced red fluorescent proteins.

PubMed

Subach, Fedor V; Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-11-29

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

Continually emerging mechanistic complexity of the multi-enzyme cellulosome complex.

PubMed

Smith, Steven P; Bayer, Edward A; Czjzek, Mirjam

2017-06-01

The robust plant cell wall polysaccharide-degrading properties of anaerobic bacteria are harnessed within elegant, marcomolecular assemblages called cellulosomes, in which proteins of complementary activities amass on scaffold protein networks. Research efforts have focused and continue to focus on providing detailed mechanistic insights into cellulosomal complex assembly, topology, and function. The accumulated information is expanding our fundamental understanding of the lignocellulosic biomass decomposition process and enhancing the potential of engineered cellulosomal systems for biotechnological purposes. Ongoing biochemical studies continue to reveal unexpected functional diversity within traditional cellulase families. Genomic, proteomic, and functional analyses have uncovered unanticipated cellulosomal proteins that augment the function of the native and designer cellulosomes. In addition, complementary structural and computational methods are continuing to provide much needed insights on the influence of cellulosomal interdomain linker regions on cellulosomal assembly and activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

PDBStat: a universal restraint converter and restraint analysis software package for protein NMR.

PubMed

Tejero, Roberto; Snyder, David; Mao, Binchen; Aramini, James M; Montelione, Gaetano T

2013-08-01

The heterogeneous array of software tools used in the process of protein NMR structure determination presents organizational challenges in the structure determination and validation processes, and creates a learning curve that limits the broader use of protein NMR in biology. These challenges, including accurate use of data in different data formats required by software carrying out similar tasks, continue to confound the efforts of novices and experts alike. These important issues need to be addressed robustly in order to standardize protein NMR structure determination and validation. PDBStat is a C/C++ computer program originally developed as a universal coordinate and protein NMR restraint converter. Its primary function is to provide a user-friendly tool for interconverting between protein coordinate and protein NMR restraint data formats. It also provides an integrated set of computational methods for protein NMR restraint analysis and structure quality assessment, relabeling of prochiral atoms with correct IUPAC names, as well as multiple methods for analysis of the consistency of atomic positions indicated by their convergence across a protein NMR ensemble. In this paper we provide a detailed description of the PDBStat software, and highlight some of its valuable computational capabilities. As an example, we demonstrate the use of the PDBStat restraint converter for restrained CS-Rosetta structure generation calculations, and compare the resulting protein NMR structure models with those generated from the same NMR restraint data using more traditional structure determination methods. These results demonstrate the value of a universal restraint converter in allowing the use of multiple structure generation methods with the same restraint data for consensus analysis of protein NMR structures and the underlying restraint data.

PDBStat: A Universal Restraint Converter and Restraint Analysis Software Package for Protein NMR

PubMed Central

Tejero, Roberto; Snyder, David; Mao, Binchen; Aramini, James M.; Montelione, Gaetano T

2013-01-01

The heterogeneous array of software tools used in the process of protein NMR structure determination presents organizational challenges in the structure determination and validation processes, and creates a learning curve that limits the broader use of protein NMR in biology. These challenges, including accurate use of data in different data formats required by software carrying out similar tasks, continue to confound the efforts of novices and experts alike. These important issues need to be addressed robustly in order to standardize protein NMR structure determination and validation. PDBStat is a C/C++ computer program originally developed as a universal coordinate and protein NMR restraint converter. Its primary function is to provide a user-friendly tool for interconverting between protein coordinate and protein NMR restraint data formats. It also provides an integrated set of computational methods for protein NMR restraint analysis and structure quality assessment, relabeling of prochiral atoms with correct IUPAC names, as well as multiple methods for analysis of the consistency of atomic positions indicated by their convergence across a protein NMR ensemble. In this paper we provide a detailed description of the PDBStat software, and highlight some of its valuable computational capabilities. As an example, we demonstrate the use of the PDBStat restraint converter for restrained CS-Rosetta structure generation calculations, and compare the resulting protein NMR structure models with those generated from the same NMR restraint data using more traditional structure determination methods. These results demonstrate the value of a universal restraint converter in allowing the use of multiple structure generation methods with the same restraint data for consensus analysis of protein NMR structures and the underlying restraint data. PMID:23897031

The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors.

PubMed

Smith, Jeffrey S; Rajagopal, Sudarshan

2016-04-22

The β-arrestins (βarrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, βarrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that βarrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for βarrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

PROFESS: a PROtein Function, Evolution, Structure and Sequence database

PubMed Central

Triplet, Thomas; Shortridge, Matthew D.; Griep, Mark A.; Stark, Jaime L.; Powers, Robert; Revesz, Peter

2010-01-01

The proliferation of biological databases and the easy access enabled by the Internet is having a beneficial impact on biological sciences and transforming the way research is conducted. There are ∼1100 molecular biology databases dispersed throughout the Internet. To assist in the functional, structural and evolutionary analysis of the abundant number of novel proteins continually identified from whole-genome sequencing, we introduce the PROFESS (PROtein Function, Evolution, Structure and Sequence) database. Our database is designed to be versatile and expandable and will not confine analysis to a pre-existing set of data relationships. A fundamental component of this approach is the development of an intuitive query system that incorporates a variety of similarity functions capable of generating data relationships not conceived during the creation of the database. The utility of PROFESS is demonstrated by the analysis of the structural drift of homologous proteins and the identification of potential pancreatic cancer therapeutic targets based on the observation of protein–protein interaction networks. Database URL: http://cse.unl.edu/∼profess/ PMID:20624718

One-step nanoimprinted hybrid micro-/nano-structure for in situ protein detection of isolated cell array via localized surface plasmon resonance

NASA Astrophysics Data System (ADS)

Ali, Riyaz Ahmad Mohamed; Villariza Espulgar, Wilfred; Aoki, Wataru; Jiang, Shu; Saito, Masato; Ueda, Mitsuyoshi; Tamiya, Eiichi

2018-03-01

Nanoplasmonic biosensors show high potentials as label-free devices for continuous monitoring in biomolecular analyses. However, most current sensors comprise multiple-dedicated layers with complicated fabrication procedures, which increases production time and manufacturing costs. In this work, we report the synergistic integration of cell-trapping microwell structures with plasmonic sensing nanopillar structures in a single-layered substrate by one-step thermal nanoimprinting. Here, microwell arrays are used for isolating cells, wherein gold-capped nanostructures sense changes in local refractive index via localized surface plasmon resonance (LSPR). Hence, proteins secreted from trapped cells can be label-freely detected as peak shifts in absorbance spectra. The fabricated device showed a detection limit of 10 ng/µL anti-IgA. In Pichia pastoris cells trial analysis, a red shift of 6.9 nm was observed over 12 h, which is likely due to the protein secretion from the cells. This approach provides an inexpensive, rapid, and reproducible alternative for mass production of biosensors for continuous biomolecular analyses.

The HPr Proteins from the Thermophile Bacillus stearothermophilus Can Form Domain-swapped Dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sridharan, Sudharsan; Razvi, Abbas; Scholtz, J. Martin

2010-07-20

The study of proteins from extremophilic organisms continues to generate interest in the field of protein folding because paradigms explaining the enhanced stability of these proteins still elude us and such studies have the potential to further our knowledge of the forces stabilizing proteins. We have undertaken such a study with our model protein HPr from a mesophile, Bacillus subtilis, and a thermophile, Bacillus stearothermophilus. We report here the high-resolution structures of the wild-type HPr protein from the thermophile and a variant, F29W. The variant proved to crystallize in two forms: a monomeric form with a structure very similar tomore » the wild-type protein as well as a domain-swapped dimer. Interestingly, the structure of the domain-swapped dimer for HPr is very different from that observed for a homologous protein, Crh, from B. subtilis. The existence of a domain-swapped dimer has implications for amyloid formation and is consistent with recent results showing that the HPr proteins can form amyloid fibrils. We also characterized the conformational stability of the thermophilic HPr proteins using thermal and solvent denaturation methods and have used the high-resolution structures in an attempt to explain the differences in stability between the different HPr proteins. Finally, we present a detailed analysis of the solution properties of the HPr proteins using a variety of biochemical and biophysical methods.« less

The value of protein structure classification information-Surveying the scientific literature

DOE PAGES

Fox, Naomi K.; Brenner, Steven E.; Chandonia, John -Marc

2015-08-27

The Structural Classification of Proteins (SCOP) and Class, Architecture, Topology, Homology (CATH) databases have been valuable resources for protein structure classification for over 20 years. Development of SCOP (version 1) concluded in June 2009 with SCOP 1.75. The SCOPe (SCOP-extended) database offers continued development of the classic SCOP hierarchy, adding over 33,000 structures. We have attempted to assess the impact of these two decade old resources and guide future development. To this end, we surveyed recent articles to learn how structure classification data are used. Of 571 articles published in 2012-2013 that cite SCOP, 439 actually use data from themore » resource. We found that the type of use was fairly evenly distributed among four top categories: A) study protein structure or evolution (27% of articles), B) train and/or benchmark algorithms (28% of articles), C) augment non-SCOP datasets with SCOP classification (21% of articles), and D) examine the classification of one protein/a small set of proteins (22% of articles). Most articles described computational research, although 11% described purely experimental research, and a further 9% included both. We examined how CATH and SCOP were used in 158 articles that cited both databases: while some studies used only one dataset, the majority used data from both resources. Protein structure classification remains highly relevant for a diverse range of problems and settings.« less

The value of protein structure classification information-Surveying the scientific literature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fox, Naomi K.; Brenner, Steven E.; Chandonia, John -Marc

The Structural Classification of Proteins (SCOP) and Class, Architecture, Topology, Homology (CATH) databases have been valuable resources for protein structure classification for over 20 years. Development of SCOP (version 1) concluded in June 2009 with SCOP 1.75. The SCOPe (SCOP-extended) database offers continued development of the classic SCOP hierarchy, adding over 33,000 structures. We have attempted to assess the impact of these two decade old resources and guide future development. To this end, we surveyed recent articles to learn how structure classification data are used. Of 571 articles published in 2012-2013 that cite SCOP, 439 actually use data from themore » resource. We found that the type of use was fairly evenly distributed among four top categories: A) study protein structure or evolution (27% of articles), B) train and/or benchmark algorithms (28% of articles), C) augment non-SCOP datasets with SCOP classification (21% of articles), and D) examine the classification of one protein/a small set of proteins (22% of articles). Most articles described computational research, although 11% described purely experimental research, and a further 9% included both. We examined how CATH and SCOP were used in 158 articles that cited both databases: while some studies used only one dataset, the majority used data from both resources. Protein structure classification remains highly relevant for a diverse range of problems and settings.« less

Ab initio protein structure assembly using continuous structure fragments and optimized knowledge-based force field.

PubMed

Xu, Dong; Zhang, Yang

2012-07-01

Ab initio protein folding is one of the major unsolved problems in computational biology owing to the difficulties in force field design and conformational search. We developed a novel program, QUARK, for template-free protein structure prediction. Query sequences are first broken into fragments of 1-20 residues where multiple fragment structures are retrieved at each position from unrelated experimental structures. Full-length structure models are then assembled from fragments using replica-exchange Monte Carlo simulations, which are guided by a composite knowledge-based force field. A number of novel energy terms and Monte Carlo movements are introduced and the particular contributions to enhancing the efficiency of both force field and search engine are analyzed in detail. QUARK prediction procedure is depicted and tested on the structure modeling of 145 nonhomologous proteins. Although no global templates are used and all fragments from experimental structures with template modeling score >0.5 are excluded, QUARK can successfully construct 3D models of correct folds in one-third cases of short proteins up to 100 residues. In the ninth community-wide Critical Assessment of protein Structure Prediction experiment, QUARK server outperformed the second and third best servers by 18 and 47% based on the cumulative Z-score of global distance test-total scores in the FM category. Although ab initio protein folding remains a significant challenge, these data demonstrate new progress toward the solution of the most important problem in the field. Copyright © 2012 Wiley Periodicals, Inc.

Robust Formation and Maintenance of Continuous Stratified Cortical Neuroepithelium by Laminin-Containing Matrix in Mouse ES Cell Culture

PubMed Central

Nasu, Makoto; Takata, Nozomu; Danjo, Teruko; Sakaguchi, Hideya; Kadoshima, Taisuke; Futaki, Sugiko; Sekiguchi, Kiyotoshi; Eiraku, Mototsugu; Sasai, Yoshiki

2012-01-01

In the mammalian cortex, the dorsal telencephalon exhibits a characteristic stratified structure. We previously reported that three-dimensional (3D) culture of mouse ES cells (mESCs) can efficiently generate cortical neuroepithelium (NE) and layer-specific cortical neurons. However, the cortical NE generated in this mESC culture was structurally unstable and broke into small neural rosettes by culture day 7, suggesting that some factors for reinforcing the structural integrity were missing. Here we report substantial supporting effects of the extracellular matrix (ECM) protein laminin on the continuous formation of properly polarized cortical NE in floating aggregate culture of mESCs. The addition of purified laminin and entactin (a laminin-associated protein), even at low concentrations, stabilized the formation of continuous cortical NE as well as the maintenance of basement membrane and prevented rosette formation. Treatment with the neutralizing ß1-integrin antibody impaired the continuous NE formation. The stabilized cortical NE exhibited typical interkinetic nuclear migration of cortical progenitors, as seen in the embryonic cortex. The laminin-treated cortical NE maintained a continuous structure even on culture days 12 and 15, and contained ventricular, basal-progenitor, cortical-plate and Cajal-Retzius cell layers. The cortical NE in this culture was flanked by cortical hem-like tissue. Furthermore, when Shh was added, ventral telencephalic structures such as lateral ganglionic eminence–like tissue formed in the region adjacent to the cortical NE. Thus, our results indicate that laminin-entactin ECM promotes the formation of structurally stable telencephalic tissues in 3D ESC culture, and supports the morphogenetic recapitulation of cortical development. PMID:23300850

Multiscale simulations on conformational dynamics and membrane interactions of the non-structural 2 (NS2) transmembrane domain.

PubMed

Hung, Huynh Minh; Hang, Tran Dieu; Nguyen, Minh Tho

2016-09-09

Hepatitis C virus (HCV) is one of the most crucial global health issues, in which the HCV non-structural protein 2 (NS2), particularly its three transmembrane segments, plays a crucial role in HCV assembly. In this context, multiscale MD simulations have been applied to investigate the preferred orientation of transmembrane domain of NS2 protein (TNS2) in a POPC bilayer, structural stability and characteristic of intramembrane protein-lipid and protein-protein interaction. Our study indicates that NS2 protein adopts three trans-membrane segments with highly stable α-helix structure in a POPC bilayer and a short helical luminal segment. While the first and second TM segment involved in continuous helical domain, the third TM segment is however cleaved into two sub-segments with different tilt angles via a kink at L87G88. Salt bridges K81-E45, R32-PO4 and R43-PO4 are determined as the key factor to stabilize the structure of TM2 and TM3 which consist of charged residues located in the hydrophobic region of the membrane. Copyright © 2016 Elsevier Inc. All rights reserved.

Spectral methods for study of the G-protein-coupled receptor rhodopsin. II. Magnetic resonance methods

NASA Astrophysics Data System (ADS)

Struts, A. V.; Barmasov, A. V.; Brown, M. F.

2016-02-01

This article continues our review of spectroscopic studies of G-protein-coupled receptors. Magnetic resonance methods including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) provide specific structural and dynamical data for the protein in conjunction with optical methods (vibrational, electronic spectroscopy) as discussed in the accompanying article. An additional advantage is the opportunity to explore the receptor proteins in the natural membrane lipid environment. Solid-state 2H and 13C NMR methods yield information about both the local structure and dynamics of the cofactor bound to the protein and its light-induced changes. Complementary site-directed spin-labeling studies monitor the structural alterations over larger distances and correspondingly longer time scales. A multiscale reaction mechanism describes how local changes of the retinal cofactor unlock the receptor to initiate large-scale conformational changes of rhodopsin. Activation of the G-protein-coupled receptor involves an ensemble of conformational substates within the rhodopsin manifold that characterize the dynamically active receptor.

The application of 3D Zernike moments for the description of "model-free" molecular structure, functional motion, and structural reliability.

PubMed

Grandison, Scott; Roberts, Carl; Morris, Richard J

2009-03-01

Protein structures are not static entities consisting of equally well-determined atomic coordinates. Proteins undergo continuous motion, and as catalytic machines, these movements can be of high relevance for understanding function. In addition to this strong biological motivation for considering shape changes is the necessity to correctly capture different levels of detail and error in protein structures. Some parts of a structural model are often poorly defined, and the atomic displacement parameters provide an excellent means to characterize the confidence in an atom's spatial coordinates. A mathematical framework for studying these shape changes, and handling positional variance is therefore of high importance. We present an approach for capturing various protein structure properties in a concise mathematical framework that allows us to compare features in a highly efficient manner. We demonstrate how three-dimensional Zernike moments can be employed to describe functions, not only on the surface of a protein but throughout the entire molecule. A number of proof-of-principle examples are given which demonstrate how this approach may be used in practice for the representation of movement and uncertainty.

Modular protein domains: an engineering approach toward functional biomaterials.

PubMed

Lin, Charng-Yu; Liu, Julie C

2016-08-01

Protein domains and peptide sequences are a powerful tool for conferring specific functions to engineered biomaterials. Protein sequences with a wide variety of functionalities, including structure, bioactivity, protein-protein interactions, and stimuli responsiveness, have been identified, and advances in molecular biology continue to pinpoint new sequences. Protein domains can be combined to make recombinant proteins with multiple functionalities. The high fidelity of the protein translation machinery results in exquisite control over the sequence of recombinant proteins and the resulting properties of protein-based materials. In this review, we discuss protein domains and peptide sequences in the context of functional protein-based materials, composite materials, and their biological applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein crystallization: Eluding the bottleneck of X-ray crystallography

PubMed Central

Holcomb, Joshua; Spellmon, Nicholas; Zhang, Yingxue; Doughan, Maysaa; Li, Chunying; Yang, Zhe

2017-01-01

To date, X-ray crystallography remains the gold standard for the determination of macromolecular structure and protein substrate interactions. However, the unpredictability of obtaining a protein crystal remains the limiting factor and continues to be the bottleneck in determining protein structures. A vast amount of research has been conducted in order to circumvent this issue with limited success. No single method has proven to guarantee the crystallization of all proteins. However, techniques using antibody fragments, lipids, carrier proteins, and even mutagenesis of crystal contacts have been implemented to increase the odds of obtaining a crystal with adequate diffraction. In addition, we review a new technique using the scaffolding ability of PDZ domains to facilitate nucleation and crystal lattice formation. Although in its infancy, such technology may be a valuable asset and another method in the crystallography toolbox to further the chances of crystallizing problematic proteins. PMID:29051919

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation.

PubMed

Sinha, Rajeshwari; Khare, Sunil K

2014-01-01

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants.

Protective role of salt in catalysis and maintaining structure of halophilic proteins against denaturation

PubMed Central

Sinha, Rajeshwari; Khare, Sunil K.

2014-01-01

Search for new industrial enzymes having novel properties continues to be a desirable pursuit in enzyme research. The halophilic organisms inhabiting under saline/ hypersaline conditions are considered as promising source of useful enzymes. Their enzymes are structurally adapted to perform efficient catalysis under saline environment wherein n0n-halophilic enzymes often lose their structure and activity. Haloenzymes have been documented to be polyextremophilic and withstand high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. Although vast amount of information have been generated on salt mediated protection and structure function relationship in halophilic proteins, their clear understanding and correct perspective still remain incoherent. Furthermore, understanding their protein architecture may give better clue for engineering stable enzymes which can withstand harsh industrial conditions. The article encompasses the current level of understanding about haloadaptations and analyzes structural basis of their enzyme stability against classical denaturants. PMID:24782853

Protein structure-structure alignment with discrete Fréchet distance.

PubMed

Jiang, Minghui; Xu, Ying; Zhu, Binhai

2008-02-01

Matching two geometric objects in two-dimensional (2D) and three-dimensional (3D) spaces is a central problem in computer vision, pattern recognition, and protein structure prediction. In particular, the problem of aligning two polygonal chains under translation and rotation to minimize their distance has been studied using various distance measures. It is well known that the Hausdorff distance is useful for matching two point sets, and that the Fréchet distance is a superior measure for matching two polygonal chains. The discrete Fréchet distance closely approximates the (continuous) Fréchet distance, and is a natural measure for the geometric similarity of the folded 3D structures of biomolecules such as proteins. In this paper, we present new algorithms for matching two polygonal chains in two dimensions to minimize their discrete Fréchet distance under translation and rotation, and an effective heuristic for matching two polygonal chains in three dimensions. We also describe our empirical results on the application of the discrete Fréchet distance to protein structure-structure alignment.

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

PubMed Central

Yamashita, Tetsuji; Hakizimana, Pierre; Wu, Siva; Hassan, Ahmed; Jacob, Stefan; Temirov, Jamshid; Fang, Jie; Mellado-Lagarde, Marcia; Gursky, Richard; Horner, Linda; Leibiger, Barbara; Leijon, Sara; Centonze, Victoria E.; Berggren, Per-Olof; Frase, Sharon; Auer, Manfred; Brownell, William E.; Fridberger, Anders; Zuo, Jian

2015-01-01

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases. PMID:26352669

Functional insights from proteome-wide structural modeling of Treponema pallidum subspecies pallidum, the causative agent of syphilis.

PubMed

Houston, Simon; Lithgow, Karen Vivien; Osbak, Kara Krista; Kenyon, Chris Richard; Cameron, Caroline E

2018-05-16

Syphilis continues to be a major global health threat with 11 million new infections each year, and a global burden of 36 million cases. The causative agent of syphilis, Treponema pallidum subspecies pallidum, is a highly virulent bacterium, however the molecular mechanisms underlying T. pallidum pathogenesis remain to be definitively identified. This is due to the fact that T. pallidum is currently uncultivatable, inherently fragile and thus difficult to work with, and phylogenetically distinct with no conventional virulence factor homologs found in other pathogens. In fact, approximately 30% of its predicted protein-coding genes have no known orthologs or assigned functions. Here we employed a structural bioinformatics approach using Phyre2-based tertiary structure modeling to improve our understanding of T. pallidum protein function on a proteome-wide scale. Phyre2-based tertiary structure modeling generated high-confidence predictions for 80% of the T. pallidum proteome (780/978 predicted proteins). Tertiary structure modeling also inferred the same function as primary structure-based annotations from genome sequencing pipelines for 525/605 proteins (87%), which represents 54% (525/978) of all T. pallidum proteins. Of the 175 T. pallidum proteins modeled with high confidence that were not assigned functions in the previously annotated published proteome, 167 (95%) were able to be assigned predicted functions. Twenty-one of the 175 hypothetical proteins modeled with high confidence were also predicted to exhibit significant structural similarity with proteins experimentally confirmed to be required for virulence in other pathogens. Phyre2-based structural modeling is a powerful bioinformatics tool that has provided insight into the potential structure and function of the majority of T. pallidum proteins and helped validate the primary structure-based annotation of more than 50% of all T. pallidum proteins with high confidence. This work represents the first T. pallidum proteome-wide structural modeling study and is one of few studies to apply this approach for the functional annotation of a whole proteome.

PASS2: an automated database of protein alignments organised as structural superfamilies.

PubMed

Bhaduri, Anirban; Pugalenthi, Ganesan; Sowdhamini, Ramanathan

2004-04-02

The functional selection and three-dimensional structural constraints of proteins in nature often relates to the retention of significant sequence similarity between proteins of similar fold and function despite poor sequence identity. Organization of structure-based sequence alignments for distantly related proteins, provides a map of the conserved and critical regions of the protein universe that is useful for the analysis of folding principles, for the evolutionary unification of protein families and for maximizing the information return from experimental structure determination. The Protein Alignment organised as Structural Superfamily (PASS2) database represents continuously updated, structural alignments for evolutionary related, sequentially distant proteins. An automated and updated version of PASS2 is, in direct correspondence with SCOP 1.63, consisting of sequences having identity below 40% among themselves. Protein domains have been grouped into 628 multi-member superfamilies and 566 single member superfamilies. Structure-based sequence alignments for the superfamilies have been obtained using COMPARER, while initial equivalencies have been derived from a preliminary superposition using LSQMAN or STAMP 4.0. The final sequence alignments have been annotated for structural features using JOY4.0. The database is supplemented with sequence relatives belonging to different genomes, conserved spatially interacting and structural motifs, probabilistic hidden markov models of superfamilies based on the alignments and useful links to other databases. Probabilistic models and sensitive position specific profiles obtained from reliable superfamily alignments aid annotation of remote homologues and are useful tools in structural and functional genomics. PASS2 presents the phylogeny of its members both based on sequence and structural dissimilarities. Clustering of members allows us to understand diversification of the family members. The search engine has been improved for simpler browsing of the database. The database resolves alignments among the structural domains consisting of evolutionarily diverged set of sequences. Availability of reliable sequence alignments of distantly related proteins despite poor sequence identity and single-member superfamilies permit better sampling of structures in libraries for fold recognition of new sequences and for the understanding of protein structure-function relationships of individual superfamilies. PASS2 is accessible at http://www.ncbs.res.in/~faculty/mini/campass/pass2.html

Ab Initio Protein Structure Assembly Using Continuous Structure Fragments and Optimized Knowledge-based Force Field

PubMed Central

Xu, Dong; Zhang, Yang

2012-01-01

Ab initio protein folding is one of the major unsolved problems in computational biology due to the difficulties in force field design and conformational search. We developed a novel program, QUARK, for template-free protein structure prediction. Query sequences are first broken into fragments of 1–20 residues where multiple fragment structures are retrieved at each position from unrelated experimental structures. Full-length structure models are then assembled from fragments using replica-exchange Monte Carlo simulations, which are guided by a composite knowledge-based force field. A number of novel energy terms and Monte Carlo movements are introduced and the particular contributions to enhancing the efficiency of both force field and search engine are analyzed in detail. QUARK prediction procedure is depicted and tested on the structure modeling of 145 non-homologous proteins. Although no global templates are used and all fragments from experimental structures with template modeling score (TM-score) >0.5 are excluded, QUARK can successfully construct 3D models of correct folds in 1/3 cases of short proteins up to 100 residues. In the ninth community-wide Critical Assessment of protein Structure Prediction (CASP9) experiment, QUARK server outperformed the second and third best servers by 18% and 47% based on the cumulative Z-score of global distance test-total (GDT-TS) scores in the free modeling (FM) category. Although ab initio protein folding remains a significant challenge, these data demonstrate new progress towards the solution of the most important problem in the field. PMID:22411565

Structural Biology of Pectin Degradation by Enterobacteriaceae

PubMed Central

Abbott, D. Wade; Boraston, Alisdair B.

2008-01-01

Pectin is a structural polysaccharide that is integral for the stability of plant cell walls. During soft rot infection, secreted virulence factors from pectinolytic bacteria such as Erwinia spp. degrade pectin, resulting in characteristic plant cell necrosis and tissue maceration. Catabolism of pectin and its breakdown products by pectinolytic bacteria occurs within distinct cellular environments. This process initiates outside the cell, continues within the periplasmic space, and culminates in the cytoplasm. Although pectin utilization is well understood at the genetic and biochemical levels, an inclusive structural description of pectinases and pectin binding proteins by both extracellular and periplasmic enzymes has been lacking, especially following the recent characterization of several periplasmic components and protein-oligogalacturonide complexes. Here we provide a comprehensive analysis of the protein folds and mechanisms of pectate lyases, polygalacturonases, and carbohydrate esterases and the binding specificities of two periplasmic pectic binding proteins from Enterobacteriaceae. This review provides a structural understanding of the molecular determinants of pectin utilization and the mechanisms driving catabolite selectivity and flow through the pathway. PMID:18535148

Structure of a designed protein cage that self-assembles into a highly porous cube

DOE PAGES

Lai, Yen-Ting; Reading, Eamonn; Hura, Greg L.; ...

2014-11-10

Natural proteins can be versatile building blocks for multimeric, self-assembling structures. Yet, creating protein-based assemblies with specific geometries and chemical properties remains challenging. Highly porous materials represent particularly interesting targets for designed assembly. Here we utilize a strategy of fusing two natural protein oligomers using a continuous alpha-helical linker to design a novel protein that self assembles into a 750 kDa, 225 Å diameter, cube-shaped cage with large openings into a 130 Å diameter inner cavity. A crystal structure of the cage showed atomic level agreement with the designed model, while electron microscopy, native mass spectrometry, and small angle x-raymore » scattering revealed alternate assembly forms in solution. These studies show that accurate design of large porous assemblies with specific shapes is feasible, while further specificity improvements will likely require limiting flexibility to select against alternative forms. Finally, these results provide a foundation for the design of advanced materials with applications in bionanotechnology, nanomedicine and material sciences.« less

The effect of increasing levels of fish oil-containing structured triglycerides on protein metabolism in parenterally fed rats stressed by burn plus endotoxin.

PubMed

Gollaher, C J; Fechner, K; Karlstad, M; Babayan, V K; Bistrian, B R

1993-01-01

This report investigates the effect of various levels of medium-chain/fish oil structured triglycerides on protein and energy metabolism in hypermetabolic rats. Male Sprague-Dawley rats (192 to 226 g) were continuously infused with isovolemic diets that provided 200 kcal/kg per day and 2 g of amino acid nitrogen per kilogram per day. The percentage of nonnitrogen calories as structured triglyceride was varied: no fat, 5%, 15%, or 30%. A 30% long-chain triglyceride diet was also provided as a control to compare the protein-sparing abilities of these two types of fat. Nitrogen excretion, plasma albumin, plasma triglycerides, and whole-body and liver and muscle protein kinetics were determined after 3 days of feeding. Whole-body protein breakdown, flux, and oxidation were similar in all groups. The 15% structured triglyceride diet maximized whole-body protein synthesis (p < .05). Liver fractional synthetic rate was significantly greater in animals receiving 5% of nonprotein calories as structured triglyceride (p < .05). Muscle fractional synthetic rate was unchanged. Plasma triglycerides were markedly elevated in the 30% structured triglyceride-fed rats. The 30% structured triglyceride diet maintained plasma albumin levels better than those diets containing no fat, 5% medium-chain triglyceride/fish oil structured triglyceride, or 30% long-chain triglycerides. Nitrogen excretion was lower in animals receiving 30% of nonnitrogen calories as a structured triglyceride than in those receiving 30% as long-chain triglycerides, but this difference did not reach statistical significance (p = .1). These data suggest that protein metabolism is optimized when structured triglyceride is provided at relatively low dietary fat intakes.

Rapid time-resolved diffraction studies of protein structures using synchrotron radiation

NASA Astrophysics Data System (ADS)

Bartunik, Hans D.; Bartunik, Lesley J.

1992-07-01

The crystal structure of intermediate states in biological reactions of proteins of multi-protein complexes may be studied by time-resolved X-ray diffraction techniques which make use of the high spectral brilliance, continuous wavelength distribution and pulsed time structure of synchrotron radiation. Laue diffraction methods provide a means of investigating intermediate structures with lifetimes in the millisecond time range at presently operational facilities. Third-generation storage rings which are under construction may permit one to reach a time resolution of one microsecond for non-cyclic and one nanosecond for cyclic reactions. The number of individual exposures required for exploring reciprocal space and hence the total time scale strongly depend on the lattice order that may be affected, e.g., by conformational changes. Time-resolved experiments require high population of a specific intermediate which has to be homogeneous over the crystal volume. A number of external excitation techniques have been developed including in situ liberation of active metabolites by laser pulse photolysis of photolabile inactive precursors. First applications to crystal structure analysis of catalytic intermediates of enzymes demonstrate the potential of time-resolved protein crystallography.

An introduction to NMR-based approaches for measuring protein dynamics

PubMed Central

Kleckner, Ian R; Foster, Mark P

2010-01-01

Proteins are inherently flexible at ambient temperature. At equilibrium, they are characterized by a set of conformations that undergo continuous exchange within a hierarchy of spatial and temporal scales ranging from nanometers to micrometers and femtoseconds to hours. Dynamic properties of proteins are essential for describing the structural bases of their biological functions including catalysis, binding, regulation and cellular structure. Nuclear magnetic resonance (NMR) spectroscopy represents a powerful technique for measuring these essential features of proteins. Here we provide an introduction to NMR-based approaches for studying protein dynamics, highlighting eight distinct methods with recent examples, contextualized within a common experimental and analytical framework. The selected methods are (1) Real-time NMR, (2) Exchange spectroscopy, (3) Lineshape analysis, (4) CPMG relaxation dispersion, (5) Rotating frame relaxation dispersion, (6) Nuclear spin relaxation, (7) Residual dipolar coupling, (8) Paramagnetic relaxation enhancement. PMID:21059410

Structure of PA1221, a nonribosomal peptide synthetase containing adenylation and peptidyl carrier protein domains.

PubMed

Mitchell, Carter A; Shi, Ce; Aldrich, Courtney C; Gulick, Andrew M

2012-04-17

Many bacteria use large modular enzymes for the synthesis of polyketide and peptide natural products. These multidomain enzymes contain integrated carrier domains that deliver bound substrates to multiple catalytic domains, requiring coordination of these chemical steps. Nonribosomal peptide synthetases (NRPSs) load amino acids onto carrier domains through the activity of an upstream adenylation domain. Our lab recently determined the structure of an engineered two-domain NRPS containing fused adenylation and carrier domains. This structure adopted a domain-swapped dimer that illustrated the interface between these two domains. To continue our investigation, we now examine PA1221, a natural two-domain protein from Pseudomonas aeruginosa. We have determined the amino acid specificity of this new enzyme and used domain specific mutations to demonstrate that loading the downstream carrier domain within a single protein molecule occurs more quickly than loading of a nonfused carrier domain intermolecularly. Finally, we have determined crystal structures of both apo- and holo-PA1221 proteins, the latter using a valine-adenosine vinylsulfonamide inhibitor that traps the adenylation domain-carrier domain interaction. The protein adopts an interface similar to that seen with the prior adenylation domain-carrier protein construct. A comparison of these structures with previous structures of multidomain NRPSs suggests that a large conformational change within the NRPS adenylation domains guides the carrier domain into the active site for thioester formation.

Antigenic characterization of bovine ephemeral fever rhabdovirus G and GNS glycoproteins expressed from recombinant baculoviruses.

PubMed

Johal, Jasjit; Gresty, Karryn; Kongsuwan, Kritaya; Walker, Peter J

2008-01-01

Recombinant baculoviruses expressing the BEFV envelope glycoprotein G and non-structural glycoprotein G(NS) were constructed. The G protein expressed in insect cells was located on the cell surface and induced spontaneous cell fusion at mildly acidic pH. The expressed G protein reacted with MAbs to continuous and conformational neutralization sites (G1, G2, G3b and G4), but not to conformational site G3a. The expressed G(NS) protein was also located on the cell surface but did not exhibit fusogenic activity. The G(NS) protein reacted with polyclonal antiserum produced from vaccinia-virus-expressed recombinant G(NS) but did not react with G protein antibodies. A His(6)-tagged, soluble form of the G protein was expressed and purified by Ni(2+)-NTA chromatography. The purified G protein reacted with BEFV-neutralizing MAbs to all continuous and conformational antigenic sites. The highly protective characteristics of the native BEFV G protein suggest that the secreted, baculovirus-expressed product may be a useful vaccine antigen.

Elastic strain and twist analysis of protein structural data and allostery of the transmembrane channel KcsA

NASA Astrophysics Data System (ADS)

Mitchell, Michael R.; Leibler, Stanislas

2018-05-01

The abundance of available static protein structural data makes the more effective analysis and interpretation of this data a valuable tool to supplement the experimental study of protein mechanics. Structural displacements can be difficult to analyze and interpret. Previously, we showed that strains provide a more natural and interpretable representation of protein deformations, revealing mechanical coupling between spatially distinct sites of allosteric proteins. Here, we demonstrate that other transformations of displacements yield additional insights. We calculate the divergence and curl of deformations of the transmembrane channel KcsA. Additionally, we introduce quantities analogous to bend, splay, and twist deformation energies of nematic liquid crystals. These transformations enable the decomposition of displacements into different modes of deformation, helping to characterize the type of deformation a protein undergoes. We apply these calculations to study the filter and gating regions of KcsA. We observe a continuous path of rotational deformations physically coupling these two regions, and, we propose, underlying the allosteric interaction between these regions. Bend, splay, and twist distinguish KcsA gate opening, filter opening, and filter-gate coupling, respectively. In general, physically meaningful representations of deformations (like strain, curl, bend, splay, and twist) can make testable predictions and yield insights into protein mechanics, augmenting experimental methods and more fully exploiting available structural data.

How does symmetry impact the flexibility of proteins?

PubMed

Schulze, Bernd; Sljoka, Adnan; Whiteley, Walter

2014-02-13

It is well known that (i) the flexibility and rigidity of proteins are central to their function, (ii) a number of oligomers with several copies of individual protein chains assemble with symmetry in the native state and (iii) added symmetry sometimes leads to added flexibility in structures. We observe that the most common symmetry classes of protein oligomers are also the symmetry classes that lead to increased flexibility in certain three-dimensional structures-and investigate the possible significance of this coincidence. This builds on the well-developed theory of generic rigidity of body-bar frameworks, which permits an analysis of the rigidity and flexibility of molecular structures such as proteins via fast combinatorial algorithms. In particular, we outline some very simple counting rules and possible algorithmic extensions that allow us to predict continuous symmetry-preserving motions in body-bar frameworks that possess non-trivial point-group symmetry. For simplicity, we focus on dimers, which typically assemble with twofold rotational axes, and often have allosteric function that requires motions to link distant sites on the two protein chains.

Ocean acidification impacts mussel control on biomineralisation

PubMed Central

Fitzer, Susan C.; Phoenix, Vernon R.; Cusack, Maggie; Kamenos, Nicholas A.

2014-01-01

Ocean acidification is altering the oceanic carbonate saturation state and threatening the survival of marine calcifying organisms. Production of their calcium carbonate exoskeletons is dependent not only on the environmental seawater carbonate chemistry but also the ability to produce biominerals through proteins. We present shell growth and structural responses by the economically important marine calcifier Mytilus edulis to ocean acidification scenarios (380, 550, 750, 1000 µatm pCO2). After six months of incubation at 750 µatm pCO2, reduced carbonic anhydrase protein activity and shell growth occurs in M. edulis. Beyond that, at 1000 µatm pCO2, biomineralisation continued but with compensated metabolism of proteins and increased calcite growth. Mussel growth occurs at a cost to the structural integrity of the shell due to structural disorientation of calcite crystals. This loss of structural integrity could impact mussel shell strength and reduce protection from predators and changing environments. PMID:25163895

Ocean acidification impacts mussel control on biomineralisation.

PubMed

Fitzer, Susan C; Phoenix, Vernon R; Cusack, Maggie; Kamenos, Nicholas A

2014-08-28

Ocean acidification is altering the oceanic carbonate saturation state and threatening the survival of marine calcifying organisms. Production of their calcium carbonate exoskeletons is dependent not only on the environmental seawater carbonate chemistry but also the ability to produce biominerals through proteins. We present shell growth and structural responses by the economically important marine calcifier Mytilus edulis to ocean acidification scenarios (380, 550, 750, 1000 µatm pCO2). After six months of incubation at 750 µatm pCO2, reduced carbonic anhydrase protein activity and shell growth occurs in M. edulis. Beyond that, at 1000 µatm pCO2, biomineralisation continued but with compensated metabolism of proteins and increased calcite growth. Mussel growth occurs at a cost to the structural integrity of the shell due to structural disorientation of calcite crystals. This loss of structural integrity could impact mussel shell strength and reduce protection from predators and changing environments.

Year 2 Report: Protein Function Prediction Platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, C E

2012-04-27

Upon completion of our second year of development in a 3-year development cycle, we have completed a prototype protein structure-function annotation and function prediction system: Protein Function Prediction (PFP) platform (v.0.5). We have met our milestones for Years 1 and 2 and are positioned to continue development in completion of our original statement of work, or a reasonable modification thereof, in service to DTRA Programs involved in diagnostics and medical countermeasures research and development. The PFP platform is a multi-scale computational modeling system for protein structure-function annotation and function prediction. As of this writing, PFP is the only existing fullymore » automated, high-throughput, multi-scale modeling, whole-proteome annotation platform, and represents a significant advance in the field of genome annotation (Fig. 1). PFP modules perform protein functional annotations at the sequence, systems biology, protein structure, and atomistic levels of biological complexity (Fig. 2). Because these approaches provide orthogonal means of characterizing proteins and suggesting protein function, PFP processing maximizes the protein functional information that can currently be gained by computational means. Comprehensive annotation of pathogen genomes is essential for bio-defense applications in pathogen characterization, threat assessment, and medical countermeasure design and development in that it can short-cut the time and effort required to select and characterize protein biomarkers.« less

Cross-over between discrete and continuous protein structure space: insights into automatic classification and networks of protein structures.

PubMed

Pascual-García, Alberto; Abia, David; Ortiz, Angel R; Bastolla, Ugo

2009-03-01

Structural classifications of proteins assume the existence of the fold, which is an intrinsic equivalence class of protein domains. Here, we test in which conditions such an equivalence class is compatible with objective similarity measures. We base our analysis on the transitive property of the equivalence relationship, requiring that similarity of A with B and B with C implies that A and C are also similar. Divergent gene evolution leads us to expect that the transitive property should approximately hold. However, if protein domains are a combination of recurrent short polypeptide fragments, as proposed by several authors, then similarity of partial fragments may violate the transitive property, favouring the continuous view of the protein structure space. We propose a measure to quantify the violations of the transitive property when a clustering algorithm joins elements into clusters, and we find out that such violations present a well defined and detectable cross-over point, from an approximately transitive regime at high structure similarity to a regime with large transitivity violations and large differences in length at low similarity. We argue that protein structure space is discrete and hierarchic classification is justified up to this cross-over point, whereas at lower similarities the structure space is continuous and it should be represented as a network. We have tested the qualitative behaviour of this measure, varying all the choices involved in the automatic classification procedure, i.e., domain decomposition, alignment algorithm, similarity score, and clustering algorithm, and we have found out that this behaviour is quite robust. The final classification depends on the chosen algorithms. We used the values of the clustering coefficient and the transitivity violations to select the optimal choices among those that we tested. Interestingly, this criterion also favours the agreement between automatic and expert classifications. As a domain set, we have selected a consensus set of 2,890 domains decomposed very similarly in SCOP and CATH. As an alignment algorithm, we used a global version of MAMMOTH developed in our group, which is both rapid and accurate. As a similarity measure, we used the size-normalized contact overlap, and as a clustering algorithm, we used average linkage. The resulting automatic classification at the cross-over point was more consistent than expert ones with respect to the structure similarity measure, with 86% of the clusters corresponding to subsets of either SCOP or CATH superfamilies and fewer than 5% containing domains in distinct folds according to both SCOP and CATH. Almost 15% of SCOP superfamilies and 10% of CATH superfamilies were split, consistent with the notion of fold change in protein evolution. These results were qualitatively robust for all choices that we tested, although we did not try to use alignment algorithms developed by other groups. Folds defined in SCOP and CATH would be completely joined in the regime of large transitivity violations where clustering is more arbitrary. Consistently, the agreement between SCOP and CATH at fold level was lower than their agreement with the automatic classification obtained using as a clustering algorithm, respectively, average linkage (for SCOP) or single linkage (for CATH). The networks representing significant evolutionary and structural relationships between clusters beyond the cross-over point may allow us to perform evolutionary, structural, or functional analyses beyond the limits of classification schemes. These networks and the underlying clusters are available at http://ub.cbm.uam.es/research/ProtNet.php.

Poly(lactic-co-glycolic acid) devices: Production and applications for sustained protein delivery.

PubMed

Lee, Parker W; Pokorski, Jonathan K

2018-03-13

Injectable or implantable poly(lactic-co-glycolic acid) (PLGA) devices for the sustained delivery of proteins have been widely studied and utilized to overcome the necessity of repeated administrations for therapeutic proteins due to poor pharmacokinetic profiles of macromolecular therapies. These devices can come in the form of microparticles, implants, or patches depending on the disease state and route of administration. Furthermore, the release rate can be tuned from weeks to months by controlling the polymer composition, geometry of the device, or introducing additives during device fabrication. Slow-release devices have become a very powerful tool for modern medicine. Production of these devices has initially focused on emulsion-based methods, relying on phase separation to encapsulate proteins within polymeric microparticles. Process parameters and the effect of additives have been thoroughly researched to ensure protein stability during device manufacturing and to control the release profile. Continuous fluidic production methods have also been utilized to create protein-laden PLGA devices through spray drying and electrospray production. Thermal processing of PLGA with solid proteins is an emerging production method that allows for continuous, high-throughput manufacturing of PLGA/protein devices. Overall, polymeric materials for protein delivery remain an emerging field of research for the creation of single administration treatments for a wide variety of disease. This review describes, in detail, methods to make PLGA devices, comparing traditional emulsion-based methods to emerging methods to fabricate protein-laden devices. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Peptide-Based Structures. © 2018 Wiley Periodicals, Inc.

Folding pathway of a multidomain protein depends on its topology of domain connectivity

PubMed Central

Inanami, Takashi; Terada, Tomoki P.; Sasai, Masaki

2014-01-01

How do the folding mechanisms of multidomain proteins depend on protein topology? We addressed this question by developing an Ising-like structure-based model and applying it for the analysis of free-energy landscapes and folding kinetics of an example protein, Escherichia coli dihydrofolate reductase (DHFR). DHFR has two domains, one comprising discontinuous N- and C-terminal parts and the other comprising a continuous middle part of the chain. The simulated folding pathway of DHFR is a sequential process during which the continuous domain folds first, followed by the discontinuous domain, thereby avoiding the rapid decrease in conformation entropy caused by the association of the N- and C-terminal parts during the early phase of folding. Our simulated results consistently explain the observed experimental data on folding kinetics and predict an off-pathway structural fluctuation at equilibrium. For a circular permutant for which the topological complexity of wild-type DHFR is resolved, the balance between energy and entropy is modulated, resulting in the coexistence of the two folding pathways. This coexistence of pathways should account for the experimentally observed complex folding behavior of the circular permutant. PMID:25267632

Astronaut Scott Parazynski works with PCG experiment on middeck

NASA Image and Video Library

1994-11-14

STS066-13-029 (3-14 Nov 1994) --- On the Space Shuttle Atlantis' mid-deck, astronaut Scott E. Parazynski, mission specialist, works at one of two areas onboard the Shuttle which support the Protein Crystal Growth (PCG) experiment. This particular section is called the Vapor Diffusion Apparatus (VDA), housed in a Single Locker Thermal Enclosure (STES). Together with the Crystal Observation System, housed in the Thermal Enclosure System (COS/TES) the VDA represents the continuing research into the structures of proteins and other macromolecules such as viruses. In addition to using the microgravity of space to grow high-quality protein crystals for structural analyses, the experiments are expected to help develop technologies and methods to improve the protein crystallization process on Earth as well as in space.

Proteins and Their Interacting Partners: An Introduction to Protein-Ligand Binding Site Prediction Methods.

PubMed

Roche, Daniel Barry; Brackenridge, Danielle Allison; McGuffin, Liam James

2015-12-15

Elucidating the biological and biochemical roles of proteins, and subsequently determining their interacting partners, can be difficult and time consuming using in vitro and/or in vivo methods, and consequently the majority of newly sequenced proteins will have unknown structures and functions. However, in silico methods for predicting protein-ligand binding sites and protein biochemical functions offer an alternative practical solution. The characterisation of protein-ligand binding sites is essential for investigating new functional roles, which can impact the major biological research spheres of health, food, and energy security. In this review we discuss the role in silico methods play in 3D modelling of protein-ligand binding sites, along with their role in predicting biochemical functionality. In addition, we describe in detail some of the key alternative in silico prediction approaches that are available, as well as discussing the Critical Assessment of Techniques for Protein Structure Prediction (CASP) and the Continuous Automated Model EvaluatiOn (CAMEO) projects, and their impact on developments in the field. Furthermore, we discuss the importance of protein function prediction methods for tackling 21st century problems.

Packing in protein cores

NASA Astrophysics Data System (ADS)

Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

2017-07-01

Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

Structural changes and fluctuations of proteins. I. A statistical thermodynamic model.

PubMed

Ikegami, A

1977-01-01

A general theory of the structural changes and fluctuations of proteins has been proposed based on statistical thermodynamic considerations at the chain level. The "structure" of protein was assumed to be characterized by the state of secondary bonds between unique pairs of specific sites on peptide chains. Every secondary bond changes between the bonded and unbonded states by thermal agitation and the "structure" is continuously fluctuating. The free energy of the "structural state" that is defined by the fraction of secondary bonds in the bonded state has been expressed by the bond energy, the cooperative interaction between bonds, the mixing entropy of bonds, and the entropy of polypeptide chains. The most probable "structural state" can be simply determined by graphical analysis and the effect of temperature or solvent composition on it is discussed. The temperature dependence of the free energy, the probability distribution of structural states and the specific heat have been calculted for two examples of structural change. The theory predicts two different types of structural changes from the ordered to disorderd state, a "structured transition" and a "gradual structural change" with rising temperature. In the "structural transition", the probability distribution has two maxima in the temperature range of transition. In the "gradual structural change", the probabilty distribution has only one maximum during the change. A considerable fraction of secondary bonds is in the unbounded state and is always fluctuating even in the ordered state at room temperature. Such structural flucutations in a single protein molecule have been discussed quantitatively. The theory is extended to include small molecules which bind to the protein molecule and affect the structural state. The changes of structural state caused by specific and non-specific binding and allosteric effects are explained in a unified manner.

EVAcon: a protein contact prediction evaluation service

PubMed Central

Graña, Osvaldo; Eyrich, Volker A.; Pazos, Florencio; Rost, Burkhard; Valencia, Alfonso

2005-01-01

Here we introduce EVAcon, an automated web service that evaluates the performance of contact prediction servers. Currently, EVAcon is monitoring nine servers, four of which are specialized in contact prediction and five are general structure prediction servers. Results are compared for all newly determined experimental structures deposited into PDB (∼5–50 per week). EVAcon allows for a precise comparison of the results based on a system of common protein subsets and the commonly accepted evaluation criteria that are also used in the corresponding category of the CASP assessment. EVAcon is a new service added to the functionality of the EVA system for the continuous evaluation of protein structure prediction servers. The new service is accesible from any of the three EVA mirrors: PDG (CNB-CSIC, Madrid) (); CUBIC (Columbia University, NYC) (); and Sali Lab (UCSF, San Francisco) (). PMID:15980486

Structural dynamics of the lac repressor-DNA complex revealed by a multiscale simulation.

PubMed

Villa, Elizabeth; Balaeff, Alexander; Schulten, Klaus

2005-05-10

A multiscale simulation of a complex between the lac repressor protein (LacI) and a 107-bp-long DNA segment is reported. The complex between the repressor and two operator DNA segments is described by all-atom molecular dynamics; the size of the simulated system comprises either 226,000 or 314,000 atoms. The DNA loop connecting the operators is modeled as a continuous elastic ribbon, described mathematically by the nonlinear Kirchhoff differential equations with boundary conditions obtained from the coordinates of the terminal base pairs of each operator. The forces stemming from the looped DNA are included in the molecular dynamics simulations; the loop structure and the forces are continuously recomputed because the protein motions during the simulations shift the operators and the presumed termini of the loop. The simulations reveal the structural dynamics of the LacI-DNA complex in unprecedented detail. The multiple domains of LacI exhibit remarkable structural stability during the simulation, moving much like rigid bodies. LacI is shown to absorb the strain from the looped DNA mainly through its mobile DNA-binding head groups. Even with large fluctuating forces applied, the head groups tilt strongly and keep their grip on the operator DNA, while the remainder of the protein retains its V-shaped structure. A simulated opening of the cleft of LacI by 500-pN forces revealed the interactions responsible for locking LacI in the V-conformation.

Protein Crystal Growth (PCG) experiment aboard mission STS-66

NASA Technical Reports Server (NTRS)

2000-01-01

On the Space Shuttle Orbiter Atlantis' middeck, Astronaut Joseph R. Tarner, mission specialist, works at an area amidst several lockers which support the Protein Crystal Growth (PCG) experiment during the STS-66 mission. This particular section is called the Crystal Observation System, housed in the Thermal Enclosure System (COS/TES). Together with the Vapor Diffusion Apparatus (VDA), housed in Single Locker Thermal Enclosure (SLTES), the COS/TES represents the continuing research into the structure of proteins and other macromolecules such as viruses.

Motif discovery with data mining in 3D protein structure databases: discovery, validation and prediction of the U-shape zinc binding ("Huf-Zinc") motif.

PubMed

Maurer-Stroh, Sebastian; Gao, He; Han, Hao; Baeten, Lies; Schymkowitz, Joost; Rousseau, Frederic; Zhang, Louxin; Eisenhaber, Frank

2013-02-01

Data mining in protein databases, derivatives from more fundamental protein 3D structure and sequence databases, has considerable unearthed potential for the discovery of sequence motif--structural motif--function relationships as the finding of the U-shape (Huf-Zinc) motif, originally a small student's project, exemplifies. The metal ion zinc is critically involved in universal biological processes, ranging from protein-DNA complexes and transcription regulation to enzymatic catalysis and metabolic pathways. Proteins have evolved a series of motifs to specifically recognize and bind zinc ions. Many of these, so called zinc fingers, are structurally independent globular domains with discontinuous binding motifs made up of residues mostly far apart in sequence. Through a systematic approach starting from the BRIX structure fragment database, we discovered that there exists another predictable subset of zinc-binding motifs that not only have a conserved continuous sequence pattern but also share a characteristic local conformation, despite being included in totally different overall folds. While this does not allow general prediction of all Zn binding motifs, a HMM-based web server, Huf-Zinc, is available for prediction of these novel, as well as conventional, zinc finger motifs in protein sequences. The Huf-Zinc webserver can be freely accessed through this URL (http://mendel.bii.a-star.edu.sg/METHODS/hufzinc/).

Modification of solubility and heat-induced gelation of amaranth 11S globulin by protein engineering.

PubMed

Carrazco-Peña, Laura; Osuna-Castro, Juan A; De León-Rodríguez, Antonio; Maruyama, Nobuyuki; Toro-Vazquez, Jorge F; Morales-Rueda, Juan A; Barba de la Rosa, Ana P

2013-04-10

The primary structure of amaranth 11S globulin (Ah11S) was engineered with the aim to improve its functional properties. Four continuous methionines were inserted in variable region V, obtaining the Ah11Sr+4M construction. Changes on protein structure and surface characteristics were analyzed in silico. Solubility and heat-induced gelation of recombinant amaranth 11S proglobulin (Ah11Sr and Ah11Sr+4M) were compared with the native protein (Ah11Sn) purified from amaranth seed flour. The Ah11Sr+4 M showed the highest surface hydrophobicity, but as consequence the solubility was reduced. At low ionic strength (μ = 0.2) and acidic pH (<4.1), the recombinant proteins Ah11Sr and Ah11Sr+4 M had the highest and lowest solubility values, respectively. All globulins samples formed gels at 90 °C and low ionic strength, but Ah11Sn produced the weakest and Ah11Sr the strongest gels. Differential scanning calorimetry analysis under gel forming conditions revealed only exothermic transitions for all amaranth 11S globulins analyzed. In conclusion, the 3D structure analysis has revealed interesting molecular features that could explain the thermal resistance and gel forming ability of amaranth 11S globulins. The incorporation of four continuous methionines in amaranth increased the hydrophobicity, and self-supporting gels formed had intermediate hardness between Ah11Sn and Ah11Sr. These functional properties could be used in the food industry for the development of new products based on amaranth proteins.

PRince: a web server for structural and physicochemical analysis of protein-RNA interface.

PubMed

Barik, Amita; Mishra, Abhishek; Bahadur, Ranjit Prasad

2012-07-01

We have developed a web server, PRince, which analyzes the structural features and physicochemical properties of the protein-RNA interface. Users need to submit a PDB file containing the atomic coordinates of both the protein and the RNA molecules in complex form (in '.pdb' format). They should also mention the chain identifiers of interacting protein and RNA molecules. The size of the protein-RNA interface is estimated by measuring the solvent accessible surface area buried in contact. For a given protein-RNA complex, PRince calculates structural, physicochemical and hydration properties of the interacting surfaces. All these parameters generated by the server are presented in a tabular format. The interacting surfaces can also be visualized with software plug-in like Jmol. In addition, the output files containing the list of the atomic coordinates of the interacting protein, RNA and interface water molecules can be downloaded. The parameters generated by PRince are novel, and users can correlate them with the experimentally determined biophysical and biochemical parameters for better understanding the specificity of the protein-RNA recognition process. This server will be continuously upgraded to include more parameters. PRince is publicly accessible and free for use. Available at http://www.facweb.iitkgp.ernet.in/~rbahadur/prince/home.html.

Twilight reloaded: the peptide experience.

PubMed

Weichenberger, Christian X; Pozharski, Edwin; Rupp, Bernhard

2017-03-01

The de facto commoditization of biomolecular crystallography as a result of almost disruptive instrumentation automation and continuing improvement of software allows any sensibly trained structural biologist to conduct crystallographic studies of biomolecules with reasonably valid outcomes: that is, models based on properly interpreted electron density. Robust validation has led to major mistakes in the protein part of structure models becoming rare, but some depositions of protein-peptide complex structure models, which generally carry significant interest to the scientific community, still contain erroneous models of the bound peptide ligand. Here, the protein small-molecule ligand validation tool Twilight is updated to include peptide ligands. (i) The primary technical reasons and potential human factors leading to problems in ligand structure models are presented; (ii) a new method used to score peptide-ligand models is presented; (iii) a few instructive and specific examples, including an electron-density-based analysis of peptide-ligand structures that do not contain any ligands, are discussed in detail; (iv) means to avoid such mistakes and the implications for database integrity are discussed and (v) some suggestions as to how journal editors could help to expunge errors from the Protein Data Bank are provided.

An alternative view of protein fold space.

PubMed

Shindyalov, I N; Bourne, P E

2000-02-15

Comparing and subsequently classifying protein structures information has received significant attention concurrent with the increase in the number of experimentally derived 3-dimensional structures. Classification schemes have focused on biological function found within protein domains and on structure classification based on topology. Here an alternative view is presented that groups substructures. Substructures are long (50-150 residue) highly repetitive near-contiguous pieces of polypeptide chain that occur frequently in a set of proteins from the PDB defined as structurally non-redundant over the complete polypeptide chain. The substructure classification is based on a previously reported Combinatorial Extension (CE) algorithm that provides a significantly different set of structure alignments than those previously described, having, for example, only a 40% overlap with FSSP. Qualitatively the algorithm provides longer contiguous aligned segments at the price of a slightly higher root-mean-square deviation (rmsd). Clustering these alignments gives a discreet and highly repetitive set of substructures not detectable by sequence similarity alone. In some cases different substructures represent all or different parts of well known folds indicative of the Russian doll effect--the continuity of protein fold space. In other cases they fall into different structure and functional classifications. It is too early to determine whether these newly classified substructures represent new insights into the evolution of a structural framework important to many proteins. What is apparent from on-going work is that these substructures have the potential to be useful probes in finding remote sequence homology and in structure prediction studies. The characteristics of the complete all-by-all comparison of the polypeptide chains present in the PDB and details of the filtering procedure by pair-wise structure alignment that led to the emergent substructure gallery are discussed. Substructure classification, alignments, and tools to analyze them are available at http://cl.sdsc.edu/ce.html.

Foldability of a Natural De Novo Evolved Protein.

PubMed

Bungard, Dixie; Copple, Jacob S; Yan, Jing; Chhun, Jimmy J; Kumirov, Vlad K; Foy, Scott G; Masel, Joanna; Wysocki, Vicki H; Cordes, Matthew H J

2017-11-07

The de novo evolution of protein-coding genes from noncoding DNA is emerging as a source of molecular innovation in biology. Studies of random sequence libraries, however, suggest that young de novo proteins will not fold into compact, specific structures typical of native globular proteins. Here we show that Bsc4, a functional, natural de novo protein encoded by a gene that evolved recently from noncoding DNA in the yeast S. cerevisiae, folds to a partially specific three-dimensional structure. Bsc4 forms soluble, compact oligomers with high β sheet content and a hydrophobic core, and undergoes cooperative, reversible denaturation. Bsc4 lacks a specific quaternary state, however, existing instead as a continuous distribution of oligomer sizes, and binds dyes indicative of amyloid oligomers or molten globules. The combination of native-like and non-native-like properties suggests a rudimentary fold that could potentially act as a functional intermediate in the emergence of new folded proteins de novo. Copyright © 2017 Elsevier Ltd. All rights reserved.

Painting proteins with covalent labels: what's in the picture?

PubMed

Fitzgerald, Michael C; West, Graham M

2009-06-01

Knowledge about the structural and biophysical properties of proteins when they are free in solution and/or in complexes with other molecules is essential for understanding the biological processes that proteins regulate. Such knowledge is also important to drug discovery efforts, particularly those focused on the development of therapeutic agents with protein targets. In the last decade a variety of different covalent labeling techniques have been used in combination with mass spectrometry to probe the solution-phase structures and biophysical properties of proteins and protein-ligand complexes. Highlighted here are five different mass spectrometry-based covalent labeling strategies including: continuous hydrogen/deuterium (H/D) exchange labeling, hydroxyl radical-mediated footprinting, SUPREX (stability of unpurified proteins from rates of H/D exchange), PLIMSTEX (protein-ligand interaction by mass spectrometry, titration, and H/D exchange), and SPROX (stability of proteins from rates of oxidation). The basic experimental protocols used in each of the above-cited methods are summarized along with the kind of biophysical information they generate. Also discussed are the relative strengths and weaknesses of the different methods for probing the wide range of conformational states that proteins and protein-ligand complexes can adopt when they are in solution.

The Mitosis and Neurodevelopment Proteins NDE1 and NDEL1 Form Dimers, Tetramers, and Polymers with a Folded Back Structure in Solution*

PubMed Central

Soares, Dinesh C.; Bradshaw, Nicholas J.; Zou, Juan; Kennaway, Christopher K.; Hamilton, Russell S.; Chen, Zhuo A.; Wear, Martin A.; Blackburn, Elizabeth A.; Bramham, Janice; Böttcher, Bettina; Millar, J. Kirsty; Barlow, Paul N.; Walkinshaw, Malcolm D.; Rappsilber, Juri; Porteous, David J.

2012-01-01

Paralogs NDE1 (nuclear distribution element 1) and NDEL1 (NDE-like 1) are essential for mitosis and neurodevelopment. Both proteins are predicted to have similar structures, based upon high sequence similarity, and they co-complex in mammalian cells. X-ray diffraction studies and homology modeling suggest that their N-terminal regions (residues 8–167) adopt continuous, extended α-helical coiled-coil structures, but no experimentally derived information on the structure of their C-terminal regions or the architecture of the full-length proteins is available. In the case of NDE1, no biophysical data exists. Here we characterize the structural architecture of both full-length proteins utilizing negative stain electron microscopy along with our established paradigm of chemical cross-linking followed by tryptic digestion, mass spectrometry, and database searching, which we enhance using isotope labeling for mixed NDE1-NDEL1. We determined that full-length NDE1 forms needle-like dimers and tetramers in solution, similar to crystal structures of NDEL1, as well as chain-like end-to-end polymers. The C-terminal domain of each protein, required for interaction with key protein partners dynein and DISC1 (disrupted-in-schizophrenia 1), includes a predicted disordered region that allows a bent back structure. This facilitates interaction of the C-terminal region with the N-terminal coiled-coil domain and is in agreement with previous results showing N- and C-terminal regions of NDEL1 and NDE1 cooperating in dynein interaction. It sheds light on recently identified mutations in the NDE1 gene that cause truncation of the encoded protein. Additionally, analysis of mixed NDE1-NDEL1 complexes demonstrates that NDE1 and NDEL1 can interact directly. PMID:22843697

Solution NMR Refinement of a Metal Ion Bound Protein Using Metal Ion Inclusive Restrained Molecular Dynamics Methods

PubMed Central

Chakravorty, Dhruva K.; Wang, Bing; Lee, Chul Won; Guerra, Alfredo J.; Giedroc, David P.; Merz, Kenneth M.

2013-01-01

Correctly calculating the structure of metal coordination sites in a protein during the process of nuclear magnetic resonance (NMR) structure determination and refinement continues to be a challenging task. In this study, we present an accurate and convenient means by which to include metal ions in the NMR structure determination process using molecular dynamics (MD) constrained by NMR-derived data to obtain a realistic and physically viable description of the metal binding site(s). This method provides the framework to accurately portray the metal ions and its binding residues in a pseudo-bond or dummy-cation like approach, and is validated by quantum mechanical/molecular mechanical (QM/MM) MD calculations constrained by NMR-derived data. To illustrate this approach, we refine the zinc coordination complex structure of the zinc sensing transcriptional repressor protein Staphylococcus aureus CzrA, generating over 130 ns of MD and QM/MM MD NMR-data compliant sampling. In addition to refining the first coordination shell structure of the Zn(II) ion, this protocol benefits from being performed in a periodically replicated solvation environment including long-range electrostatics. We determine that unrestrained (not based on NMR data) MD simulations correlated to the NMR data in a time-averaged ensemble. The accurate solution structure ensemble of the metal-bound protein accurately describes the role of conformational dynamics in allosteric regulation of DNA binding by zinc and serves to validate our previous unrestrained MD simulations of CzrA. This methodology has potentially broad applicability in the structure determination of metal ion bound proteins, protein folding and metal template protein-design studies. PMID:23609042

Spring-loaded model revisited: paramyxovirus fusion requires engagement of a receptor binding protein beyond initial triggering of the fusion protein.

PubMed

Porotto, Matteo; Devito, Ilaria; Palmer, Samantha G; Jurgens, Eric M; Yee, Jia L; Yokoyama, Christine C; Pessi, Antonello; Moscona, Anne

2011-12-01

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry.

Role of individual cysteine residues in the processing and antigenicity of the measles virus haemagglutinin protein.

PubMed

Hu, A; Norrby, E

1994-09-01

The haemagglutinin (H) protein is the dominant envelope glycoprotein of measles virus. The protein contains 13 cysteine residues among its 617 amino acids and all are located in its ectodomain. In previous studies, the capacity of a panel of monoclonal antibodies (MAbs) to react with continuous and discontinuous epitopes was defined. It was shown that the absence of disulphide bonds impaired the capacity of the protein to react with MAbs specific for the discontinuous epitopes. In the present study, our objective was to determine the contribution of individual cysteine residues to the folding of H protein into its native conformation. Site-directed oligonucleotide mutagenesis was used to create 13 mutants, each with a serine replacing a cysteine. The mutated genes were directly expressed in the BHK-21 cells by use of a vaccinia virus-driven T7 polymerase system. Investigations of the antigenic structure and intracellular processing properties of the mutant proteins reveal the following outcome. (i) Replacements of cysteine residues 139, 154, 188, 386, 570 or 606 had no detectable effect on the antigenic structure and intracellular processing of the H protein. However, a mutant with a replaced cysteine residue 154 displayed modified migration properties. (ii) Alterations of cysteine residues 381 or 494 displayed a moderate effect on H protein properties. The two mutants expressed discontinuous epitopes, indicating that they were partially folded, but they did not oligomerize, did not reach the medial Golgi complex and failed to be transported to the cell surface. (iii) Substitutions of cysteine residues 287, 300, 394, 579 or 583 resulted in a complete loss of binding of the MAbs that recognize the discontinuous epitopes, with no effect on the binding of a MAb reacting with a continuous epitope. No dimeric form of the proteins was observed and only high mannose oligosaccharides were demonstrated in these mutants, suggesting that the modified proteins did not oligomerize and were retained in the endoplasmic reticulum. In conclusion, cysteine residues 287, 300, 381, 394, 494, 579 and 583 appear to play a particularly critical role in the antigenic structure and processing of the H molecules and they probably participate in the inter- or intramolecular disulphide bonding.

Bhageerath-H: A homology/ab initio hybrid server for predicting tertiary structures of monomeric soluble proteins

PubMed Central

2014-01-01

Background The advent of human genome sequencing project has led to a spurt in the number of protein sequences in the databanks. Success of structure based drug discovery severely hinges on the availability of structures. Despite significant progresses in the area of experimental protein structure determination, the sequence-structure gap is continually widening. Data driven homology based computational methods have proved successful in predicting tertiary structures for sequences sharing medium to high sequence similarities. With dwindling similarities of query sequences, advanced homology/ ab initio hybrid approaches are being explored to solve structure prediction problem. Here we describe Bhageerath-H, a homology/ ab initio hybrid software/server for predicting protein tertiary structures with advancing drug design attempts as one of the goals. Results Bhageerath-H web-server was validated on 75 CASP10 targets which showed TM-scores ≥0.5 in 91% of the cases and Cα RMSDs ≤5Å from the native in 58% of the targets, which is well above the CASP10 water mark. Comparison with some leading servers demonstrated the uniqueness of the hybrid methodology in effectively sampling conformational space, scoring best decoys and refining low resolution models to high and medium resolution. Conclusion Bhageerath-H methodology is web enabled for the scientific community as a freely accessible web server. The methodology is fielded in the on-going CASP11 experiment. PMID:25521245

Recrystallization inhibition in ice due to ice binding protein activity detected by nuclear magnetic resonance.

PubMed

Brown, Jennifer R; Seymour, Joseph D; Brox, Timothy I; Skidmore, Mark L; Wang, Chen; Christner, Brent C; Luo, Bing-Hao; Codd, Sarah L

2014-09-01

Liquid water present in polycrystalline ice at the interstices between ice crystals results in a network of liquid-filled veins and nodes within a solid ice matrix, making ice a low porosity porous media. Here we used nuclear magnetic resonance (NMR) relaxation and time dependent self-diffusion measurements developed for porous media applications to monitor three dimensional changes to the vein network in ices with and without a bacterial ice binding protein (IBP). Shorter effective diffusion distances were detected as a function of increased irreversible ice binding activity, indicating inhibition of ice recrystallization and persistent small crystal structure. The modification of ice structure by the IBP demonstrates a potential mechanism for the microorganism to enhance survivability in ice. These results highlight the potential of NMR techniques in evaluation of the impact of IBPs on vein network structure and recrystallization processes; information useful for continued development of ice-interacting proteins for biotechnology applications.

A global optimization algorithm for protein surface alignment

PubMed Central

2010-01-01

Background A relevant problem in drug design is the comparison and recognition of protein binding sites. Binding sites recognition is generally based on geometry often combined with physico-chemical properties of the site since the conformation, size and chemical composition of the protein surface are all relevant for the interaction with a specific ligand. Several matching strategies have been designed for the recognition of protein-ligand binding sites and of protein-protein interfaces but the problem cannot be considered solved. Results In this paper we propose a new method for local structural alignment of protein surfaces based on continuous global optimization techniques. Given the three-dimensional structures of two proteins, the method finds the isometric transformation (rotation plus translation) that best superimposes active regions of two structures. We draw our inspiration from the well-known Iterative Closest Point (ICP) method for three-dimensional (3D) shapes registration. Our main contribution is in the adoption of a controlled random search as a more efficient global optimization approach along with a new dissimilarity measure. The reported computational experience and comparison show viability of the proposed approach. Conclusions Our method performs well to detect similarity in binding sites when this in fact exists. In the future we plan to do a more comprehensive evaluation of the method by considering large datasets of non-redundant proteins and applying a clustering technique to the results of all comparisons to classify binding sites. PMID:20920230

Do homologous thermophilic-mesophilic proteins exhibit similar structures and dynamics at optimal growth temperatures? A molecular dynamics simulation study.

PubMed

Basu, Sohini; Sen, Srikanta

2013-02-25

Structure and dynamics both are known to be important for the activity of a protein. A fundamental question is whether a thermophilic protein and its mesophilic homologue exhibit similar dynamics at their respective optimal growth temperatures. We have addressed this question by performing molecular dynamics (MD) simulations of a natural mesophilic-thermophilic homologue pair at their respective optimal growth temperatures to compare their structural, dynamical, and solvent properties. The MD simulations were done in explicit aqueous solvent under periodic boundary and constant pressure and temperature (CPT) conditions and continued for 10.0 ns using the same protocol for the two proteins, excepting the temperatures. The trajectories were analyzed to compare the properties of the two proteins. Results indicated that the dynamical behaviors of the two proteins at the respective optimal growth temperatures were remarkably similar. For the common residues in the thermophilic protein, the rms fluctuations have a general trend to be slightly higher compared to that in the mesophilic counterpart. Lindemann parameter values indicated that only a few residues exhibited solid-like dynamics while the protein as a whole appeared as a molten globule in each case. Interestingly, the water-water interaction was found to be strikingly similar in spite of the difference in temperatures while, the protein-water interaction was significantly different in the two simulations.

Mass spectrometric analyses of organophosphate insecticide oxon protein adducts.

PubMed

Thompson, Charles M; Prins, John M; George, Kathleen M

2010-01-01

Organophosphate (OP) insecticides continue to be used to control insect pests. Acute and chronic exposures to OP insecticides have been documented to cause adverse health effects, but few OP-adducted proteins have been correlated with these illnesses at the molecular level. Our aim was to review the literature covering the current state of the art in mass spectrometry (MS) used to identify OP protein biomarkers. We identified general and specific research reports related to OP insecticides, OP toxicity, OP structure, and protein MS by searching PubMed and Chemical Abstracts for articles published before December 2008. A number of OP-based insecticides share common structural elements that result in predictable OP-protein adducts. The resultant OP-protein adducts show an increase in molecular mass that can be identified by MS and correlated with the OP agent. Customized OP-containing probes have also been used to tag and identify protein targets that can be identified by MS. MS is a useful and emerging tool for the identification of proteins that are modified by activated organophosphate insecticides. MS can characterize the structure of the OP adduct and also the specific amino acid residue that forms the key bond with the OP. Each protein that is modified in a unique way by an OP represents a unique molecular biomarker that with further research can lead to new correlations with exposure.

Mass Spectrometric Analyses of Organophosphate Insecticide Oxon Protein Adducts

PubMed Central

Thompson, Charles M.; Prins, John M.; George, Kathleen M.

2010-01-01

Objective Organophosphate (OP) insecticides continue to be used to control insect pests. Acute and chronic exposures to OP insecticides have been documented to cause adverse health effects, but few OP-adducted proteins have been correlated with these illnesses at the molecular level. Our aim was to review the literature covering the current state of the art in mass spectrometry (MS) used to identify OP protein biomarkers. Data sources and extraction We identified general and specific research reports related to OP insecticides, OP toxicity, OP structure, and protein MS by searching PubMed and Chemical Abstracts for articles published before December 2008. Data synthesis A number of OP-based insecticides share common structural elements that result in predictable OP–protein adducts. The resultant OP–protein adducts show an increase in molecular mass that can be identified by MS and correlated with the OP agent. Customized OP-containing probes have also been used to tag and identify protein targets that can be identified by MS. Conclusions MS is a useful and emerging tool for the identification of proteins that are modified by activated organophosphate insecticides. MS can characterize the structure of the OP adduct and also the specific amino acid residue that forms the key bond with the OP. Each protein that is modified in a unique way by an OP represents a unique molecular biomarker that with further research can lead to new correlations with exposure. PMID:20056576

Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahms, Sven O., E-mail: sdahms@fli-leibniz.de; Mayer, Magnus C.; Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow

2015-03-01

Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects variousmore » (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.« less

Real-time HD Exchange Kinetics of Proteins from Buffered Aqueous Solution with Electrothermal Supercharging and Top-Down Tandem Mass Spectrometry

NASA Astrophysics Data System (ADS)

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

2016-06-01

Electrothermal supercharging (ETS) with electrospray ionization produces highly charged protein ions from buffered aqueous solutions in which proteins have native folded structures. ETS increases the charge of ribonuclease A by 34%, whereas only a 6% increase in charge occurs for a reduced-alkylated form of this protein, which is unfolded and its structure is ~66% random coil in this solution. These results indicate that protein denaturation that occurs in the ESI droplets is the primary mechanism for ETS. ETS does not affect the extent of solution-phase hydrogen-deuterium exchange (HDX) that occurs for four proteins that have significantly different structures in solution, consistent with a droplet lifetime that is considerably shorter than observable rates of HDX. Rate constants for HDX of ubiquitin are obtained with a spatial resolution of ~1.3 residues with ETS and electron transfer dissociation of the 10+ charge-state using a single capillary containing a few μL of protein solution in which HDX continuously occurs. HDX protection at individual residues with ETS HDX is similar to that with reagent supercharging HDX and with solution-phase NMR, indicating that the high spray potentials required to induce ETS do not lead to HD scrambling.

Spring-Loaded Model Revisited: Paramyxovirus Fusion Requires Engagement of a Receptor Binding Protein beyond Initial Triggering of the Fusion Protein▿

PubMed Central

Porotto, Matteo; DeVito, Ilaria; Palmer, Samantha G.; Jurgens, Eric M.; Yee, Jia L.; Yokoyama, Christine C.; Pessi, Antonello; Moscona, Anne

2011-01-01

During paramyxovirus entry into a host cell, receptor engagement by a specialized binding protein triggers conformational changes in the adjacent fusion protein (F), leading to fusion between the viral and cell membranes. According to the existing paradigm of paramyxovirus membrane fusion, the initial activation of F by the receptor binding protein sets off a spring-loaded mechanism whereby the F protein progresses independently through the subsequent steps in the fusion process, ending in membrane merger. For human parainfluenza virus type 3 (HPIV3), the receptor binding protein (hemagglutinin-neuraminidase [HN]) has three functions: receptor binding, receptor cleaving, and activating F. We report that continuous receptor engagement by HN activates F to advance through the series of structural rearrangements required for fusion. In contrast to the prevailing model, the role of HN-receptor engagement in the fusion process is required beyond an initiating step, i.e., it is still required even after the insertion of the fusion peptide into the target cell membrane, enabling F to mediate membrane merger. We also report that for Nipah virus, whose receptor binding protein has no receptor-cleaving activity, the continuous stimulation of the F protein by a receptor-engaged binding protein is key for fusion. We suggest a general model for paramyxovirus fusion activation in which receptor engagement plays an active role in F activation, and the continued engagement of the receptor binding protein is essential to F protein function until the onset of membrane merger. This model has broad implications for the mechanism of paramyxovirus fusion and for strategies to prevent viral entry. PMID:21976650

The nucleotide-free state of heterotrimeric G proteins α-subunit adopts a highly stable conformation.

PubMed

Andhirka, Sai Krishna; Vignesh, Ravichandran; Aradhyam, Gopala Krishna

2017-08-01

Deciphering the mechanism of activation of heterotrimeric G proteins by their cognate receptors continues to be an intriguing area of research. The recently solved crystal structure of the ternary complex captured the receptor-bound α-subunit in an open conformation, without bound nucleotide has improved our understanding of the activation process. Despite these advancements, the mechanism by which the receptor causes GDP release from the α-subunit remains elusive. To elucidate the mechanism of activation, we studied guanine nucleotide-induced structural stability of the α-subunit (in response to thermal/chaotrope-mediated stress). Inherent stabilities of the inactive (GDP-bound) and active (GTP-bound) forms contribute antagonistically to the difference in conformational stability whereas the GDP-bound protein is able to switch to a stable intermediate state, GTP-bound protein loses this ability. Partial perturbation of the protein fold reveals the underlying influence of the bound nucleotide providing an insight into the mechanism of activation. An extra stable, pretransition intermediate, 'empty pocket' state (conformationally active-state like) in the unfolding pathway of GDP-bound protein mimics a gating system - the activation process having to overcome this stable intermediate state. We demonstrate that a relatively more complex conformational fold of the GDP-bound protein is at the core of the gating system. We report capturing this threshold, 'metastable empty pocket' conformation (the gate) of α-subunit of G protein and hypothesize that the receptor activates the G protein by enabling it to achieve this structure through mild structural perturbation. © 2017 Federation of European Biochemical Societies.

The protein structure prediction problem could be solved using the current PDB library

PubMed Central

Zhang, Yang; Skolnick, Jeffrey

2005-01-01

For single-domain proteins, we examine the completeness of the structures in the current Protein Data Bank (PDB) library for use in full-length model construction of unknown sequences. To address this issue, we employ a comprehensive benchmark set of 1,489 medium-size proteins that cover the PDB at the level of 35% sequence identity and identify templates by structure alignment. With homologous proteins excluded, we can always find similar folds to native with an average rms deviation (RMSD) from native of 2.5 Å with ≈82% alignment coverage. These template structures often contain a significant number of insertions/deletions. The tasser algorithm was applied to build full-length models, where continuous fragments are excised from the top-scoring templates and reassembled under the guide of an optimized force field, which includes consensus restraints taken from the templates and knowledge-based statistical potentials. For almost all targets (except for 2/1,489), the resultant full-length models have an RMSD to native below 6 Å (97% of them below 4 Å). On average, the RMSD of full-length models is 2.25 Å, with aligned regions improved from 2.5 Å to 1.88 Å, comparable with the accuracy of low-resolution experimental structures. Furthermore, starting from state-of-the-art structural alignments, we demonstrate a methodology that can consistently bring template-based alignments closer to native. These results are highly suggestive that the protein-folding problem can in principle be solved based on the current PDB library by developing efficient fold recognition algorithms that can recover such initial alignments. PMID:15653774

CORAL: aligning conserved core regions across domain families.

PubMed

Fong, Jessica H; Marchler-Bauer, Aron

2009-08-01

Homologous protein families share highly conserved sequence and structure regions that are frequent targets for comparative analysis of related proteins and families. Many protein families, such as the curated domain families in the Conserved Domain Database (CDD), exhibit similar structural cores. To improve accuracy in aligning such protein families, we propose a profile-profile method CORAL that aligns individual core regions as gap-free units. CORAL computes optimal local alignment of two profiles with heuristics to preserve continuity within core regions. We benchmarked its performance on curated domains in CDD, which have pre-defined core regions, against COMPASS, HHalign and PSI-BLAST, using structure superpositions and comprehensive curator-optimized alignments as standards of truth. CORAL improves alignment accuracy on core regions over general profile methods, returning a balanced score of 0.57 for over 80% of all domain families in CDD, compared with the highest balanced score of 0.45 from other methods. Further, CORAL provides E-values to aid in detecting homologous protein families and, by respecting block boundaries, produces alignments with improved 'readability' that facilitate manual refinement. CORAL will be included in future versions of the NCBI Cn3D/CDTree software, which can be downloaded at http://www.ncbi.nlm.nih.gov/Structure/cdtree/cdtree.shtml. Supplementary data are available at Bioinformatics online.

Mutual adaptation of a membrane protein and its lipid bilayer during conformational changes.

PubMed

Sonntag, Yonathan; Musgaard, Maria; Olesen, Claus; Schiøtt, Birgit; Møller, Jesper Vuust; Nissen, Poul; Thøgersen, Lea

2011-01-01

The structural elucidation of membrane proteins continues to gather pace, but we know little about their molecular interactions with the lipid environment or how they interact with the surrounding bilayer. Here, with the aid of low-resolution X-ray crystallography, we present direct structural information on membrane interfaces as delineated by lipid phosphate groups surrounding the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in its phosphorylated and dephosphorylated Ca(2+)-free forms. The protein-lipid interactions are further analysed using molecular dynamics simulations. We find that SERCA adapts to membranes of different hydrophobic thicknesses by inducing local deformations in the lipid bilayers and by undergoing small rearrangements of the amino-acid side chains and helix tilts. These mutually adaptive interactions allow smooth transitions through large conformational changes associated with the transport cycle of SERCA, a strategy that may be of general nature for many membrane proteins.

Study of structure-function relationships in proteins: Techniques and applications ot cytochrome c: Final report January 15, 1988--January 14, 1989

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldstein, D.A.; Rackovsky, S.R.

1989-08-01

During the initial period of this work we explored the differential geometry results which had been used to explain the structure-function relationships in the set of yeast iso-1-cytochrome c mutants studied under the initial contract. In addition we continued the development of techniques which would permit the structural characterization and comparison of proteins in a very efficient manner. We have expanded the studies based on the characterization of the structural preferences of various residues in a sample of twenty six globular proteins. It has been demonstrated that the overall structural preferences and the amino acid specific preferences seen in themore » analysis carried out at the five alpha carbon level can not be explained by the results of the analysis carried out at the four alpha carbon level. Thus the structural preferences seen must be described by considering groups of five or more residues. We do no yet have enough data to extend the analysis to the six alpha carbon unit level. We have also verified that the yeast/tuna structural analogy which we used before was justified, and have performed a conformational energy minimization of the reduced yeast cytochrome c crystal data in order to have a baseline for the study of mutant proteins. 6 refs.« less

Crenactin from Pyrobaculum calidifontis is closely related to actin in structure and forms steep helical filaments

PubMed Central

Izoré, Thierry; Duman, Ramona; Kureisaite-Ciziene, Danguole; Löwe, Jan

2014-01-01

Polymerising proteins of the actin family are nearly ubiquitous. Crenactins, restricted to Crenarchaea, are more closely related to actin than bacterial MreB. Crenactins occur in gene clusters hinting at an unknown, but conserved function. We solved the crystal structure of crenactin at 3.2 Å resolution. The protein crystallises as a continuous right-handed helix with 8 subunits per complete turn, spanning 419 Å. The structure of crenactin shows several loops that are longer than in actin, but overall, crenactin is closely related to eukaryotic actin, with an RMSD of 1.6 Å. Crenactin filaments imaged by electron microscopy showed polymers with very similar helical parameters. PMID:24486010

Fast and Accurate Multivariate Gaussian Modeling of Protein Families: Predicting Residue Contacts and Protein-Interaction Partners

PubMed Central

Feinauer, Christoph; Procaccini, Andrea; Zecchina, Riccardo; Weigt, Martin; Pagnani, Andrea

2014-01-01

In the course of evolution, proteins show a remarkable conservation of their three-dimensional structure and their biological function, leading to strong evolutionary constraints on the sequence variability between homologous proteins. Our method aims at extracting such constraints from rapidly accumulating sequence data, and thereby at inferring protein structure and function from sequence information alone. Recently, global statistical inference methods (e.g. direct-coupling analysis, sparse inverse covariance estimation) have achieved a breakthrough towards this aim, and their predictions have been successfully implemented into tertiary and quaternary protein structure prediction methods. However, due to the discrete nature of the underlying variable (amino-acids), exact inference requires exponential time in the protein length, and efficient approximations are needed for practical applicability. Here we propose a very efficient multivariate Gaussian modeling approach as a variant of direct-coupling analysis: the discrete amino-acid variables are replaced by continuous Gaussian random variables. The resulting statistical inference problem is efficiently and exactly solvable. We show that the quality of inference is comparable or superior to the one achieved by mean-field approximations to inference with discrete variables, as done by direct-coupling analysis. This is true for (i) the prediction of residue-residue contacts in proteins, and (ii) the identification of protein-protein interaction partner in bacterial signal transduction. An implementation of our multivariate Gaussian approach is available at the website http://areeweb.polito.it/ricerca/cmp/code. PMID:24663061

Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin*

PubMed Central

Courson, David S.; Rock, Ronald S.

2010-01-01

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and α-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. α-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. PMID:20551315

Thermal stability, storage and release of proteins with tailored fit in silica

NASA Astrophysics Data System (ADS)

Chen, Yun-Chu; Smith, Tristan; Hicks, Robert H.; Doekhie, Aswin; Koumanov, Francoise; Wells, Stephen A.; Edler, Karen J.; van den Elsen, Jean; Holman, Geoffrey D.; Marchbank, Kevin J.; Sartbaeva, Asel

2017-04-01

Biological substances based on proteins, including vaccines, antibodies, and enzymes, typically degrade at room temperature over time due to denaturation, as proteins unfold with loss of secondary and tertiary structure. Their storage and distribution therefore relies on a “cold chain” of continuous refrigeration; this is costly and not always effective, as any break in the chain leads to rapid loss of effectiveness and potency. Efforts have been made to make vaccines thermally stable using treatments including freeze-drying (lyophilisation), biomineralisation, and encapsulation in sugar glass and organic polymers. Here for the first time we show that proteins can be enclosed in a deposited silica “cage”, rendering them stable against denaturing thermal treatment and long-term ambient-temperature storage, and subsequently released into solution with their structure and function intact. This “ensilication” method produces a storable solid protein-loaded material without the need for desiccation or freeze-drying. Ensilication offers the prospect of a solution to the “cold chain” problem for biological materials, in particular for vaccines.

Thermal stability, storage and release of proteins with tailored fit in silica.

PubMed

Chen, Yun-Chu; Smith, Tristan; Hicks, Robert H; Doekhie, Aswin; Koumanov, Francoise; Wells, Stephen A; Edler, Karen J; van den Elsen, Jean; Holman, Geoffrey D; Marchbank, Kevin J; Sartbaeva, Asel

2017-04-24

Biological substances based on proteins, including vaccines, antibodies, and enzymes, typically degrade at room temperature over time due to denaturation, as proteins unfold with loss of secondary and tertiary structure. Their storage and distribution therefore relies on a "cold chain" of continuous refrigeration; this is costly and not always effective, as any break in the chain leads to rapid loss of effectiveness and potency. Efforts have been made to make vaccines thermally stable using treatments including freeze-drying (lyophilisation), biomineralisation, and encapsulation in sugar glass and organic polymers. Here for the first time we show that proteins can be enclosed in a deposited silica "cage", rendering them stable against denaturing thermal treatment and long-term ambient-temperature storage, and subsequently released into solution with their structure and function intact. This "ensilication" method produces a storable solid protein-loaded material without the need for desiccation or freeze-drying. Ensilication offers the prospect of a solution to the "cold chain" problem for biological materials, in particular for vaccines.

Innovative computer-aided methods for the discovery of new kinase ligands.

PubMed

Abuhammad, Areej; Taha, Mutasem

2016-04-01

Recent evidence points to significant roles played by protein kinases in cell signaling and cellular proliferation. Faulty protein kinases are involved in cancer, diabetes and chronic inflammation. Efforts are continuously carried out to discover new inhibitors for selected protein kinases. In this review, we discuss two new computer-aided methodologies we developed to mine virtual databases for new bioactive compounds. One method is ligand-based exploration of the pharmacophoric space of inhibitors of any particular biotarget followed by quantitative structure-activity relationship-based selection of the best pharmacophore(s). The second approach is structure-based assuming that potent ligands come into contact with binding site spots distinct from those contacted by weakly potent ligands. Both approaches yield pharmacophores useful as 3D search queries for the discovery of new bioactive (kinase) inhibitors.

Structural disorder in plant proteins: where plasticity meets sessility.

PubMed

Covarrubias, Alejandra A; Cuevas-Velazquez, Cesar L; Romero-Pérez, Paulette S; Rendón-Luna, David F; Chater, Caspar C C

2017-09-01

Plants are sessile organisms. This intriguing nature provokes the question of how they survive despite the continual perturbations caused by their constantly changing environment. The large amount of knowledge accumulated to date demonstrates the fascinating dynamic and plastic mechanisms, which underpin the diverse strategies selected in plants in response to the fluctuating environment. This phenotypic plasticity requires an efficient integration of external cues to their growth and developmental programs that can only be achieved through the dynamic and interactive coordination of various signaling networks. Given the versatility of intrinsic structural disorder within proteins, this feature appears as one of the leading characters of such complex functional circuits, critical for plant adaptation and survival in their wild habitats. In this review, we present information of those intrinsically disordered proteins (IDPs) from plants for which their high level of predicted structural disorder has been correlated with a particular function, or where there is experimental evidence linking this structural feature with its protein function. Using examples of plant IDPs involved in the control of cell cycle, metabolism, hormonal signaling and regulation of gene expression, development and responses to stress, we demonstrate the critical importance of IDPs throughout the life of the plant.

Protein folding simulations: from coarse-grained model to all-atom model.

PubMed

Zhang, Jian; Li, Wenfei; Wang, Jun; Qin, Meng; Wu, Lei; Yan, Zhiqiang; Xu, Weixin; Zuo, Guanghong; Wang, Wei

2009-06-01

Protein folding is an important and challenging problem in molecular biology. During the last two decades, molecular dynamics (MD) simulation has proved to be a paramount tool and was widely used to study protein structures, folding kinetics and thermodynamics, and structure-stability-function relationship. It was also used to help engineering and designing new proteins, and to answer even more general questions such as the minimal number of amino acid or the evolution principle of protein families. Nowadays, the MD simulation is still undergoing rapid developments. The first trend is to toward developing new coarse-grained models and studying larger and more complex molecular systems such as protein-protein complex and their assembling process, amyloid related aggregations, and structure and motion of chaperons, motors, channels and virus capsides; the second trend is toward building high resolution models and explore more detailed and accurate pictures of protein folding and the associated processes, such as the coordination bond or disulfide bond involved folding, the polarization, charge transfer and protonate/deprotonate process involved in metal coupled folding, and the ion permeation and its coupling with the kinetics of channels. On these new territories, MD simulations have given many promising results and will continue to offer exciting views. Here, we review several new subjects investigated by using MD simulations as well as the corresponding developments of appropriate protein models. These include but are not limited to the attempt to go beyond the topology based Gō-like model and characterize the energetic factors in protein structures and dynamics, the study of the thermodynamics and kinetics of disulfide bond involved protein folding, the modeling of the interactions between chaperonin and the encapsulated protein and the protein folding under this circumstance, the effort to clarify the important yet still elusive folding mechanism of protein BBL, the development of discrete MD and its application in studying the alpha-beta conformational conversion and oligomer assembling process, and the modeling of metal ion involved protein folding. (c) 2009 IUBMB.

Expanding the scale of molecular biophysics.

PubMed

Levine, Herbert

2016-10-07

Here, I argue that some of the secrets of complex biological function rely on assemblies of many heterogeneous proteins that together enable sophisticated sensing and actuating processes. Evolution seems to delight in making these structures and in continually elaborating upon their capabilities. Developing tools that can go beyond the few protein limit, both on the experimental frontier and from a theoretical, conceptual framework, should be an extremely high priority for the next generation of molecular biophysicists.

Interrogating Spatio-Mechanical EphA2 Signaling in Cancer

DTIC Science & Technology

2012-03-01

and membrane phase structure modulation by protein binding”, Martin B. Forstner, Chanel K. Lee, Atul N. Parikh, and Jay T. Groves. PMID: 17117874 55...Continuation Format Page anchored proteins and their behavior in supported lipid bilayers", Margot G. Paulick, Amber R. Wise, Martin B. Forstner, Jay T... Martin B. Forstner, Jay T. Groves and Carolyn R. Bertozzi. PMC: 2154431 67. Current Opinion in Immunology, 2007, 19, 6, 722 - 727: "Interrogating

Microgravity

NASA Image and Video Library

2000-11-03

On the Space Shuttle Orbiter Atlantis' middeck, Astronaut Joseph R. Tarner, mission specialist, works at an area amidst several lockers which support the Protein Crystal Growth (PCG) experiment during the STS-66 mission. This particular section is called the Crystal Observation System, housed in the Thermal Enclosure System (COS/TES). Together with the Vapor Diffusion Apparatus (VDA), housed in Single Locker Thermal Enclosure (SLTES), the COS/TES represents the continuing research into the structure of proteins and other macromolecules such as viruses.

A probabilistic and continuous model of protein conformational space for template-free modeling.

PubMed

Zhao, Feng; Peng, Jian; Debartolo, Joe; Freed, Karl F; Sosnick, Tobin R; Xu, Jinbo

2010-06-01

One of the major challenges with protein template-free modeling is an efficient sampling algorithm that can explore a huge conformation space quickly. The popular fragment assembly method constructs a conformation by stringing together short fragments extracted from the Protein Data Base (PDB). The discrete nature of this method may limit generated conformations to a subspace in which the native fold does not belong. Another worry is that a protein with really new fold may contain some fragments not in the PDB. This article presents a probabilistic model of protein conformational space to overcome the above two limitations. This probabilistic model employs directional statistics to model the distribution of backbone angles and 2(nd)-order Conditional Random Fields (CRFs) to describe sequence-angle relationship. Using this probabilistic model, we can sample protein conformations in a continuous space, as opposed to the widely used fragment assembly and lattice model methods that work in a discrete space. We show that when coupled with a simple energy function, this probabilistic method compares favorably with the fragment assembly method in the blind CASP8 evaluation, especially on alpha or small beta proteins. To our knowledge, this is the first probabilistic method that can search conformations in a continuous space and achieves favorable performance. Our method also generated three-dimensional (3D) models better than template-based methods for a couple of CASP8 hard targets. The method described in this article can also be applied to protein loop modeling, model refinement, and even RNA tertiary structure prediction.

Structure of Toxoplasma gondii fructose-1,6-bisphosphate aldolase.

PubMed

Boucher, Lauren E; Bosch, Jürgen

2014-09-01

The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolase has been crystallized in space group P22121, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0 Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.

Estimation of relative order tensors, and reconstruction of vectors in space using unassigned RDC data and its application

NASA Astrophysics Data System (ADS)

Miao, Xijiang; Mukhopadhyay, Rishi; Valafar, Homayoun

2008-10-01

Advances in NMR instrumentation and pulse sequence design have resulted in easier acquisition of Residual Dipolar Coupling (RDC) data. However, computational and theoretical analysis of this type of data has continued to challenge the international community of investigators because of their complexity and rich information content. Contemporary use of RDC data has required a-priori assignment, which significantly increases the overall cost of structural analysis. This article introduces a novel algorithm that utilizes unassigned RDC data acquired from multiple alignment media ( nD-RDC, n ⩾ 3) for simultaneous extraction of the relative order tensor matrices and reconstruction of the interacting vectors in space. Estimation of the relative order tensors and reconstruction of the interacting vectors can be invaluable in a number of endeavors. An example application has been presented where the reconstructed vectors have been used to quantify the fitness of a template protein structure to the unknown protein structure. This work has other important direct applications such as verification of the novelty of an unknown protein and validation of the accuracy of an available protein structure model in drug design. More importantly, the presented work has the potential to bridge the gap between experimental and computational methods of structure determination.

Design and Structure-Guided Development of Novel Inhibitors of the Xeroderma Pigmentosum Group A (XPA) Protein-DNA Interaction.

PubMed

Gavande, Navnath S; VanderVere-Carozza, Pamela; Mishra, Akaash K; Vernon, Tyler L; Pawelczak, Katherine S; Turchi, John J

2017-10-12

XPA is a unique and essential protein required for the nucleotide excision DNA repair pathway and represents a therapeutic target in oncology. Herein, we are the first to develop novel inhibitors of the XPA-DNA interaction through structure-guided drug design efforts. Ester derivatives of the compounds 1 (X80), 22, and 24 displayed excellent inhibitory activity (IC 50 of 0.82 ± 0.18 μM and 1.3 ± 0.22 μM, respectively) but poor solubility. We have synthesized novel amide derivatives that retain potency and have much improved solubility. Furthermore, compound 1 analogs exhibited good specificity for XPA over RPA (replication protein A), another DNA-binding protein that participates in the nucleotide excision repair (NER) pathway. Importantly, there were no significant interactions observed by the X80 class of compounds directly with DNA. Molecular docking studies revealed a mechanistic model for the interaction, and these studies could serve as the basis for continued analysis of structure-activity relationships and drug development efforts of this novel target.

Macromolecular diffractive imaging using imperfect crystals

PubMed Central

Ayyer, Kartik; Yefanov, Oleksandr; Oberthür, Dominik; Roy-Chowdhury, Shatabdi; Galli, Lorenzo; Mariani, Valerio; Basu, Shibom; Coe, Jesse; Conrad, Chelsie E.; Fromme, Raimund; Schaffer, Alexander; Dörner, Katerina; James, Daniel; Kupitz, Christopher; Metz, Markus; Nelson, Garrett; Lourdu Xavier, Paulraj; Beyerlein, Kenneth R.; Schmidt, Marius; Sarrou, Iosifina; Spence, John C. H.; Weierstall, Uwe; White, Thomas A.; Yang, Jay-How; Zhao, Yun; Liang, Mengning; Aquila, Andrew; Hunter, Mark S.; Robinson, Joseph S.; Koglin, Jason E.; Boutet, Sébastien; Fromme, Petra; Barty, Anton; Chapman, Henry N.

2016-01-01

The three-dimensional structures of macromolecules and their complexes are predominantly elucidated by X-ray protein crystallography. A major limitation is access to high-quality crystals, to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields sufficiently high-resolution information that the crystal structure can be solved. The observation that crystals with shrunken unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 hints that crystallographic resolution for some macromolecules may be limited not by their heterogeneity but rather by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern, equal to the incoherent sum of diffraction from rigid single molecular complexes aligned along several discrete crystallographic orientations and hence with an increased information content3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4 —they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 Å limit of measurable Bragg peaks, which allows us to directly phase5 the pattern. With the molecular envelope conventionally determined at 4.5 Å as a constraint, we then obtain a static image of the photosystem II dimer at 3.5 Å resolution. This result shows that continuous diffraction can be used to overcome long-supposed resolution limits of macromolecular crystallography, with a method that puts great value in commonly encountered imperfect crystals and opens up the possibility for model-free phasing6,7. PMID:26863980

The interactions of peripheral membrane proteins with biological membranes

DOE PAGES

Johs, Alexander; Whited, A. M.

2015-07-29

The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

Nuclear Proteomics Reveals the Role of Protein Synthesis and Chromatin Structure in Root Tip of Soybean during the Initial Stage of Flooding Stress.

PubMed

Yin, Xiaojian; Komatsu, Setsuko

2016-07-01

To identify the upstream events controlling the regulation of flooding-responsive proteins in soybean, proteomic analysis of nuclear proteins in root tip was performed. By using nuclear fractions, which were highly enriched, a total of 365 nuclear proteins were changed in soybean root tip at initial stage of flooding stress. Four exon-junction complex-related proteins and NOP1/NOP56, which function in upstream of 60S preribosome biogenesis, were decreased in flooded soybean. Furthermore, proteomic analysis of crude protein extract revealed that the protein translation was suppressed by continuous flooding stress. Seventeen chromatin structure-related nuclear proteins were decreased in response to flooding stress. Out of them, histone H3 was clearly decreased with protein abundance and mRNA expression levels at the initial flooding stress. Additionally, a number of protein synthesis-, RNA-, and DNA-related nuclear proteins were decreased in a time-dependent manner. mRNA expressions of genes encoding the significantly changed flooding-responsive nuclear proteins were inhibited by the transcriptional inhibitor, actinomycin D. These results suggest that protein translation is suppressed through inhibition of preribosome biogenesis- and mRNA processing-related proteins in nuclei of soybean root tip at initial flooding stress. In addition, flooding stress may regulate histone variants with gene expression in root tip.

How does symmetry impact the flexibility of proteins?

PubMed Central

Schulze, Bernd; Sljoka, Adnan; Whiteley, Walter

2014-01-01

It is well known that (i) the flexibility and rigidity of proteins are central to their function, (ii) a number of oligomers with several copies of individual protein chains assemble with symmetry in the native state and (iii) added symmetry sometimes leads to added flexibility in structures. We observe that the most common symmetry classes of protein oligomers are also the symmetry classes that lead to increased flexibility in certain three-dimensional structures—and investigate the possible significance of this coincidence. This builds on the well-developed theory of generic rigidity of body–bar frameworks, which permits an analysis of the rigidity and flexibility of molecular structures such as proteins via fast combinatorial algorithms. In particular, we outline some very simple counting rules and possible algorithmic extensions that allow us to predict continuous symmetry-preserving motions in body–bar frameworks that possess non-trivial point-group symmetry. For simplicity, we focus on dimers, which typically assemble with twofold rotational axes, and often have allosteric function that requires motions to link distant sites on the two protein chains. PMID:24379431

Continuous bioproduction of short-chain fatty acids from sludge enhanced by the combined use of surfactant and alkaline pH.

PubMed

Chen, Yinguang; Liu, Kun; Su, Yinglong; Zheng, Xiong; Wang, Qin

2013-07-01

This work reported the enhancement of continuous SCFA production from sludge by the combined use of surfactant (sodium dodecylbenzene sulfonate (SDBS)) and pH 10 (i.e., SDBS & pH 10). The maximal SCFA production (2056 mg COD/L) was achieved under the SDBS & pH 10 condition at a sludge retention time (SRT) of 12d, which was much higher than that of the blank, sole SDBS, or pH 10. The mechanisms investigation showed that the combined strategy had greater sludge solubilization, higher protein hydrolysis, and lower activity of methanogens. Fluorescence in situ hybridization analysis revealed that the abundance of bacteria was increased, whereas that of archaea was decreased by SDBS & pH 10. The excitation emission matrix fluorescence spectroscopy assay further suggested that SBDS caused protein structure change, which benefited protein hydrolysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Approach to characterization of the higher order structure of disulfide-containing proteins using hydrogen/deuterium exchange and top-down mass spectrometry.

PubMed

Wang, Guanbo; Kaltashov, Igor A

2014-08-05

Top-down hydrogen/deuterium exchange (HDX) with mass spectrometric (MS) detection has recently matured to become a potent biophysical tool capable of providing valuable information on higher order structure and conformational dynamics of proteins at an unprecedented level of structural detail. However, the scope of the proteins amenable to the analysis by top-down HDX MS still remains limited, with the protein size and the presence of disulfide bonds being the two most important limiting factors. While the limitations imposed by the physical size of the proteins gradually become more relaxed as the sensitivity, resolution and dynamic range of modern MS instrumentation continue to improve at an ever accelerating pace, the presence of the disulfide linkages remains a much less forgiving limitation even for the proteins of relatively modest size. To circumvent this problem, we introduce an online chemical reduction step following completion and quenching of the HDX reactions and prior to the top-down MS measurements of deuterium occupancy of individual backbone amides. Application of the new methodology to the top-down HDX MS characterization of a small (99 residue long) disulfide-containing protein β2-microglobulin allowed the backbone amide protection to be probed with nearly a single-residue resolution across the entire sequence. The high-resolution backbone protection pattern deduced from the top-down HDX MS measurements carried out under native conditions is in excellent agreement with the crystal structure of the protein and high-resolution NMR data, suggesting that introduction of the chemical reduction step to the top-down routine does not trigger hydrogen scrambling either during the electrospray ionization process or in the gas phase prior to the protein ion dissociation.

Structural mapping of the coiled-coil domain of a bacterial condensin and comparative analyses across all domains of life suggest conserved features of SMC proteins.

PubMed

Waldman, Vincent M; Stanage, Tyler H; Mims, Alexandra; Norden, Ian S; Oakley, Martha G

2015-06-01

The structural maintenance of chromosomes (SMC) proteins form the cores of multisubunit complexes that are required for the segregation and global organization of chromosomes in all domains of life. These proteins share a common domain structure in which N- and C- terminal regions pack against one another to form a globular ATPase domain. This "head" domain is connected to a central, globular, "hinge" or dimerization domain by a long, antiparallel coiled coil. To date, most efforts for structural characterization of SMC proteins have focused on the globular domains. Recently, however, we developed a method to map interstrand interactions in the 50-nm coiled-coil domain of MukB, the divergent SMC protein found in γ-proteobacteria. Here, we apply that technique to map the structure of the Bacillus subtilis SMC (BsSMC) coiled-coil domain. We find that, in contrast to the relatively complicated coiled-coil domain of MukB, the BsSMC domain is nearly continuous, with only two detectable coiled-coil interruptions. Near the middle of the domain is a break in coiled-coil structure in which there are three more residues on the C-terminal strand than on the N-terminal strand. Close to the head domain, there is a second break with a significantly longer insertion on the same strand. These results provide an experience base that allows an informed interpretation of the output of coiled-coil prediction algorithms for this family of proteins. A comparison of such predictions suggests that these coiled-coil deviations are highly conserved across SMC types in a wide variety of organisms, including humans. © 2015 Wiley Periodicals, Inc.

Discrete, continuous, and stochastic models of protein sorting in the Golgi apparatus

PubMed Central

Gong, Haijun; Guo, Yusong; Linstedt, Adam

2017-01-01

The Golgi apparatus plays a central role in processing and sorting proteins and lipids in eukaryotic cells. Golgi compartments constantly exchange material with each other and with other cellular components, allowing them to maintain and reform distinct identities despite dramatic changes in structure and size during cell division, development, and osmotic stress. We have developed three minimal models of membrane and protein exchange in the Golgi—a discrete, stochastic model, a continuous ordinary differential equation model, and a continuous stochastic differential equation model—each based on two fundamental mechanisms: vesicle-coat-mediated selective concentration of cargoes and soluble N-ethylmaleimide-sensitive factor attachment protein receptor SNARE proteins during vesicle formation and SNARE-mediated selective fusion of vesicles. By exploring where the models differ, we hope to discover whether the discrete, stochastic nature of vesicle-mediated transport is likely to have appreciable functional consequences for the Golgi. All three models show similar ability to restore and maintain distinct identities over broad parameter ranges. They diverge, however, in conditions corresponding to collapse and reassembly of the Golgi. The results suggest that a continuum model provides a good description of Golgi maintenance but that considering the discrete nature of vesicle-based traffic is important to understanding assembly and disassembly of the Golgi. Experimental analysis validates a prediction of the models that altering guanine nucleotide exchange factor expression levels will modulate Golgi size. PMID:20365406

A new method to improve network topological similarity search: applied to fold recognition

PubMed Central

Lhota, John; Hauptman, Ruth; Hart, Thomas; Ng, Clara; Xie, Lei

2015-01-01

Motivation: Similarity search is the foundation of bioinformatics. It plays a key role in establishing structural, functional and evolutionary relationships between biological sequences. Although the power of the similarity search has increased steadily in recent years, a high percentage of sequences remain uncharacterized in the protein universe. Thus, new similarity search strategies are needed to efficiently and reliably infer the structure and function of new sequences. The existing paradigm for studying protein sequence, structure, function and evolution has been established based on the assumption that the protein universe is discrete and hierarchical. Cumulative evidence suggests that the protein universe is continuous. As a result, conventional sequence homology search methods may be not able to detect novel structural, functional and evolutionary relationships between proteins from weak and noisy sequence signals. To overcome the limitations in existing similarity search methods, we propose a new algorithmic framework—Enrichment of Network Topological Similarity (ENTS)—to improve the performance of large scale similarity searches in bioinformatics. Results: We apply ENTS to a challenging unsolved problem: protein fold recognition. Our rigorous benchmark studies demonstrate that ENTS considerably outperforms state-of-the-art methods. As the concept of ENTS can be applied to any similarity metric, it may provide a general framework for similarity search on any set of biological entities, given their representation as a network. Availability and implementation: Source code freely available upon request Contact: lxie@iscb.org PMID:25717198

Homologous ligands accommodated by discrete conformations of a buried cavity.

PubMed

Merski, Matthew; Fischer, Marcus; Balius, Trent E; Eidam, Oliv; Shoichet, Brian K

2015-04-21

Conformational change in protein-ligand complexes is widely modeled, but the protein accommodation expected on binding a congeneric series of ligands has received less attention. Given their use in medicinal chemistry, there are surprisingly few substantial series of congeneric ligand complexes in the Protein Data Bank (PDB). Here we determine the structures of eight alkyl benzenes, in single-methylene increases from benzene to n-hexylbenzene, bound to an enclosed cavity in T4 lysozyme. The volume of the apo cavity suffices to accommodate benzene but, even with toluene, larger cavity conformations become observable in the electron density, and over the series two other major conformations are observed. These involve discrete changes in main-chain conformation, expanding the site; few continuous changes in the site are observed. In most structures, two discrete protein conformations are observed simultaneously, and energetic considerations suggest that these conformations are low in energy relative to the ground state. An analysis of 121 lysozyme cavity structures in the PDB finds that these three conformations dominate the previously determined structures, largely modeled in a single conformation. An investigation of the few congeneric series in the PDB suggests that discrete changes are common adaptations to a series of growing ligands. The discrete, but relatively few, conformational states observed here, and their energetic accessibility, may have implications for anticipating protein conformational change in ligand design.

The alphabet of intrinsic disorder

PubMed Central

Uversky, Vladimir N

2013-01-01

The ability of a protein to fold into unique functional state or to stay intrinsically disordered is encoded in its amino acid sequence. Both ordered and intrinsically disordered proteins (IDPs) are natural polypeptides that use the same arsenal of 20 proteinogenic amino acid residues as their major building blocks. The exceptional structural plasticity of IDPs, their capability to exist as heterogeneous structural ensembles and their wide array of important disorder-based biological functions that complements functional repertoire of ordered proteins are all rooted within the peculiar differential usage of these building blocks by ordered proteins and IDPs. In fact, some residues (so-called disorder-promoting residues) are noticeably more common in IDPs than in sequences of ordered proteins, which, in their turn, are enriched in several order-promoting residues. Furthermore, residues can be arranged according to their “disorder promoting potencies,” which are evaluated based on the relative abundances of various amino acids in ordered and disordered proteins. This review continues a series of publications on the roles of different amino acids in defining the phenomenon of protein intrinsic disorder and concerns glutamic acid, which is the second most disorder-promoting residue. PMID:28516010

Equilibrium of adsorption of mixed milk protein/surfactant solutions at the water/air interface.

PubMed

Kotsmar, C; Grigoriev, D O; Xu, F; Aksenenko, E V; Fainerman, V B; Leser, M E; Miller, R

2008-12-16

Ellipsometry and surface profile analysis tensiometry were used to study and compare the adsorption behavior of beta-lactoglobulin (BLG)/C10DMPO, beta-casein (BCS)/C10DMPO and BCS/C12DMPO mixtures at the air/solution interface. The adsorption from protein/surfactant mixed solutions is of competitive nature. The obtained adsorption isotherms suggest a gradual replacement of the protein molecules at the interface with increasing surfactant concentration for all studied mixed systems. The thickness, refractive index, and the adsorbed amount of the respective adsorption layers, determined by ellipsometry, decrease monotonically and reach values close to those for a surface covered only by surfactant molecules, indicating the absence of proteins from a certain surfactant concentration on. These results correlate with the surface tension data. A continuous increase of adsorption layer thickness was observed up to this concentration, caused by the desorption of segments of the protein and transforming the thin surface layer into a rather diffuse and thick one. Replacement and structural changes of the protein molecules are discussed in terms of protein structure and surface activity of surfactant molecules. Theoretical models derived recently were used for the quantitative description of the equilibrium state of the mixed surface layers.

Discovering the intelligence in molecular biology.

PubMed

Uberbacher, E

1995-12-01

The Third International Conference on Intelligent Systems in Molecular Biology was truly an outstanding event. Computational methods in molecular biology have reached a new level of maturity and utility, resulting in many high-impact applications. The success of this meeting bodes well for the rapid and continuing development of computational methods, intelligent systems and information-based approaches for the biosciences. The basic technology, originally most often applied to 'feasibility' problems, is now dealing effectively with the most difficult real-world problems. Significant progress has been made in understanding protein-structure information, structural classification, and how functional information and the relevant features of active-site geometry can be gleaned from structures by automated computational approaches. The value and limits of homology-based methods, and the ability to classify proteins by structure in the absence of homology, have reached a new level of sophistication. New methods for covariation analysis in the folding of large structures such as RNAs have shown remarkably good results, indicating the long-term potential to understand very complicated molecules and multimolecular complexes using computational means. Novel methods, such as HMMs, context-free grammars and the uses of mutual information theory, have taken center stage as highly valuable tools in our quest to represent and characterize biological information. A focus on creative uses of intelligent systems technologies and the trend toward biological application will undoubtedly continue and grow at the 1996 ISMB meeting in St Louis.

Twilight reloaded: the peptide experience

PubMed Central

Weichenberger, Christian X.; Pozharski, Edwin; Rupp, Bernhard

2017-01-01

The de facto commoditization of biomolecular crystallography as a result of almost disruptive instrumentation automation and continuing improvement of software allows any sensibly trained structural biologist to conduct crystallo­graphic studies of biomolecules with reasonably valid outcomes: that is, models based on properly interpreted electron density. Robust validation has led to major mistakes in the protein part of structure models becoming rare, but some depositions of protein–peptide complex structure models, which generally carry significant interest to the scientific community, still contain erroneous models of the bound peptide ligand. Here, the protein small-molecule ligand validation tool Twilight is updated to include peptide ligands. (i) The primary technical reasons and potential human factors leading to problems in ligand structure models are presented; (ii) a new method used to score peptide-ligand models is presented; (iii) a few instructive and specific examples, including an electron-density-based analysis of peptide-ligand structures that do not contain any ligands, are discussed in detail; (iv) means to avoid such mistakes and the implications for database integrity are discussed and (v) some suggestions as to how journal editors could help to expunge errors from the Protein Data Bank are provided. PMID:28291756

Protein Structural Studies by Traveling Wave Ion Mobility Spectrometry: A Critical Look at Electrospray Sources and Calibration Issues

NASA Astrophysics Data System (ADS)

Sun, Yu; Vahidi, Siavash; Sowole, Modupeola A.; Konermann, Lars

2016-01-01

The question whether electrosprayed protein ions retain solution-like conformations continues to be a matter of debate. One way to address this issue involves comparisons of collision cross sections (Ω) measured by ion mobility spectrometry (IMS) with Ω values calculated for candidate structures. Many investigations in this area employ traveling wave IMS (TWIMS). It is often implied that nanoESI is more conducive for the retention of solution structure than regular ESI. Focusing on ubiquitin, cytochrome c, myoglobin, and hemoglobin, we demonstrate that Ω values and collisional unfolding profiles are virtually indistinguishable under both conditions. These findings suggest that gas-phase structures and ion internal energies are independent of the type of electrospray source. We also note that TWIMS calibration can be challenging because differences in the extent of collisional activation relative to drift tube reference data may lead to ambiguous peak assignments. It is demonstrated that this problem can be circumvented by employing collisionally heated calibrant ions. Overall, our data are consistent with the view that exposure of native proteins to electrospray conditions can generate kinetically trapped ions that retain solution-like structures on the millisecond time scale of TWIMS experiments.

Prediction of protein-peptide interactions: application of the XPairIt API to anthrax lethal factor and substrates

NASA Astrophysics Data System (ADS)

Hurley, Margaret M.; Sellers, Michael S.

2013-05-01

As software and methodology develop, key aspects of molecular interactions such as detailed energetics and flexibility are continuously better represented in docking simulations. In the latest iteration of the XPairIt API and Docking Protocol, we perform a blind dock of a peptide into the cleavage site of the Anthrax lethal factor (LF) metalloprotein. Molecular structures are prepared from RCSB:1JKY and we demonstrate a reasonably accurate docked peptide through analysis of protein motion and, using NCI Plot, visualize and characterize the forces leading to binding. We compare our docked structure to the 1JKY crystal structure and the more recent 1PWV structure, and discuss both captured and overlooked interactions. Our results offer a more detailed look at secondary contact and show that both van der Waals and electrostatic interactions from peptide residues further from the enzyme's catalytic site are significant.

FireProt: web server for automated design of thermostable proteins

PubMed Central

Musil, Milos; Stourac, Jan; Brezovsky, Jan; Prokop, Zbynek; Zendulka, Jaroslav; Martinek, Tomas

2017-01-01

Abstract There is a continuous interest in increasing proteins stability to enhance their usability in numerous biomedical and biotechnological applications. A number of in silico tools for the prediction of the effect of mutations on protein stability have been developed recently. However, only single-point mutations with a small effect on protein stability are typically predicted with the existing tools and have to be followed by laborious protein expression, purification, and characterization. Here, we present FireProt, a web server for the automated design of multiple-point thermostable mutant proteins that combines structural and evolutionary information in its calculation core. FireProt utilizes sixteen tools and three protein engineering strategies for making reliable protein designs. The server is complemented with interactive, easy-to-use interface that allows users to directly analyze and optionally modify designed thermostable mutants. FireProt is freely available at http://loschmidt.chemi.muni.cz/fireprot. PMID:28449074

Integration of QUARK and I-TASSER for Ab Initio Protein Structure Prediction in CASP11.

PubMed

Zhang, Wenxuan; Yang, Jianyi; He, Baoji; Walker, Sara Elizabeth; Zhang, Hongjiu; Govindarajoo, Brandon; Virtanen, Jouko; Xue, Zhidong; Shen, Hong-Bin; Zhang, Yang

2016-09-01

We tested two pipelines developed for template-free protein structure prediction in the CASP11 experiment. First, the QUARK pipeline constructs structure models by reassembling fragments of continuously distributed lengths excised from unrelated proteins. Five free-modeling (FM) targets have the model successfully constructed by QUARK with a TM-score above 0.4, including the first model of T0837-D1, which has a TM-score = 0.736 and RMSD = 2.9 Å to the native. Detailed analysis showed that the success is partly attributed to the high-resolution contact map prediction derived from fragment-based distance-profiles, which are mainly located between regular secondary structure elements and loops/turns and help guide the orientation of secondary structure assembly. In the Zhang-Server pipeline, weakly scoring threading templates are re-ordered by the structural similarity to the ab initio folding models, which are then reassembled by I-TASSER based structure assembly simulations; 60% more domains with length up to 204 residues, compared to the QUARK pipeline, were successfully modeled by the I-TASSER pipeline with a TM-score above 0.4. The robustness of the I-TASSER pipeline can stem from the composite fragment-assembly simulations that combine structures from both ab initio folding and threading template refinements. Despite the promising cases, challenges still exist in long-range beta-strand folding, domain parsing, and the uncertainty of secondary structure prediction; the latter of which was found to affect nearly all aspects of FM structure predictions, from fragment identification, target classification, structure assembly, to final model selection. Significant efforts are needed to solve these problems before real progress on FM could be made. Proteins 2016; 84(Suppl 1):76-86. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Protein structure based prediction of catalytic residues.

PubMed

Fajardo, J Eduardo; Fiser, Andras

2013-02-22

Worldwide structural genomics projects continue to release new protein structures at an unprecedented pace, so far nearly 6000, but only about 60% of these proteins have any sort of functional annotation. We explored a range of features that can be used for the prediction of functional residues given a known three-dimensional structure. These features include various centrality measures of nodes in graphs of interacting residues: closeness, betweenness and page-rank centrality. We also analyzed the distance of functional amino acids to the general center of mass (GCM) of the structure, relative solvent accessibility (RSA), and the use of relative entropy as a measure of sequence conservation. From the selected features, neural networks were trained to identify catalytic residues. We found that using distance to the GCM together with amino acid type provide a good discriminant function, when combined independently with sequence conservation. Using an independent test set of 29 annotated protein structures, the method returned 411 of the initial 9262 residues as the most likely to be involved in function. The output 411 residues contain 70 of the annotated 111 catalytic residues. This represents an approximately 14-fold enrichment of catalytic residues on the entire input set (corresponding to a sensitivity of 63% and a precision of 17%), a performance competitive with that of other state-of-the-art methods. We found that several of the graph based measures utilize the same underlying feature of protein structures, which can be simply and more effectively captured with the distance to GCM definition. This also has the added the advantage of simplicity and easy implementation. Meanwhile sequence conservation remains by far the most influential feature in identifying functional residues. We also found that due the rapid changes in size and composition of sequence databases, conservation calculations must be recalibrated for specific reference databases.

MultitaskProtDB-II: an update of a database of multitasking/moonlighting proteins

PubMed Central

Franco-Serrano, Luís; Hernández, Sergio; Calvo, Alejandra; Severi, María A; Ferragut, Gabriela; Pérez-Pons, JosepAntoni; Piñol, Jaume; Pich, Òscar; Mozo-Villarias, Ángel; Amela, Isaac

2018-01-01

Abstract Multitasking, or moonlighting, is the capability of some proteins to execute two or more biological functions. MultitaskProtDB-II is a database of multifunctional proteins that has been updated. In the previous version, the information contained was: NCBI and UniProt accession numbers, canonical and additional biological functions, organism, monomeric/oligomeric states, PDB codes and bibliographic references. In the present update, the number of entries has been increased from 288 to 694 moonlighting proteins. MultitaskProtDB-II is continually being curated and updated. The new database also contains the following information: GO descriptors for the canonical and moonlighting functions, three-dimensional structure (for those proteins lacking PDB structure, a model was made using Itasser and Phyre), the involvement of the proteins in human diseases (78% of human moonlighting proteins) and whether the protein is a target of a current drug (48% of human moonlighting proteins). These numbers highlight the importance of these proteins for the analysis and explanation of human diseases and target-directed drug design. Moreover, 25% of the proteins of the database are involved in virulence of pathogenic microorganisms, largely in the mechanism of adhesion to the host. This highlights their importance for the mechanism of microorganism infection and vaccine design. MultitaskProtDB-II is available at http://wallace.uab.es/multitaskII. PMID:29136215

Distant plant homologues: don't throw out the baby.

PubMed

Gardiner, John; Overall, Robyn; Marc, Jan

2012-03-01

Plants and metazoans share many similarities in terms of conserved proteins. Antibodies have been used extensively to detect remote homologues, many of which are yet to be identified conclusively. Genome sequencing and the creation of novel sequence or structure comparison programs have assisted greatly in the identification of distant protein homologues. The continuing development of new software algorithms and the combining of bioinformatics with proteomics offer hope that remaining homologues will be soon identified. Copyright © 2011 Elsevier Ltd. All rights reserved.

Astronaut Joseph R. Tanner works with PCG experiment on middeck

NASA Image and Video Library

1994-11-14

On the Space Shuttle Atlantis' mid-deck, astronaut Joseph R. Tanner, mission specialist, works at area amidst several lockers onboard the Shuttle which support the Protein Crystal Growth (PCG) experiment. This particular section is called the Crystal Observation System, housed in the Thermal Enclosure System (COS/TES). Together with the Vapor Diffusion Apparatus (VDA), housed in a Single Locker Thermal Enclosure (SLTES) which is out of frame, the Cos/TES represents the continuing research into the structures of proteins and other macromolecules such as viruses.

The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs.

PubMed

Groves, M R; Hanlon, N; Turowski, P; Hemmings, B A; Barford, D

1999-01-08

The PR65/A subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit, generating functionally diverse heterotrimers. Mutations of the beta isoform of PR65 are associated with lung and colon tumors. The crystal structure of the PR65/Aalpha subunit, at 2.3 A resolution, reveals the conformation of its 15 tandemly repeated HEAT sequences, degenerate motifs of approximately 39 amino acids present in a variety of proteins, including huntingtin and importin beta. Individual motifs are composed of a pair of antiparallel alpha helices that assemble in a mainly linear, repetitive fashion to form an elongated molecule characterized by a double layer of alpha helices. Left-handed rotations at three interrepeat interfaces generate a novel left-hand superhelical conformation. The protein interaction interface is formed from the intrarepeat turns that are aligned to form a continuous ridge.

Activators of G-protein signaling 3: a drug addiction molecular gateway.

PubMed

Bowers, Michael Scott

2010-09-01

Drug addiction is marked by continued drug-seeking behavior despite deleterious consequences and a heightened propensity to relapse not withstanding long, drug-free periods. The enduring nature of addiction has been hypothesized to arise from perturbations in intracellular signaling, gene expression, and brain circuitry induced by substance abuse. Ameliorating some of these aberrations should abate behavioral and neurochemical markers associated with an 'addiction phenotype'. This review summarizes data showing that protein expression and signaling through the nonreceptor activator of G-protein signaling 3 (AGS3) are altered by commonly abused substances in rat and in in-vitro addiction models. AGS3 structure and function are unrelated to the more broadly studied regulator of G-protein signaling family. Thus, the unique role of AGS3 is the focus of this review. Intriguingly, AGS3 protein changes persist into drug abstinence. Accordingly, studies probing the role of AGS3 in the neurochemistry of drug-seeking behavior and relapse are studied in detail. To illuminate this study, AGS3 structure, cellular localization, and function are covered so that an idealized AGS3-targeted pharmacotherapy can be proposed.

Activators of G-protein Signaling 3: A drug addiction molecular gateway

PubMed Central

Bowers, M. Scott

2010-01-01

Drug addiction is marked by continued drug-seeking behavior despite deleterious consequences and a heightened propensity to relapse notwithstanding long, drug-free periods. The enduring nature of addiction has been hypothesized to arise from perturbations in intracellular signaling, gene expression, and brain circuitry induced by substance abuse. Ameliorating some of these aberrations should abate behavioral and neurochemical markers associated with an “addiction phenotype”. This review summarizes data showing that protein expression and signaling through the non-receptor Activator of heterotrimeric G-protein Signaling 3 (AGS3) is altered by commonly abused substances in rat and in vitro addiction models. AGS3 structure and function are unrelated to the more broadly studied Regulator of G-protein Signaling (RGS) family. Thus, the unique role of AGS3 is the focus of this review. Intriguingly, AGS3 protein changes persist into drug abstinence. Accordingly, studies probing the role of AGS3 in the neurochemistry of drug-seeking behavior and relapse are reviewed in detail. To illuminate this work, AGS3 structure, cellular localization, and function are covered so that an idealized AGS3-targeted pharmacotherapy can be proposed. PMID:20700046

Sequence/structural analysis of xylem proteome emphasizes pathogenesis-related proteins, chitinases and β-1, 3-glucanases as key players in grapevine defense against Xylella fastidiosa.

PubMed

Chakraborty, Sandeep; Nascimento, Rafael; Zaini, Paulo A; Gouran, Hossein; Rao, Basuthkar J; Goulart, Luiz R; Dandekar, Abhaya M

2016-01-01

Background. Xylella fastidiosa, the causative agent of various plant diseases including Pierce's disease in the US, and Citrus Variegated Chlorosis in Brazil, remains a continual source of concern and economic losses, especially since almost all commercial varieties are sensitive to this Gammaproteobacteria. Differential expression of proteins in infected tissue is an established methodology to identify key elements involved in plant defense pathways. Methods. In the current work, we developed a methodology named CHURNER that emphasizes relevant protein functions from proteomic data, based on identification of proteins with similar structures that do not necessarily have sequence homology. Such clustering emphasizes protein functions which have multiple copies that are up/down-regulated, and highlights similar proteins which are differentially regulated. As a working example we present proteomic data enumerating differentially expressed proteins in xylem sap from grapevines that were infected with X. fastidiosa. Results. Analysis of this data by CHURNER highlighted pathogenesis related PR-1 proteins, reinforcing this as the foremost protein function in xylem sap involved in the grapevine defense response to X. fastidiosa. β-1, 3-glucanase, which has both anti-microbial and anti-fungal activities, is also up-regulated. Simultaneously, chitinases are found to be both up and down-regulated by CHURNER, and thus the net gain of this protein function loses its significance in the defense response. Discussion. We demonstrate how structural data can be incorporated in the pipeline of proteomic data analysis prior to making inferences on the importance of individual proteins to plant defense mechanisms. We expect CHURNER to be applicable to any proteomic data set.

Protein crystal growth (5-IML-1)

NASA Technical Reports Server (NTRS)

Bugg, Charles E.

1992-01-01

Proteins (enzymes, hormones, immunoglobulins) account for 50 pct. or more of the dry weight of most living systems. A detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting projects have terminated at the crystal growth stage. In principle, there are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor is the elimination of density driven convective flow. Other factors that can be controlled in the absence of gravity is the sedimentation of growing crystals in a gravitational field, and the potential advantage of doing containerless crystal growth. As a result of these theories and facts, one can readily understand why the microgravity environment of an Earth orbiting vehicle seems to offer unique opportunities for the protein crystallographer. This perception has led to the establishment of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project. The results of experiments already performed during STS missions have in many cases resulted in large protein crystals which are structurally correct. Thus, the near term objective of the PCG/ME project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

Structural dynamics of free proteins in diffraction.

PubMed

Lin, Milo M; Shorokhov, Dmitry; Zewail, Ahmed H

2011-10-26

Among the macromolecular patterns of biological significance, right-handed α-helices are perhaps the most abundant structural motifs. Here, guided by experimental findings, we discuss both ultrafast initial steps and longer-time-scale structural dynamics of helix-coil transitions induced by a range of temperature jumps in large, isolated macromolecular ensembles of an α-helical protein segment thymosin β(9) (Tβ(9)), and elucidate the comprehensive picture of (un)folding. In continuation of an earlier theoretical work from this laboratory that utilized a simplistic structure-scrambling algorithm combined with a variety of self-avoidance thresholds to approximately model helix-coil transitions in Tβ(9), in the present contribution we focus on the actual dynamics of unfolding as obtained from massively distributed ensemble-convergent MD simulations which provide an unprecedented scope of information on the nature of transient macromolecular structures, and with atomic-scale spatiotemporal resolution. In addition to the use of radial distribution functions of ultrafast electron diffraction (UED) simulations in gaining an insight into the elementary steps of conformational interconversions, we also investigate the structural dynamics of the protein via the native (α-helical) hydrogen bonding contact metric which is an intuitive coarse graining approach. Importantly, the decay of α-helical motifs and the (globular) conformational annealing in Tβ(9) occur consecutively or competitively, depending on the magnitude of temperature jump.

Structural characterization of more potent alternatives to HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.

PubMed

Nemashkalova, Ekaterina L; Kazakov, Alexei S; Khasanova, Leysan M; Permyakov, Eugene A; Permyakov, Sergei E

2013-09-10

HAMLET is a complex of human α-lactalbumin (hLA) with oleic acid (OA) that kills various tumor cells and strains of Streptococcus pneumoniae. More potent protein-OA complexes were previously reported for bovine α-lactalbumin (bLA) and β-lactoglobulin (bLG), and pike parvalbumin (pPA), and here we explore their structural features. The concentration dependencies of the tryptophan fluorescence of hLA, bLA, and bLG complexes with OA reveal their disintegration at protein concentrations below the micromolar level. Chemical cross-linking experiments provide evidence that association with OA shifts the distribution of oligomeric forms of hLA, bLA, bLG, and pPA toward higher-order oligomers. This effect is confirmed for bLA and bLG using the dynamic light scattering method, while pPA is shown to associate with OA vesicles. Like hLA binding, OA binding increases the affinity of bLG for small unilamellar dipalmitoylphosphatidylcholine vesicles, while pPA efficiently binds to the vesicles irrespective of OA binding. The association of OA with bLG and pPA increases their α-helix and cross-β-sheet content and resistance to enzymatic proteolysis, which is indicative of OA-induced protein structuring. The lack of excess heat sorption during melting of bLG and pPA in complex with OA and the presence of a cooperative thermal transition at the level of their secondary structure suggest that the OA-bound forms of bLG and pPA lack a fixed tertiary structure but exhibit a continuous thermal transition. Overall, despite marked differences, the HAMLET-like complexes that were studied exhibit a common feature: a tendency toward protein oligomerization. Because OA-induced oligomerization has been reported for other proteins, this phenomenon is inherent to many proteins.

Recent Advances in Transferable Coarse-Grained Modeling of Proteins

PubMed Central

Kar, Parimal; Feig, Michael

2017-01-01

Computer simulations are indispensable tools for studying the structure and dynamics of biological macromolecules. Biochemical processes occur on different scales of length and time. Atomistic simulations cannot cover the relevant spatiotemporal scales at which the cellular processes occur. To address this challenge, coarse-grained (CG) modeling of the biological systems are employed. Over the last few years, many CG models for proteins continue to be developed. However, many of them are not transferable with respect to different systems and different environments. In this review, we discuss those CG protein models that are transferable and that retain chemical specificity. We restrict ourselves to CG models of soluble proteins only. We also briefly review recent progress made in the multi-scale hybrid all-atom/coarse-grained simulations of proteins. PMID:25443957

Solid-state NMR studies of proteins immobilized on inorganic surfaces

DOE PAGES

Shaw, Wendy J.

2014-10-29

Solid state NMR is the primary tool for studying the quantitative, site-specific structure, orientation, and dynamics of biomineralization proteins under biologically relevant conditions. Two calcium phosphate proteins, statherin and leucine rich amelogenin protein (LRAP), have been studied in depth and have different features, challenging our ability to extract design principles. More recent studies of the significantly larger full-length amelogenin represent a challenging but necessary step to ultimately investigate the full diversity of biomineralization proteins. Interactions of amino acids and silaffin peptide with silica are also being studied, along with qualitative studies of proteins interacting with calcium carbonate. Dipolar recoupling techniquesmore » have formed the core of the quantitative studies, yet, the need for isolated spin pairs makes this approach costly and time intensive. The use of multi-dimensional techniques is advancing, methodology which, despite its challenges with these difficult-to-study proteins, will continue to drive future advancements in this area.« less

Prions in Yeast

PubMed Central

Liebman, Susan W.; Chernoff, Yury O.

2012-01-01

The concept of a prion as an infectious self-propagating protein isoform was initially proposed to explain certain mammalian diseases. It is now clear that yeast also has heritable elements transmitted via protein. Indeed, the “protein only” model of prion transmission was first proven using a yeast prion. Typically, known prions are ordered cross-β aggregates (amyloids). Recently, there has been an explosion in the number of recognized prions in yeast. Yeast continues to lead the way in understanding cellular control of prion propagation, prion structure, mechanisms of de novo prion formation, specificity of prion transmission, and the biological roles of prions. This review summarizes what has been learned from yeast prions. PMID:22879407

Structure of Toxoplasma gondii fructose-1,6-bisphosphate aldolase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boucher, Lauren E.; Bosch, Jürgen, E-mail: jbosch@jhu.edu; Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205

The structure of T. gondii fructose-1,6-bisphosphate aldolase, a glycolytic enzyme and structural component of the invasion machinery, was determined to a resolution of 2.0 Å. The apicomplexan parasite Toxoplasma gondii must invade host cells to continue its lifecycle. It invades different cell types using an actomyosin motor that is connected to extracellular adhesins via the bridging protein fructose-1,6-@@bisphosphate aldolase. During invasion, aldolase serves in the role of a structural bridging protein, as opposed to its normal enzymatic role in the glycolysis pathway. Crystal structures of the homologous Plasmodium falciparum fructose-1,6-bisphosphate aldolase have been described previously. Here, T. gondii fructose-1,6-bisphosphate aldolasemore » has been crystallized in space group P22{sub 1}2{sub 1}, with the biologically relevant tetramer in the asymmetric unit, and the structure has been determined via molecular replacement to a resolution of 2.0 Å. An analysis of the quality of the model and of the differences between the four chains in the asymmetric unit and a comparison between the T. gondii and P. falciparum aldolase structures is presented.« less

High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy

NASA Astrophysics Data System (ADS)

Demers, Jean-Philippe; Habenstein, Birgit; Loquet, Antoine; Kumar Vasa, Suresh; Giller, Karin; Becker, Stefan; Baker, David; Lange, Adam; Sgourakis, Nikolaos G.

2014-09-01

We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state nuclear magnetic resonance (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-Å cryo-EM density map and 996 ssNMR distance constraints, the structure of the type-III secretion system needle of Shigella flexneri is determined to a precision of 0.4 Å. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.

New in protein structure and function annotation: hotspots, single nucleotide polymorphisms and the 'Deep Web'.

PubMed

Bromberg, Yana; Yachdav, Guy; Ofran, Yanay; Schneider, Reinhard; Rost, Burkhard

2009-05-01

The rapidly increasing quantity of protein sequence data continues to widen the gap between available sequences and annotations. Comparative modeling suggests some aspects of the 3D structures of approximately half of all known proteins; homology- and network-based inferences annotate some aspect of function for a similar fraction of the proteome. For most known protein sequences, however, there is detailed knowledge about neither their function nor their structure. Comprehensive efforts towards the expert curation of sequence annotations have failed to meet the demand of the rapidly increasing number of available sequences. Only the automated prediction of protein function in the absence of homology can close the gap between available sequences and annotations in the foreseeable future. This review focuses on two novel methods for automated annotation, and briefly presents an outlook on how modern web software may revolutionize the field of protein sequence annotation. First, predictions of protein binding sites and functional hotspots, and the evolution of these into the most successful type of prediction of protein function from sequence will be discussed. Second, a new tool, comprehensive in silico mutagenesis, which contributes important novel predictions of function and at the same time prepares for the onset of the next sequencing revolution, will be described. While these two new sub-fields of protein prediction represent the breakthroughs that have been achieved methodologically, it will then be argued that a different development might further change the way biomedical researchers benefit from annotations: modern web software can connect the worldwide web in any browser with the 'Deep Web' (ie, proprietary data resources). The availability of this direct connection, and the resulting access to a wealth of data, may impact drug discovery and development more than any existing method that contributes to protein annotation.

SEORious business: structural proteins in sieve tubes and their involvement in sieve element occlusion.

PubMed

Knoblauch, Michael; Froelich, Daniel R; Pickard, William F; Peters, Winfried S

2014-04-01

The phloem provides a network of sieve tubes for long-distance translocation of photosynthates. For over a century, structural proteins in sieve tubes have presented a conundrum since they presumably increase the hydraulic resistance of the tubes while no potential function other than sieve tube or wound sealing in the case of injury has been suggested. Here we summarize and critically evaluate current speculations regarding the roles of these proteins. Our understanding suffers from the suggestive power of images; what looks like a sieve tube plug on micrographs may not actually impede translocation very much. Recent reports of an involvement of SEOR (sieve element occlusion-related) proteins, a class of P-proteins, in the sealing of injured sieve tubes are inconclusive; various lines of evidence suggest that, in neither intact nor injured plants, are SEORs determinative of translocation stoppage. Similarly, the popular notion that P-proteins serve in the defence against phloem sap-feeding insects is unsupported by empirical facts; it is conceivable that in functional sieve tubes, aphids actually could benefit from inducing a plug. The idea that rising cytosolic Ca(2+) generally triggers sieve tube blockage by P-proteins appears widely accepted, despite lacking experimental support. Even in forisomes, P-protein assemblages restricted to one single plant family and the only Ca(2+)-responsive P-proteins known, the available evidence does not unequivocally suggest that plug formation is the cause rather than a consequence of translocation stoppage. We conclude that the physiological roles of structural P-proteins remain elusive, and that in vivo studies of their dynamics in continuous sieve tube networks combined with flow velocity measurements will be required to (hopefully) resolve this scientific roadblock.

An approach to functionally relevant clustering of the protein universe: Active site profile-based clustering of protein structures and sequences.

PubMed

Knutson, Stacy T; Westwood, Brian M; Leuthaeuser, Janelle B; Turner, Brandon E; Nguyendac, Don; Shea, Gabrielle; Kumar, Kiran; Hayden, Julia D; Harper, Angela F; Brown, Shoshana D; Morris, John H; Ferrin, Thomas E; Babbitt, Patricia C; Fetrow, Jacquelyn S

2017-04-01

Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification-amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two-Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure-Function Linkage Database, SFLD) self-identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self-identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well-curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP-identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F-measure and performance analysis on the enolase search results and comparison to GEMMA and SCI-PHY demonstrate that TuLIP avoids the over-division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

The continuing conundrum of the LEA proteins.

PubMed

Tunnacliffe, Alan; Wise, Michael J

2007-10-01

Research into late embryogenesis abundant (LEA) proteins has been ongoing for more than 20 years but, although there is a strong association of LEA proteins with abiotic stress tolerance particularly dehydration and cold stress, for most of that time, their function has been entirely obscure. After their initial discovery in plant seeds, three major groups (numbered 1, 2 and 3) of LEA proteins have been described in a range of different plants and plant tissues. Homologues of groups 1 and 3 proteins have also been found in bacteria and in certain invertebrates. In this review, we present some new data, survey the biochemistry, biophysics and bioinformatics of the LEA proteins and highlight several possible functions. These include roles as antioxidants and as membrane and protein stabilisers during water stress, either by direct interaction or by acting as molecular shields. Along with other hydrophilic proteins and compatible solutes, LEA proteins might also serve as "space fillers" to prevent cellular collapse at low water activities. This multifunctional capacity of the LEA proteins is probably attributable in part to their structural plasticity, as they are largely lacking in secondary structure in the fully hydrated state, but can become more folded during water stress and/or through association with membrane surfaces. The challenge now facing researchers investigating these enigmatic proteins is to make sense of the various in vitro defined functions in the living cell: Are the LEA proteins truly multi-talented, or are they still just misunderstood?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wemmer, D.E.; Kumar, N.V.; Metrione, R.M.

Toxin II from Radianthus paumotensis (Rp/sub II/) has been investigated by high-resolution NMR and chemical sequencing methods. Resonance assignments have been obtained for this protein by the sequential approach. NMR assignments could not be made consistent with the previously reported primary sequence for this protein, and chemical methods have been used to determine a sequence with which the NMR data are consistent. Analysis of the 2D NOE spectra shows that the protein secondary structure is comprised of two sequences of ..beta..-sheet, probably joined into a distorted continuous sheet, connected by turns and extended loops, without any regular ..cap alpha..-helical segments.more » The residues previously implicated in activity in this class of proteins, D8 and R13, occur in a loop region.« less

Hydrogen Exchange and Mass Spectrometry: A Historical Perspective

PubMed Central

Englander, S. Walter

2012-01-01

Protein molecules naturally emit streams of information-rich signals in the language of hydrogen exchange concerning the intimate details of their stability, dynamics, function, changes therein, and effects thereon, all resolved to the level of their individual amino acids. The effort to measure protein hydrogen exchange behavior, understand the underlying chemistry and structural physics of hydrogen exchange processes, and use this information to learn about protein properties and function has continued for 50 years. Recent work uses mass spectrometric analysis together with an earlier proteolytic fragmentation method to extend the hydrogen exchange capability to large biologically interesting proteins. This article briefly reviews the advances that have led us to this point and the understanding that has so far been achieved. PMID:16876429

Revealing the global map of protein folding space by large-scale simulations

NASA Astrophysics Data System (ADS)

Sinner, Claude; Lutz, Benjamin; Verma, Abhinav; Schug, Alexander

2015-12-01

The full characterization of protein folding is a remarkable long-standing challenge both for experiment and simulation. Working towards a complete understanding of this process, one needs to cover the full diversity of existing folds and identify the general principles driving the process. Here, we want to understand and quantify the diversity in folding routes for a large and representative set of protein topologies covering the full range from all alpha helical topologies towards beta barrels guided by the key question: Does the majority of the observed routes contribute to the folding process or only a particular route? We identified a set of two-state folders among non-homologous proteins with a sequence length of 40-120 residues. For each of these proteins, we ran native-structure based simulations both with homogeneous and heterogeneous contact potentials. For each protein, we simulated dozens of folding transitions in continuous uninterrupted simulations and constructed a large database of kinetic parameters. We investigate folding routes by tracking the formation of tertiary structure interfaces and discuss whether a single specific route exists for a topology or if all routes are equiprobable. These results permit us to characterize the complete folding space for small proteins in terms of folding barrier ΔG‡, number of routes, and the route specificity RT.

A history of neutrons in biology: the development of neutron protein crystallography at BNL and LANL.

PubMed

Schoenborn, Benno P

2010-11-01

The first neutron diffraction data were collected from crystals of myoglobin almost 42 years ago using a step-scan diffractometer with a single detector. Since then, major advances have been made in neutron sources, instrumentation and data collection and analysis, and in biochemistry. Fundamental discoveries about enzyme mechanisms, biological complex structures, protein hydration and H-atom positions have been and continue to be made using neutron diffraction. The promise of neutrons has not changed since the first crystal diffraction data were collected. Today, with the developments of beamlines at spallation neutron sources and the use of the Laue method for data collection, the field of neutrons in structural biology has renewed vitality.

CompaRNA: a server for continuous benchmarking of automated methods for RNA secondary structure prediction

PubMed Central

Puton, Tomasz; Kozlowski, Lukasz P.; Rother, Kristian M.; Bujnicki, Janusz M.

2013-01-01

We present a continuous benchmarking approach for the assessment of RNA secondary structure prediction methods implemented in the CompaRNA web server. As of 3 October 2012, the performance of 28 single-sequence and 13 comparative methods has been evaluated on RNA sequences/structures released weekly by the Protein Data Bank. We also provide a static benchmark generated on RNA 2D structures derived from the RNAstrand database. Benchmarks on both data sets offer insight into the relative performance of RNA secondary structure prediction methods on RNAs of different size and with respect to different types of structure. According to our tests, on the average, the most accurate predictions obtained by a comparative approach are generated by CentroidAlifold, MXScarna, RNAalifold and TurboFold. On the average, the most accurate predictions obtained by single-sequence analyses are generated by CentroidFold, ContextFold and IPknot. The best comparative methods typically outperform the best single-sequence methods if an alignment of homologous RNA sequences is available. This article presents the results of our benchmarks as of 3 October 2012, whereas the rankings presented online are continuously updated. We will gladly include new prediction methods and new measures of accuracy in the new editions of CompaRNA benchmarks. PMID:23435231

Surface energetics and protein-protein interactions: analysis and mechanistic implications

PubMed Central

Peri, Claudio; Morra, Giulia; Colombo, Giorgio

2016-01-01

Understanding protein-protein interactions (PPI) at the molecular level is a fundamental task in the design of new drugs, the prediction of protein function and the clarification of the mechanisms of (dis)regulation of biochemical pathways. In this study, we use a novel computational approach to investigate the energetics of aminoacid networks located on the surface of proteins, isolated and in complex with their respective partners. Interestingly, the analysis of individual proteins identifies patches of surface residues that, when mapped on the structure of their respective complexes, reveal regions of residue-pair couplings that extend across the binding interfaces, forming continuous motifs. An enhanced effect is visible across the proteins of the dataset forming larger quaternary assemblies. The method indicates the presence of energetic signatures in the isolated proteins that are retained in the bound form, which we hypothesize to determine binding orientation upon complex formation. We propose our method, BLUEPRINT, as a complement to different approaches ranging from the ab-initio characterization of PPIs, to protein-protein docking algorithms, for the physico-chemical and functional investigation of protein-protein interactions. PMID:27050828

Fibrous Protein Structures: Hierarchy, History and Heroes.

PubMed

Squire, John M; Parry, David A D

2017-01-01

During the 1930s and 1940s the technique of X-ray diffraction was applied widely by William Astbury and his colleagues to a number of naturally-occurring fibrous materials. On the basis of the diffraction patterns obtained, he observed that the structure of each of the fibres was dominated by one of a small number of different types of molecular conformation. One group of fibres, known as the k-m-e-f group of proteins (keratin - myosin - epidermin - fibrinogen), gave rise to diffraction characteristics that became known as the α-pattern. Others, such as those from a number of silks, gave rise to a different pattern - the β-pattern, while connective tissues yielded a third unique set of diffraction characteristics. At the time of Astbury's work, the structures of these materials were unknown, though the spacings of the main X-ray reflections gave an idea of the axial repeats and the lateral packing distances. In a breakthrough in the early 1950s, the basic structures of all of these fibrous proteins were determined. It was found that the long protein chains, composed of strings of amino acids, could be folded up in a systematic manner to generate a limited number of structures that were consistent with the X-ray data. The most important of these were known as the α-helix, the β-sheet, and the collagen triple helix. These studies provided information about the basic building blocks of all proteins, both fibrous and globular. They did not, however, provide detailed information about how these molecules packed together in three-dimensions to generate the fibres found in vivo. A number of possible packing arrangements were subsequently deduced from the X-ray diffraction and other data, but it is only in the last few years, through the continued improvements of electron microscopy, that the packing details within some fibrous proteins can now be seen directly. Here we outline briefly some of the milestones in fibrous protein structure determination, the role of the amino acid sequences and how new techniques, including electron microscopy, are helping to define fibrous protein structures in three-dimensions. We also introduce the idea that, from the known sequence characteristics of different fibrous proteins, new molecules can be designed and synthesized, thereby generating new biological materials with specific structural properties. Some of these, for example, are planned for use in drug delivery systems. Along the way we also introduce the various Chapters of the book, where individual fibrous proteins are discussed in detail.

Functional and evolutionary relationships between bacteriorhodopsin and halorhodopsin in the archaebacterium, halobacterium halobium

NASA Technical Reports Server (NTRS)

Lanyi, J. K.

1986-01-01

The archaebacteria occupy a unique place in phylogenetic trees constructed from analyses of sequences from key informational macromolecules, and their study continues to yield interesting ideas on the early evolution and divergence of biological forms. It is now known that the halobacteria among these species contain various retinal-proteins, resembling eukaryotic rhodopsins, but with different functions. Two of these pigments, located in the cytoplasmic membranes of the bacteria, are bacteriorhodopsin (a light-driven proton pump) and halorhodopsin (a light-driven chloride pump). Comparison of these systems is expected to reveal structure/function relationships in these simple (primitive?) energy transducing membrane components and evolutionary relationships which had produced the structural features which allow the divergent functions. Findings indicate that very different primary structures are needed for these proteins to accomplish their different functions. Indeed, analysis of partial amino acid sequences from halo-opsin shows already that few if any long segments exist which are homologous to bacterio-opsin. Either these proteins diverged a very long time ago to allow for the observed differences, or the evolutionary clock in the halobacteria runs faster than usual.

Understanding Marine Mussel Adhesion

PubMed Central

Roberto, Francisco F.

2007-01-01

In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion. PMID:17990038

Growing protein crystals in microgravity - The NASA Microgravity Science and Applications Division (MSAD) Protein Crystal Growth (PCG) program

NASA Technical Reports Server (NTRS)

Herren, B.

1992-01-01

In collaboration with a medical researcher at the University of Alabama at Birmingham, NASA's Marshall Space Flight Center in Huntsville, Alabama, under the sponsorship of the Microgravity Science and Applications Division (MSAD) at NASA Headquarters, is continuing a series of space experiments in protein crystal growth which could lead to innovative new drugs as well as basic science data on protein molecular structures. From 1985 through 1992, Protein Crystal Growth (PCG) experiments will have been flown on the Space Shuttle a total of 14 times. The first four hand-held experiments were used to test hardware concepts; later flights incorporated these concepts for vapor diffusion protein crystal growth with temperature control. This article provides an overview of the PCG program: its evolution, objectives, and plans for future experiments on NASA's Space Shuttle and Space Station Freedom.

Princeton_TIGRESS 2.0: High refinement consistency and net gains through support vector machines and molecular dynamics in double-blind predictions during the CASP11 experiment.

PubMed

Khoury, George A; Smadbeck, James; Kieslich, Chris A; Koskosidis, Alexandra J; Guzman, Yannis A; Tamamis, Phanourios; Floudas, Christodoulos A

2017-06-01

Protein structure refinement is the challenging problem of operating on any protein structure prediction to improve its accuracy with respect to the native structure in a blind fashion. Although many approaches have been developed and tested during the last four CASP experiments, a majority of the methods continue to degrade models rather than improve them. Princeton_TIGRESS (Khoury et al., Proteins 2014;82:794-814) was developed previously and utilizes separate sampling and selection stages involving Monte Carlo and molecular dynamics simulations and classification using an SVM predictor. The initial implementation was shown to consistently refine protein structures 76% of the time in our own internal benchmarking on CASP 7-10 targets. In this work, we improved the sampling and selection stages and tested the method in blind predictions during CASP11. We added a decomposition of physics-based and hybrid energy functions, as well as a coordinate-free representation of the protein structure through distance-binning Cα-Cα distances to capture fine-grained movements. We performed parameter estimation to optimize the adjustable SVM parameters to maximize precision while balancing sensitivity and specificity across all cross-validated data sets, finding enrichment in our ability to select models from the populations of similar decoys generated for targets in CASPs 7-10. The MD stage was enhanced such that larger structures could be further refined. Among refinement methods that are currently implemented as web-servers, Princeton_TIGRESS 2.0 demonstrated the most consistent and most substantial net refinement in blind predictions during CASP11. The enhanced refinement protocol Princeton_TIGRESS 2.0 is freely available as a web server at http://atlas.engr.tamu.edu/refinement/. Proteins 2017; 85:1078-1098. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Homologous ligands accommodated by discrete conformations of a buried cavity

PubMed Central

Merski, Matthew; Fischer, Marcus; Balius, Trent E.; Eidam, Oliv; Shoichet, Brian K.

2015-01-01

Conformational change in protein–ligand complexes is widely modeled, but the protein accommodation expected on binding a congeneric series of ligands has received less attention. Given their use in medicinal chemistry, there are surprisingly few substantial series of congeneric ligand complexes in the Protein Data Bank (PDB). Here we determine the structures of eight alkyl benzenes, in single-methylene increases from benzene to n-hexylbenzene, bound to an enclosed cavity in T4 lysozyme. The volume of the apo cavity suffices to accommodate benzene but, even with toluene, larger cavity conformations become observable in the electron density, and over the series two other major conformations are observed. These involve discrete changes in main-chain conformation, expanding the site; few continuous changes in the site are observed. In most structures, two discrete protein conformations are observed simultaneously, and energetic considerations suggest that these conformations are low in energy relative to the ground state. An analysis of 121 lysozyme cavity structures in the PDB finds that these three conformations dominate the previously determined structures, largely modeled in a single conformation. An investigation of the few congeneric series in the PDB suggests that discrete changes are common adaptations to a series of growing ligands. The discrete, but relatively few, conformational states observed here, and their energetic accessibility, may have implications for anticipating protein conformational change in ligand design. PMID:25847998

Mutations that Cause Human Disease: A Computational/Experimental Approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beernink, P; Barsky, D; Pesavento, B

International genome sequencing projects have produced billions of nucleotides (letters) of DNA sequence data, including the complete genome sequences of 74 organisms. These genome sequences have created many new scientific opportunities, including the ability to identify sequence variations among individuals within a species. These genetic differences, which are known as single nucleotide polymorphisms (SNPs), are particularly important in understanding the genetic basis for disease susceptibility. Since the report of the complete human genome sequence, over two million human SNPs have been identified, including a large-scale comparison of an entire chromosome from twenty individuals. Of the protein coding SNPs (cSNPs), approximatelymore » half leads to a single amino acid change in the encoded protein (non-synonymous coding SNPs). Most of these changes are functionally silent, while the remainder negatively impact the protein and sometimes cause human disease. To date, over 550 SNPs have been found to cause single locus (monogenic) diseases and many others have been associated with polygenic diseases. SNPs have been linked to specific human diseases, including late-onset Parkinson disease, autism, rheumatoid arthritis and cancer. The ability to predict accurately the effects of these SNPs on protein function would represent a major advance toward understanding these diseases. To date several attempts have been made toward predicting the effects of such mutations. The most successful of these is a computational approach called ''Sorting Intolerant From Tolerant'' (SIFT). This method uses sequence conservation among many similar proteins to predict which residues in a protein are functionally important. However, this method suffers from several limitations. First, a query sequence must have a sufficient number of relatives to infer sequence conservation. Second, this method does not make use of or provide any information on protein structure, which can be used to understand how an amino acid change affects the protein. The experimental methods that provide the most detailed structural information on proteins are X-ray crystallography and NMR spectroscopy. However, these methods are labor intensive and currently cannot be carried out on a genomic scale. Nonetheless, Structural Genomics projects are being pursued by more than a dozen groups and consortia worldwide and as a result the number of experimentally determined structures is rising exponentially. Based on the expectation that protein structures will continue to be determined at an ever-increasing rate, reliable structure prediction schemes will become increasingly valuable, leading to information on protein function and disease for many different proteins. Given known genetic variability and experimentally determined protein structures, can we accurately predict the effects of single amino acid substitutions? An objective assessment of this question would involve comparing predicted and experimentally determined structures, which thus far has not been rigorously performed. The completed research leveraged existing expertise at LLNL in computational and structural biology, as well as significant computing resources, to address this question.« less

Inferring network structure in non-normal and mixed discrete-continuous genomic data.

PubMed

Bhadra, Anindya; Rao, Arvind; Baladandayuthapani, Veerabhadran

2018-03-01

Inferring dependence structure through undirected graphs is crucial for uncovering the major modes of multivariate interaction among high-dimensional genomic markers that are potentially associated with cancer. Traditionally, conditional independence has been studied using sparse Gaussian graphical models for continuous data and sparse Ising models for discrete data. However, there are two clear situations when these approaches are inadequate. The first occurs when the data are continuous but display non-normal marginal behavior such as heavy tails or skewness, rendering an assumption of normality inappropriate. The second occurs when a part of the data is ordinal or discrete (e.g., presence or absence of a mutation) and the other part is continuous (e.g., expression levels of genes or proteins). In this case, the existing Bayesian approaches typically employ a latent variable framework for the discrete part that precludes inferring conditional independence among the data that are actually observed. The current article overcomes these two challenges in a unified framework using Gaussian scale mixtures. Our framework is able to handle continuous data that are not normal and data that are of mixed continuous and discrete nature, while still being able to infer a sparse conditional sign independence structure among the observed data. Extensive performance comparison in simulations with alternative techniques and an analysis of a real cancer genomics data set demonstrate the effectiveness of the proposed approach. © 2017, The International Biometric Society.

Inferring network structure in non-normal and mixed discrete-continuous genomic data

PubMed Central

Bhadra, Anindya; Rao, Arvind; Baladandayuthapani, Veerabhadran

2017-01-01

Inferring dependence structure through undirected graphs is crucial for uncovering the major modes of multivariate interaction among high-dimensional genomic markers that are potentially associated with cancer. Traditionally, conditional independence has been studied using sparse Gaussian graphical models for continuous data and sparse Ising models for discrete data. However, there are two clear situations when these approaches are inadequate. The first occurs when the data are continuous but display non-normal marginal behavior such as heavy tails or skewness, rendering an assumption of normality inappropriate. The second occurs when a part of the data is ordinal or discrete (e.g., presence or absence of a mutation) and the other part is continuous (e.g., expression levels of genes or proteins). In this case, the existing Bayesian approaches typically employ a latent variable framework for the discrete part that precludes inferring conditional independence among the data that are actually observed. The current article overcomes these two challenges in a unified framework using Gaussian scale mixtures. Our framework is able to handle continuous data that are not normal and data that are of mixed continuous and discrete nature, while still being able to infer a sparse conditional sign independence structure among the observed data. Extensive performance comparison in simulations with alternative techniques and an analysis of a real cancer genomics data set demonstrate the effectiveness of the proposed approach. PMID:28437848

2.2 Å resolution cryo-EM structure of β-galactosidase in complex with a cell-permeant inhibitor.

PubMed

Bartesaghi, Alberto; Merk, Alan; Banerjee, Soojay; Matthies, Doreen; Wu, Xiongwu; Milne, Jacqueline L S; Subramaniam, Sriram

2015-06-05

Cryo-electron microscopy (cryo-EM) is rapidly emerging as a powerful tool for protein structure determination at high resolution. Here we report the structure of a complex between Escherichia coli β-galactosidase and the cell-permeant inhibitor phenylethyl β-D-thiogalactopyranoside (PETG), determined by cryo-EM at an average resolution of ~2.2 angstroms (Å). Besides the PETG ligand, we identified densities in the map for ~800 water molecules and for magnesium and sodium ions. Although it is likely that continued advances in detector technology may further enhance resolution, our findings demonstrate that preparation of specimens of adequate quality and intrinsic protein flexibility, rather than imaging or image-processing technologies, now represent the major bottlenecks to routinely achieving resolutions close to 2 Å using single-particle cryo-EM. Copyright © 2015, American Association for the Advancement of Science.

Optimization of a Bioluminescence Resonance Energy Transfer-Based Assay for Screening of Trypanosoma cruzi Protein/Protein Interaction Inhibitors.

PubMed

Mild, Jesica G; Fernandez, Lucia R; Gayet, Odile; Iovanna, Juan; Dusetti, Nelson; Edreira, Martin M

2018-05-01

Chagas disease, a parasitic disease caused by Trypanosoma cruzi, is a major public health burden in poor rural populations of Central and South America and a serious emerging threat outside the endemic region, since the number of infections in non-endemic countries continues to rise. In order to develop more efficient anti-trypanosomal treatments to replace the outdated therapies, new molecular targets need to be explored and new drugs discovered. Trypanosoma cruzi has distinctive structural and functional characteristics with respect to the human host. These exclusive features could emerge as interesting drug targets. In this work, essential and differential protein-protein interactions for the parasite, including the ribosomal P proteins and proteins involved in mRNA processing, were evaluated in a bioluminescence resonance energy transfer-based assay as a starting point for drug screening. Suitable conditions to consider using this simple and robust methodology to screening compounds and natural extracts able to inhibit protein-protein interactions were set in living cells and lysates.

Detecting Coevolution in and among Protein Domains

PubMed Central

Yeang, Chen-Hsiang; Haussler, David

2007-01-01

Correlated changes of nucleic or amino acids have provided strong information about the structures and interactions of molecules. Despite the rich literature in coevolutionary sequence analysis, previous methods often have to trade off between generality, simplicity, phylogenetic information, and specific knowledge about interactions. Furthermore, despite the evidence of coevolution in selected protein families, a comprehensive screening of coevolution among all protein domains is still lacking. We propose an augmented continuous-time Markov process model for sequence coevolution. The model can handle different types of interactions, incorporate phylogenetic information and sequence substitution, has only one extra free parameter, and requires no knowledge about interaction rules. We employ this model to large-scale screenings on the entire protein domain database (Pfam). Strikingly, with 0.1 trillion tests executed, the majority of the inferred coevolving protein domains are functionally related, and the coevolving amino acid residues are spatially coupled. Moreover, many of the coevolving positions are located at functionally important sites of proteins/protein complexes, such as the subunit linkers of superoxide dismutase, the tRNA binding sites of ribosomes, the DNA binding region of RNA polymerase, and the active and ligand binding sites of various enzymes. The results suggest sequence coevolution manifests structural and functional constraints of proteins. The intricate relations between sequence coevolution and various selective constraints are worth pursuing at a deeper level. PMID:17983264

Immunoglobulin light chains, glycosaminoglycans and amyloid.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stevens, F. J.; Kisilevsky, R.; Biosciences Division

2000-03-01

Immunoglobulin light chains are the precursor proteins for fibrils that are formed during primary amyloidosis and in amyloidosis associated with multiple myeloma. As found for the approximately 20 currently described forms of focal, localized, or systemic amyloidoses, light chain-related fibrils extracted from physiological deposits are invariably associated with glycosaminoglycans, predominantly heparan sulfate. Other amyloid-related proteins are either structurally normal, such as g2-microglobulin and islet amyloid polypeptide, fragments of normal proteins such as serum amyloid A protein or the precursor protein of the g peptide involved in Alzheimer's disease, or are inherited forms of single amino acid variants of a normalmore » protein such as found in the familial forms of amyloid associated with transthyretin. In contrast, the primary structures of light chains involved in fibril formation exhibit extensive mutational diversity rendering some proteins highly amyloidogenic and others non-pathological. The interactions between light chains and glycosaminoglycans are also affected by amino acid variation and may influence the clinical course of disease by enhancing fibril stability and contributing to resistance to protease degradation. Relatively little is currently known about the mechanisms by which glycosaminoglycans interact with light chains and light-chain fibrils. It is probable that future studies of this uniquely diverse family of proteins will continue o shed light on the processes of amyloidosis, and contribute as well to a greater understanding of the normal physiological roles of glycosaminoglycans.« less

Recombinant spider silk from aqueous solutions via a bio-inspired microfluidic chip

NASA Astrophysics Data System (ADS)

Peng, Qingfa; Zhang, Yaopeng; Lu, Li; Shao, Huili; Qin, Kankan; Hu, Xuechao; Xia, Xiaoxia

2016-11-01

Spiders achieve superior silk fibres by controlling the molecular assembly of silk proteins and the hierarchical structure of fibres. However, current wet-spinning process for recombinant spidroins oversimplifies the natural spinning process. Here, water-soluble recombinant spider dragline silk protein (with a low molecular weight of 47 kDa) was adopted to prepare aqueous spinning dope. Artificial spider silks were spun via microfluidic wet-spinning, using a continuous post-spin drawing process (WS-PSD). By mimicking the natural spinning apparatus, shearing and elongational sections were integrated in the microfluidic spinning chip to induce assembly, orientation of spidroins, and fibril structure formation. The additional post-spin drawing process following the wet-spinning section partially mimics the spinning process of natural spider silk and substantially contributes to the compact aggregation of microfibrils. Subsequent post-stretching further improves the hierarchical structure of the fibres, including the crystalline structure, orientation, and fibril melting. The tensile strength and elongation of post-treated fibres reached up to 510 MPa and 15%, respectively.

Protein crystallography and infectious diseases.

PubMed Central

Verlinde, C. L.; Merritt, E. A.; Van den Akker, F.; Kim, H.; Feil, I.; Delboni, L. F.; Mande, S. C.; Sarfaty, S.; Petra, P. H.; Hol, W. G.

1994-01-01

The current rapid growth in the number of known 3-dimensional protein structures is producing a database of structures that is increasingly useful as a starting point for the development of new medically relevant molecules such as drugs, therapeutic proteins, and vaccines. This development is beautifully illustrated in the recent book, Protein structure: New approaches to disease and therapy (Perutz, 1992). There is a great and growing promise for the design of molecules for the treatment or prevention of a wide variety of diseases, an endeavor made possible by the insights derived from the structure and function of crucial proteins from pathogenic organisms and from man. We present here 2 illustrations of structure-based drug design. The first is the prospect of developing antitrypanosomal drugs based on crystallographic, ligand-binding, and molecular modeling studies of glycolytic glycosomal enzymes from Trypanosomatidae. These unicellular organisms are responsible for several tropical diseases, including African and American trypanosomiases, as well as various forms of leishmaniasis. Because the target enzymes are also present in the human host, this project is a pioneering study in selective design. The second illustrative case is the prospect of designing anti-cholera drugs based on detailed analysis of the structure of cholera toxin and the closely related Escherichia coli heat-labile enterotoxin. Such potential drugs can be targeted either at inhibiting the toxin's receptor binding site or at blocking the toxin's intracellular catalytic activity. Study of the Vibrio cholerae and E. coli toxins serves at the same time as an example of a general approach to structure-based vaccine design. These toxins exhibit a remarkable ability to stimulate the mucosal immune system, and early results have suggested that this property can be maintained by engineered fusion proteins based on the native toxin structure. The challenge is thus to incorporate selected epitopes from foreign pathogens into the native framework of the toxin such that crucial features of both the epitope and the toxin are maintained. That is, the modified toxin must continue to evoke a strong mucosal immune response, and this response must be directed against an epitope conformation characteristic of the original pathogen. PMID:7849584

Thiazolidine-2,4-dione derivatives: programmed chemical weapons for key protein targets of various pathological conditions.

PubMed

Chadha, Navriti; Bahia, Malkeet Singh; Kaur, Maninder; Silakari, Om

2015-07-01

Thiazolidine-2,4-dione is an extensively explored heterocyclic nucleus for designing of novel agents implicated for a wide variety of pathophysiological conditions, that is, diabetes, diabetic complications, cancer, arthritis, inflammation, microbial infection, and melanoma, etc. The current paradigm of drug development has shifted to the structure-based drug design, since high-throughput screenings have continued to generate disappointing results. The gap between hit generation and drug establishment can be narrowed down by investigation of ligand interactions with its receptor protein. Therefore, it would always be highly beneficial to gain knowledge of molecular level interactions between specific protein target and developed ligands; since this information can be maneuvered to design new molecules with improved protein fitting. Thus, considering this aspect, we have corroborated the information about molecular (target) level implementations of thiazolidine-2,4-diones (TZD) derivatives having therapeutic implementations such as, but not limited to, anti-diabetic (glitazones), anti-cancer, anti-arthritic, anti-inflammatory, anti-oxidant and anti-microbial, etc. The structure based SAR of TZD derivatives for various protein targets would serve as a benchmark for the alteration of existing ligands to design new ones with better binding interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.

From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes.

PubMed

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-10

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes.

Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

PubMed

Bezerra, Gustavo Arruda; Ohara-Nemoto, Yuko; Cornaciu, Irina; Fedosyuk, Sofiya; Hoffmann, Guillaume; Round, Adam; Márquez, José A; Nemoto, Takayuki K; Djinović-Carugo, Kristina

2017-06-06

Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

Dissociation of Recombinant Prion Protein Fibrils into Short Protofilaments: Implications for the Endocytic Pathway and Involvement of the N-Terminal Domain

PubMed Central

Qi, Xu; Moore, Roger A.; McGuirl, Michele A.

2012-01-01

Fibril dissociation is necessary for efficient conversion of normal prion protein to its misfolded state and continued propagation into amyloid. Recent studies have revealed that conversion occurs along the endocytic pathway. To better understand the dissociation process, we have investigated the effect of low pH on the stability of recombinant prion fibrils. We show that under conditions that mimic the endocytic environment, amyloid fibrils made from full length prion protein dissociate both laterally and axially to form protofilaments. About 5% of the protofilaments are short enough to be considered soluble and contain ~100–300 monomers per structure; these also retain the biophysical characteristics of the filaments. We propose that protonation of His residues and charge repulsion in the N-terminal domain trigger fibril dissociation. Our data suggest that lysosomes and late endosomes are competent milieus for propagating the misfolded state not only by destabilizing the normal prion protein, but by accelerating fibril dissociation into smaller structures that may act as seeds. PMID:22591453

Organic Matrix-related mineralization of sea urchin spicules, spines, test and teeth

PubMed Central

Veis, Arthur

2012-01-01

The camarodont echinoderms have five distinct mineralized skeletal elements: the embryonic spicules and mature test; spines, lantern stereom and teeth. The embryonic spicules are transient structural elements of the larval skeleton whereas the spines and test plates are permanent structural elements. The teeth are continuously growing structures, matching wear at the incisal adoral end to the rate of new production at the aboral plumula. The mineral in all cases is a high magnesium calcite, but the magnesium content, crystal shape and growth pattern is different in each type of skeletal element. The crystal shape and organization into macro structures depends on the presence of an organic matrix which creates the spaces and controls the environments for crystal initiation and growth. The detailed mechanisms of crystal regulation are not known, but much work has been done on defining the proteins which appear to be involved. Phosphorylated matrix proteins may be of special importance. Biochemical isolation of proteins, construction and analysis of cDNA libraries, and most recently high-throughput proteomic analysis in conjunction with the sequencing of the complete genome have yielded a detailed list of protein components likely to be involved in the mineralization processes. However, the proteome-genome analyses have not yet provided insight into the mechanisms of crystallization, calcite composition, and orientation applicable to all skeletal elements. Although the embryonic pluteus and their spicules are the best studied system, it appears that spicule is not representative of the mature skeletal elements. Now armed with the compositions of most of the proteins involved, the next phase of research will have to focus on the specific localization of the proteins and individual biochemistries of each system with regard to mineral content and placement. PMID:21622194

Diarylthiophenes as inhibitors of the pore-forming protein perforin.

PubMed

Miller, Christian K; Huttunen, Kristiina M; Denny, William A; Jaiswal, Jagdish K; Ciccone, Annette; Browne, Kylie A; Trapani, Joseph A; Spicer, Julie A

2016-01-15

Evolution from a furan-containing high-throughput screen (HTS) hit (1) resulted in isobenzofuran-1(3H)-one (2) as a potent inhibitor of the function of both isolated perforin protein and perforin delivered in situ by intact KHYG-1 NK cells. In the current study, structure-activity relationship (SAR) development towards a novel series of diarylthiophene analogues has continued through the use of substituted-benzene and -pyridyl moieties as bioisosteres for 2-thioxoimidazolidin-4-one (A) on a thiophene (B) -isobenzofuranone (C) scaffold. The resulting compounds were tested for their ability to inhibit perforin lytic activity in vitro. Carboxamide (23) shows a 4-fold increase over (2) in lytic activity against isolated perforin and provides good rationale for continued development within this class. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Structural markers of the evolution of whey protein isolate powder during aging and effects on foaming properties.

PubMed

Norwood, E-A; Le Floch-Fouéré, C; Briard-Bion, V; Schuck, P; Croguennec, T; Jeantet, R

2016-07-01

The market for dairy powders, including high added-value products (e.g., infant formulas, protein isolates) has increased continuously over the past decade. However, the processing and storage of whey protein isolate (WPI) powders can result in changes in their structural and functional properties. It is therefore of great importance to understand the mechanisms and to identify the structural markers involved in the aging of WPI powders to control their end use properties. This study was performed to determine the effects of different storage conditions on protein lactosylations, protein denaturation in WPI, and in parallel on their foaming and interfacial properties. Six storage conditions involving different temperatures (θ) and water activities (aw) were studied for periods of up to 12mo. The results showed that for θ≤20°C, foaming properties of powders did not significantly differ from nonaged whey protein isolates (reference), regardless of the aw. On the other hand, powders presented significant levels of denaturation/aggregation and protein modification involving first protein lactosylation and then degradation of Maillard reaction products, resulting in a higher browning index compared with the reference, starting from the early stage of storage at 60°C. These changes resulted in a higher foam density and a slightly better foam stability (whisking) at 6mo. At 40°C, powders showed transitional evolution. The findings of this study will make it possible to define maximum storage durations and to recommend optimal storage conditions in accordance with WPI powder end-use properties. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Administration of structured lipid composed of MCT and fish oil reduces net protein catabolism in enterally fed burned rats.

PubMed Central

Teo, T C; DeMichele, S J; Selleck, K M; Babayan, V K; Blackburn, G L; Bistrian, B R

1989-01-01

The effects of enteral feeding with safflower oil or a structured lipid (SL) derived from 60% medium-chain triglyceride (MCT) and 40% fish oil (MCT/fish oil) on protein and energy metabolism were compared in gastrostomy-fed burned rats (30% body surface area) by measuring oxygen consumption, carbon dioxide production, nitrogen balance, total liver protein, whole-body leucine kinetics, and rectus muscle and liver protein fractional synthetic rates (FSR, %/day). Male Sprague-Dawley rats (195 +/- 5g) received 50 ml/day of an enteral regimen containing 50 kcal, 2 g amino acids, and 40% nonprotein calories as lipid for three days. Protein kinetics were estimated by using a continuous L-[1-14C] leucine infusion technique on day 2. Thermally injured rats enterally fed MCT/fish oil yielded significantly higher daily and cumulative nitrogen balances (p less than or equal to 0.025) and rectus muscle (39%) FSR (p less than or equal to 0.05) when compared with safflower oil. MCT/fish oil showed a 22% decrease (p less than or equal to 0.005) in per cent flux oxidized and a 7% (p less than or equal to 0.05) decrease in total energy expenditure (TEE) versus safflower oil. A 15% increase in liver FSR was accompanied by a significant elevation (p less than or equal to 0.025) in total liver protein with MCT/fish oil. This novel SL shares the properties of other structured lipids in that it reduces the net protein catabolic effects of burn injury, in part, by influencing tissue protein synthetic rates. The reduction in TEE is unique to MCT/fish oil and may relate to the ability of fish oil to diminish the injury response. PMID:2500898

Changes in Quaternary Structure in the Signaling Mechanisms of PAS Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ayers, Rebecca A.; Moffat, Keith

2008-12-15

FixL from Bradyrhizobium japonicum is a PAS sensor protein in which two PAS domains covalently linked to a histidine kinase domain are responsible for regulating nitrogen fixation in an oxygen-dependent manner. The more C-terminal PAS domain, denoted bjFixLH, contains a heme cofactor that binds diatomic molecules such as carbon monoxide and oxygen and regulates the activity of the FixL histidine kinase as part of a two-component signaling system. We present the structures of ferric, deoxy, and carbon monoxide-bound bjFixLH in a new space group (P1) and at resolutions (1.5--1.8 {angstrom}) higher than the resolutions of those previously obtained. Interestingly, bjFixLHmore » can form two different dimers (in P1 and R32 crystal forms) in the same crystallization solution, where the monomers in one dimer are rotated {approx}175 deg. relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. We use screw rotation analysis to carry out a quantitative pairwise comparison of PAS quaternary structures, which identifies five different relative orientations adopted by isolated PAS monomers. We conclude that PAS monomer arrangement is context-dependent and could differ depending on whether the PAS domains are isolated or are part of a full-length protein. Structurally homologous residues comprise a conserved dimer interface. Using network analysis, we find that the architecture of the PAS dimer interface is continuous rather than modular; the network of residues comprising the interface is strongly connected. A continuous dimer interface is consistent with the low dimer-monomer dissociation equilibrium constant. Finally, we quantitate quaternary structural changes induced by carbon monoxide binding to a bjFixLH dimer, in which monomers rotate by up to 2 deg. relative to each other. We relate these changes to those in other dimeric PAS domains and discuss the role of quaternary structural changes in the signaling mechanisms of PAS sensor proteins.« less

Protein structure based prediction of catalytic residues

PubMed Central

2013-01-01

Background Worldwide structural genomics projects continue to release new protein structures at an unprecedented pace, so far nearly 6000, but only about 60% of these proteins have any sort of functional annotation. Results We explored a range of features that can be used for the prediction of functional residues given a known three-dimensional structure. These features include various centrality measures of nodes in graphs of interacting residues: closeness, betweenness and page-rank centrality. We also analyzed the distance of functional amino acids to the general center of mass (GCM) of the structure, relative solvent accessibility (RSA), and the use of relative entropy as a measure of sequence conservation. From the selected features, neural networks were trained to identify catalytic residues. We found that using distance to the GCM together with amino acid type provide a good discriminant function, when combined independently with sequence conservation. Using an independent test set of 29 annotated protein structures, the method returned 411 of the initial 9262 residues as the most likely to be involved in function. The output 411 residues contain 70 of the annotated 111 catalytic residues. This represents an approximately 14-fold enrichment of catalytic residues on the entire input set (corresponding to a sensitivity of 63% and a precision of 17%), a performance competitive with that of other state-of-the-art methods. Conclusions We found that several of the graph based measures utilize the same underlying feature of protein structures, which can be simply and more effectively captured with the distance to GCM definition. This also has the added the advantage of simplicity and easy implementation. Meanwhile sequence conservation remains by far the most influential feature in identifying functional residues. We also found that due the rapid changes in size and composition of sequence databases, conservation calculations must be recalibrated for specific reference databases. PMID:23433045

A multiphase approach to model ultrafiltration of deformable colloids

NASA Astrophysics Data System (ADS)

Haribabu, Malavika; Dunstan, Dave; Davidson, Malcolm; Harvie, Dalton

2017-11-01

Ultrafiltration (UF) is widely used in the dairy industry to fractionate and concentrate proteins, during the manufacture of milk protein concentrate and cheese. The protein build-up, comprising casein micelles (CM) and whey proteins, at the membrane surface during UF increases the resistance of the membrane system, thereby decreasing the performance of the process unit. CM have a complex structure that hydrodynamically behaves as a hard-sphere when dilute, but deforms beyond the random packing limit, forming a shear-thinning gel. This study employs a mixture model, based on the mixture phase continuity, Navier-Stokes equations, and solids continuity equation, to predict the solid concentration and velocity distribution during UF of CM. Micelle deformation is modelled as a function of volume fraction and dependent on its elastic modulus and particle size. The effect of deformation on gel permeability is implemented via Happel's permeability for hard spheres. Under crossflow conditions, the gel thickness is observed to increase along the membrane length, followed by a decrease towards the end of the membrane, resulting in an increase in flux at the latter section of the membrane. This study demonstrates that the membrane end-effects are important in determining UF performance.

Thermodynamic study of the native and phosphorylated regulatory domain of the CFTR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marasini, Carlotta, E-mail: marasini@ge.ibf.cnr.it; Galeno, Lauretta; Moran, Oscar

2012-07-06

Highlights: Black-Right-Pointing-Pointer CFTR mutations produce cystic fibrosis. Black-Right-Pointing-Pointer Chloride transport depends on the regulatory domain phosphorylation. Black-Right-Pointing-Pointer Regulatory domain is intrinsically disordered. Black-Right-Pointing-Pointer Secondary structure and protein stability change upon phosphorylation. -- Abstract: The regulatory domain (RD) of the cystic fibrosis transmembrane conductance regulator (CFTR), the defective protein in cystic fibrosis, is the region of the channel that regulates the CFTR activity with multiple phosphorylation sites. This domain is an intrinsically disordered protein, characterized by lack of stable or unique tertiary structure. The disordered character of a protein is directly correlated with its function. The flexibility of RD may bemore » important for its regulatory role: the continuous conformational change may be necessary for the progressive phosphorylation, and thus activation, of the channel. However, the lack of a defined and stable structure results in a considerable limitation when trying to in build a unique molecular model for the RD. Moreover, several evidences indicate significant structural differences between the native, non-phosphorylated state, and the multiple phosphorylated state of the protein. The aim of our work is to provide data to describe the conformations and the thermodynamic properties in these two functional states of RD. We have done the circular dichroism (CD) spectra in samples with a different degree of phosphorylation, from the non-phosphorylated state to a bona fide completely phosphorylated state. Analysis of CD spectra showed that the random coil and {beta}-sheets secondary structure decreased with the polypeptide phosphorylation, at expenses of an increase of {alpha}-helix. This observation lead to interpret phosphorylation as a mechanism favoring a more structured state. We also studied the thermal denaturation curves of the protein in the two conditions, monitoring the changes of the mean residue ellipticity measured at 222 nm as a function of temperature, between 20 and 95 Degree-Sign C. The thermodynamic analysis of the denaturation curves shows that phosphorylation of the protein induces a state of lower stability of R domain, characterized by a lower transition temperature, and by a smaller Gibbs free energy difference between the native and the unfolded states.« less

Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

NASA Technical Reports Server (NTRS)

Boo, Yong Chool; Jo, Hanjoong

2003-01-01

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

Protein and calorie prescription for children and young adults receiving continuous renal replacement therapy: a report from the Prospective Pediatric Continuous Renal Replacement Therapy Registry Group.

PubMed

Zappitelli, Michael; Goldstein, Stuart L; Symons, Jordan M; Somers, Michael J G; Baum, Michelle A; Brophy, Patrick D; Blowey, Douglas; Fortenberry, James D; Chua, Annabelle N; Flores, Francisco X; Benfield, Mark R; Alexander, Steven R; Askenazi, David; Hackbarth, Richard; Bunchman, Timothy E

2008-12-01

Few published reports describe nutrition provision for critically ill children and young adults with acute kidney injury receiving continuous renal replacement therapy. The goals of this study were to describe feeding practices in pediatric continuous renal replacement therapy and to evaluate factors associated with over- and under-prescription of protein and calories. Retrospective database study. Multicenter study in pediatric critical care units. Patients with acute kidney injury (estimated glomerular filtration rate < 75 mL/min/1.73 m at continuous renal replacement therapy initiation) enrolled in the Prospective Pediatric Continuous Renal Replacement Therapy Registry. None. Nutrition variables: initial and maximal protein (g/kg/day) and caloric (kcal/kg/day) prescription and predicted resting energy expenditure (kcal/kg/day). We determined factors predicting initial and maximal protein and caloric prescription by multivariate analysis. One hundred ninety-five patients (median [interquartile range] age = 8.1 [12.8] yrs, 56.9% men) were studied. Mean protein and caloric prescriptions at continuous renal replacement therapy initiation were 1.3 +/- 1.5 g/kg/day (median, 1.0; range, 0-10) and 37 +/- 27 kcal/kg/day (median, 32; range, 0-107). Mean maximal protein and caloric prescriptions during continuous renal replacement therapy were 2.0 +/- 1.5 g/kg/day (median, 1.7; range, 0-12) and 48 +/- 32 kcal/kg/day (median, 43; range, 0-117). Thirty-four percent of patients were initially prescribed < 1 g/kg/day protein; 23% never attained > 1 g/kg/day protein prescription. By continuous renal replacement therapy day 5, median protein prescribed was > 2 g/kg/day. Protein prescription practices differed substantially between medical centers with 5 of 10 centers achieving maximal protein prescription of > 2 g/kg/day in > or = 40% of patients. Caloric prescription exceeded predicted resting energy expenditure by 30%-100%. Factors independently associated with maximal protein and caloric prescription while on continuous renal replacement therapy were younger age, initial protein and caloric prescription and number of continuous renal replacement therapy treatment days (p < 0.05). Protein prescription in pediatric continuous renal replacement therapy may be inadequate. Inter-center variation exists with respect to nutrition prescription. Feeding practice standardization and research in pediatric acute kidney injury nutrition are essential to begin providing evidence-based feeding recommendations.

Protein crystal growth

NASA Technical Reports Server (NTRS)

Bugg, Charles E.

1993-01-01

Proteins account for 50% or more of the dry weight of most living systems and play a crucial role in virtually all biological processes. Since the specific functions of essentially all biological molecules are determined by their three-dimensional structures, it is obvious that a detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. At the present time, protein crystallography has no substitute, it is the only technique available for elucidating the atomic arrangements within complicated biological molecules. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting and promising projects have terminated at the crystal growth stage. There is a pressing need to better understand protein crystal growth, and to develop new techniques that can be used to enhance the size and quality of protein crystals. There are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor that might be expected to alter crystal growth processes in space is the elimination of density-driven convective flow. Another factor that can be readily controlled in the absence of gravity is the sedimentation of growing crystal in a gravitational field. Another potential advantage of microgravity for protein crystal growth is the option of doing containerless crystal growth. One can readily understand why the microgravity environment established by Earth-orbiting vehicles is perceived to offer unique opportunities for the protein crystallographer. The near term objectives of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

The Use of Affinity Tags to Overcome Obstacles in Recombinant Protein Expression and Purification.

PubMed

Amarasinghe, Chinthaka; Jin, Jian-Ping

2015-01-01

Research and industrial demands for recombinant proteins continue to increase over time for their broad applications in structural and functional studies and as therapeutic agents. These applications often require large quantities of recombinant protein at desirable purity, which highlights the importance of developing and improving production approaches that provide high level expression and readily achievable purity of recombinant protein. E. coli is the most widely used host for the expression of a diverse range of proteins at low cost. However, there are common pitfalls that can severely limit the expression of exogenous proteins, such as stability, low solubility and toxicity to the host cell. To overcome these obstacles, one strategy that has found to be promising is the use of affinity tags or carrier peptide to aid in the folding of the target protein, increase solubility, lower toxicity and increase the level of expression. In the meantime, the tags and fusion proteins can be designed to facilitate affinity purification. Since the fusion protein may not exhibit the native conformation of the target protein, various strategies have been developed to remove the tag during or after purification to avoid potential complications in structural and functional studies and to obtain native biological activities. Despite extensive research and rapid development along these lines, there are unsolved problems and imperfect applications. This focused review compares and contrasts various strategies that employ affinity tags to improve bacterial expression and to facilitate purification of recombinant proteins. The pros and cons of the approaches are discussed for more effective applications and new directions of future improvement.

Exact Length Distribution of Filamentous Structures Assembled from a Finite Pool of Subunits.

PubMed

Harbage, David; Kondev, Jané

2016-07-07

Self-assembling filamentous structures made of protein subunits are ubiquitous in cell biology. These structures are often highly dynamic, with subunits in a continuous state of flux, binding to and falling off of filaments. In spite of this constant turnover of their molecular parts, many cellular structures seem to maintain a well-defined size over time, which is often required for their proper functioning. One widely discussed mechanism of size regulation involves the cell maintaining a finite pool of protein subunits available for assembly. This finite pool mechanism can control the length of a single filament by having assembly proceed until the pool of free subunits is depleted to the point when assembly and disassembly are balanced. Still, this leaves open the question of whether the same mechanism can provide size control for multiple filamentous structures that are assembled from a common pool of protein subunits, as is often the case in cells. We address this question by solving the steady-state master equation governing the stochastic assembly and disassembly of multifilament structures made from a shared finite pool of subunits. We find that, while the total number of subunits within a multifilament structure is well-defined, individual filaments within the structure have a wide, power-law distribution of lengths. We also compute the phase diagram for two multifilament structures competing for the same pool of subunits and identify conditions for coexistence when both have a well-defined size. These predictions can be tested in cell experiments in which the size of the subunit pool or the number of filament nucleators is tuned.

Utilisation of adsorption and desorption for simultaneously improving protein crystallisation success rate and crystal quality

NASA Astrophysics Data System (ADS)

Guo, Yun-Zhu; Sun, Li-Hua; Oberthuer, Dominik; Zhang, Chen-Yan; Shi, Jian-Yu; di, Jiang-Lei; Zhang, Bao-Liang; Cao, Hui-Ling; Liu, Yong-Ming; Li, Jian; Wang, Qian; Huang, Huan-Huan; Liu, Jun; Schulz, Jan-Mirco; Zhang, Qiu-Yu; Zhao, Jian-Lin; Betzel, Christian; He, Jian-Hua; Yin, Da-Chuan

2014-12-01

High-quality protein crystals of suitable size are an important prerequisite for applying X-ray crystallography to determine the 3-dimensional structure of proteins. However, it is often difficult to obtain protein crystals of appropriate size and quality because nucleation and growth processes can be unsuccessful. Here, we show that by adsorbing proteins onto porous polystyrene-divinylbenzene microspheres (SDB) floating on the surface of the crystallisation solution, a localised high supersaturation region at the surface of the microspheres and a low supersaturation region below the microspheres can coexist in a single solution. The crystals will easily nucleate in the region of high supersaturation, but when they grow to a certain size, they will sediment to the region of low supersaturation and continue to grow. In this way, the probability of crystallisation and crystal quality can be simultaneously increased in a single solution without changing other crystallisation parameters.

The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

NASA Astrophysics Data System (ADS)

Hu, Xiaohu; Hong, Liang; Dean Smith, Micholas; Neusius, Thomas; Cheng, Xiaolin; Smith, Jeremy C.

2016-02-01

Internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behaviour with effective relaxation times existing over many decades in time, from ps up to ~102 s (refs ,,,). Here, using molecular dynamics simulations, we show that, on timescales from 10-12 to 10-5 s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of the energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behaviour persists up to timescales approaching the in vivo lifespan of individual protein molecules.

Animal Mitochondrial DNA Replication

PubMed Central

Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.

2016-01-01

Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

Construction and Structural Analysis of Tethered Lipid Bilayer Containing Photosynthetic Antenna Proteins for Functional Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sumino, Ayumi; Dewa, Takehisa; Takeuchi, Toshikazu

2011-07-11

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAY), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RCmore » within the tethered membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.« less

Protein aggregation as bacterial inclusion bodies is reversible.

PubMed

Carrió, M M; Villaverde, A

2001-01-26

Inclusion bodies are refractile, intracellular protein aggregates usually observed in bacteria upon targeted gene overexpression. Since their occurrence has a major economical impact in protein production bio-processes, in vitro refolding strategies are under continuous exploration. In this work, we prove spontaneous in vivo release of both beta-galactosidase and P22 tailspike polypeptides from inclusion bodies resulting in their almost complete disintegration and in the concomitant appearance of soluble, properly folded native proteins with full biological activity. Since, in particular, the tailspike protein exhibits an unusually slow and complex folding pathway involving deep interdigitation of beta-sheet structures, its in vivo refolding indicates that bacterial inclusion body proteins are not collapsed into an irreversible unfolded state. Then, inclusion bodies can be observed as transient deposits of folding-prone polypeptides, resulting from an unbalanced equilibrium between in vivo protein precipitation and refolding that can be actively displaced by arresting protein synthesis. The observation that the formation of big inclusion bodies is reversible in vivo can be also relevant in the context of amyloid diseases, in which deposition of important amounts of aggregated protein initiates the pathogenic process.

Theoretical investigation of interaction of sorbitol molecules with alcohol dehydrogenase in aqueous solution using molecular dynamics simulation.

PubMed

Bahrami, Homayoon; Zahedi, Mansour; Moosavi-Movahedi, Ali Akbar; Azizian, Homa; Amanlou, Massoud

2011-03-01

The nature of protein-sorbitol-water interaction in solution at the molecular level, has been investigated using molecular dynamics simulations. In order to do this task, two molecular dynamics simulations of the protein ADH in solution at room temperature have been carried out, one in the presence (about 0.9 M) and another in the absence of sorbitol. The results show that the sorbitol molecules cluster and move toward the protein, and form hydrogen bonds with protein. Also, coating by sorbitol reduces the conformational fluctuations of the protein compared to the sorbitol-free system. Thus, it is concluded that at moderate concentration of sorbitol solution, sorbitol molecules interact with ADH via many H-bonds that prevent the protein folding. In fact, at more concentrated sorbitol solution, water and sorbitol molecules accumulate around the protein surface and form a continuous space-filling network to reduce the protein flexibility. Namely, in such solution, sorbitol molecules can stabilize a misfolded state of ADH, and prevent the protein from folding to its native structure.

Analysis of the Intrinsically Disordered N-Terminus of the DNA Junction-Resolving Enzyme T7 Endonuclease I: Identification of Structure Formed upon DNA Binding

PubMed Central

2016-01-01

The four-way (Holliday) DNA junction of homologous recombination is processed by the symmetrical cleavage of two strands by a nuclease. These junction-resolving enzymes bind to four-way junctions in dimeric form, distorting the structure of the junction in the process. Crystal structures of T7 endonuclease I have been determined as free protein, and the complex with a DNA junction. In neither crystal structure was the N-terminal 16-amino acid peptide visible, yet deletion of this peptide has a marked effect on the resolution process. Here we have investigated the N-terminal peptide by inclusion of spin-label probes at unique sites within this region, studied by electron paramagnetic resonance. Continuous wave experiments show that these labels are mobile in the free protein but become constrained on binding a DNA junction, with the main interaction occurring for residues 7–10 and 12. Distance measurements between equivalent positions within the two peptides of a dimer using PELDOR showed that the intermonomeric distances for residues 2–12 are long and broadly distributed in the free protein but are significantly shortened and become more defined on binding to DNA. These results suggest that the N-terminal peptides become more organized on binding to the DNA junction and nestle into the minor grooves at the branchpoint, consistent with the biochemical data indicating an important role in the resolution process. This study demonstrates the presence of structure within a protein region that cannot be viewed by crystallography. PMID:27387136

Protein folding by NMR.

PubMed

Zhuravleva, Anastasia; Korzhnev, Dmitry M

2017-05-01

Protein folding is a highly complex process proceeding through a number of disordered and partially folded nonnative states with various degrees of structural organization. These transiently and sparsely populated species on the protein folding energy landscape play crucial roles in driving folding toward the native conformation, yet some of these nonnative states may also serve as precursors for protein misfolding and aggregation associated with a range of devastating diseases, including neuro-degeneration, diabetes and cancer. Therefore, in vivo protein folding is often reshaped co- and post-translationally through interactions with the ribosome, molecular chaperones and/or other cellular components. Owing to developments in instrumentation and methodology, solution NMR spectroscopy has emerged as the central experimental approach for the detailed characterization of the complex protein folding processes in vitro and in vivo. NMR relaxation dispersion and saturation transfer methods provide the means for a detailed characterization of protein folding kinetics and thermodynamics under native-like conditions, as well as modeling high-resolution structures of weakly populated short-lived conformational states on the protein folding energy landscape. Continuing development of isotope labeling strategies and NMR methods to probe high molecular weight protein assemblies, along with advances of in-cell NMR, have recently allowed protein folding to be studied in the context of ribosome-nascent chain complexes and molecular chaperones, and even inside living cells. Here we review solution NMR approaches to investigate the protein folding energy landscape, and discuss selected applications of NMR methodology to studying protein folding in vitro and in vivo. Together, these examples highlight a vast potential of solution NMR in providing atomistic insights into molecular mechanisms of protein folding and homeostasis in health and disease. Copyright © 2016 Elsevier B.V. All rights reserved.

Integration of QUARK and I-TASSER for ab initio protein structure prediction in CASP11

PubMed Central

Zhang, Wenxuan; Yang, Jianyi; He, Baoji; Walker, Sara Elizabeth; Zhang, Hongjiu; Govindarajoo, Brandon; Virtanen, Jouko; Xue, Zhidong; Shen, Hong-Bin; Zhang, Yang

2015-01-01

We tested two pipelines developed for template-free protein structure prediction in the CASP11 experiment. First, the QUARK pipeline constructs structure models by reassembling fragments of continuously distributed lengths excised from unrelated proteins. Five free-modeling (FM) targets have the model successfully constructed by QUARK with a TM-score above 0.4, including the first model of T0837-D1, which has a TM-score=0.736 and RMSD=2.9 Å to the native. Detailed analysis showed that the success is partly attributed to the high-resolution contact map prediction derived from fragment-based distance-profiles, which are mainly located between regular secondary structure elements and loops/turns and help guide the orientation of secondary structure assembly. In the Zhang-Server pipeline, weakly scoring threading templates are re-ordered by the structural similarity to the ab initio folding models, which are then reassembled by I-TASSER based structure assembly simulations; 60% more domains with length up to 204 residues, compared to the QUARK pipeline, were successfully modeled by the I-TASSER pipeline with a TM-score above 0.4. The robustness of the I-TASSER pipeline can stem from the composite fragment-assembly simulations that combine structures from both ab initio folding and threading template refinements. Despite the promising cases, challenges still exist in long-range beta-strand folding, domain parsing, and the uncertainty of secondary structure prediction; the latter of which was found to affect nearly all aspects of FM structure predictions, from fragment identification, target classification, structure assembly, to final model selection. Significant efforts are needed to solve these problems before real progress on FM could be made. PMID:26370505

Actinous enigma or enigmatic actin

PubMed Central

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

2014-01-01

Being the most abundant protein of the eukaryotic cell, actin continues to keep its secrets for more than 60 years. Everything about this protein, its structure, functions, and folding, is mysteriously counterintuitive, and this review represents an attempt to solve some of the riddles and conundrums commonly found in the field of actin research. In fact, actin is a promiscuous binder with a wide spectrum of biological activities. It can exist in at least three structural forms, globular, fibrillar, and inactive (G-, F-, and I-actin, respectively). G-actin represents a thermodynamically instable, quasi-stationary state, which is formed in vivo as a result of the energy-intensive, complex posttranslational folding events controlled and driven by cellular folding machinery. The G-actin structure is dependent on the ATP and Mg2+ binding (which in vitro is typically substituted by Ca2+) and protein is easily converted to the I-actin by the removal of metal ions and by action of various denaturing agents (pH, temperature, and chemical denaturants). I-actin cannot be converted back to the G-form. Foldable and “natively folded” forms of actin are always involved in interactions either with the specific protein partners, such as Hsp70 chaperone, prefoldin, and the CCT chaperonin during the actin folding in vivo or with Mg2+ and ATP as it takes place in the G-form. We emphasize that the solutions for the mysteries of actin multifunctionality, multistructurality, and trapped unfolding can be found in the quasi-stationary nature of this enigmatic protein, which clearly possesses many features attributed to both globular and intrinsically disordered proteins. PMID:28232879

Supported Lipid Bilayer/Carbon Nanotube Hybrids

NASA Astrophysics Data System (ADS)

Zhou, Xinjian; Moran-Mirabal, Jose; Craighead, Harold; McEuen, Paul

2007-03-01

We form supported lipid bilayers on single-walled carbon nanotubes and use this hybrid structure to probe the properties of lipid membranes and their functional constituents. We first demonstrate membrane continuity and lipid diffusion over the nanotube. A membrane-bound tetanus toxin protein, on the other hand, sees the nanotube as a diffusion barrier whose strength depends on the diameter of the nanotube. Finally, we present results on the electrical detection of specific binding of streptavidin to biotinylated lipids with nanotube field effect transistors. Possible techniques to extract dynamic information about the protein binding events will also be discussed.

Proteome complexity and the forces that drive proteome imbalance.

PubMed

Harper, J Wade; Bennett, Eric J

2016-09-15

The cellular proteome is a complex microcosm of structural and regulatory networks that requires continuous surveillance and modification to meet the dynamic needs of the cell. It is therefore crucial that the protein flux of the cell remains in balance to ensure proper cell function. Genetic alterations that range from chromosome imbalance to oncogene activation can affect the speed, fidelity and capacity of protein biogenesis and degradation systems, which often results in proteome imbalance. An improved understanding of the causes and consequences of proteome imbalance is helping to reveal how these systems can be targeted to treat diseases such as cancer.

Continuous mutual improvement of macromolecular structure models in the PDB and of X-ray crystallographic software: the dual role of deposited experimental data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terwilliger, Thomas C., E-mail: terwilliger@lanl.gov; Bricogne, Gerard, E-mail: terwilliger@lanl.gov; Los Alamos National Laboratory, Mail Stop M888, Los Alamos, NM 87507

Macromolecular structures deposited in the PDB can and should be continually reinterpreted and improved on the basis of their accompanying experimental X-ray data, exploiting the steady progress in methods and software that the deposition of such data into the PDB on a massive scale has made possible. Accurate crystal structures of macromolecules are of high importance in the biological and biomedical fields. Models of crystal structures in the Protein Data Bank (PDB) are in general of very high quality as deposited. However, methods for obtaining the best model of a macromolecular structure from a given set of experimental X-ray datamore » continue to progress at a rapid pace, making it possible to improve most PDB entries after their deposition by re-analyzing the original deposited data with more recent software. This possibility represents a very significant departure from the situation that prevailed when the PDB was created, when it was envisioned as a cumulative repository of static contents. A radical paradigm shift for the PDB is therefore proposed, away from the static archive model towards a much more dynamic body of continuously improving results in symbiosis with continuously improving methods and software. These simultaneous improvements in methods and final results are made possible by the current deposition of processed crystallographic data (structure-factor amplitudes) and will be supported further by the deposition of raw data (diffraction images). It is argued that it is both desirable and feasible to carry out small-scale and large-scale efforts to make this paradigm shift a reality. Small-scale efforts would focus on optimizing structures that are of interest to specific investigators. Large-scale efforts would undertake a systematic re-optimization of all of the structures in the PDB, or alternatively the redetermination of groups of structures that are either related to or focused on specific questions. All of the resulting structures should be made generally available, along with the precursor entries, with various views of the structures being made available depending on the types of questions that users are interested in answering.« less

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

PubMed Central

Maadooliat, Mehdi; Huang, Jianhua Z.

2013-01-01

Despite considerable progress in the past decades, protein structure prediction remains one of the major unsolved problems in computational biology. Angular-sampling-based methods have been extensively studied recently due to their ability to capture the continuous conformational space of protein structures. The literature has focused on using a variety of parametric models of the sequential dependencies between angle pairs along the protein chains. In this article, we present a thorough review of angular-sampling-based methods by assessing three main questions: What is the best distribution type to model the protein angles? What is a reasonable number of components in a mixture model that should be considered to accurately parameterize the joint distribution of the angles? and What is the order of the local sequence–structure dependency that should be considered by a prediction method? We assess the model fits for different methods using bivariate lag-distributions of the dihedral/planar angles. Moreover, the main information across the lags can be extracted using a technique called Lag singular value decomposition (LagSVD), which considers the joint distribution of the dihedral/planar angles over different lags using a nonparametric approach and monitors the behavior of the lag-distribution of the angles using singular value decomposition. As a result, we developed graphical tools and numerical measurements to compare and evaluate the performance of different model fits. Furthermore, we developed a web-tool (http://www.stat.tamu.edu/∼madoliat/LagSVD) that can be used to produce informative animations. PMID:22926831

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

PubMed Central

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca

2018-01-01

Abstract The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases. PMID:29718412

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures.

PubMed

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca; Franco, Elisa

2018-06-01

The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

PubMed

Schmitter, Daniel; Wachowicz, Paulina; Sage, Daniel; Chasapi, Anastasia; Xenarios, Ioannis; Simanis; Unser, Michael

2013-01-01

The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. (Continued on next page) (Continued from previous page). "RodCellJ" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html.

Single-cell protein from waste cellulose

NASA Technical Reports Server (NTRS)

Dunlap, C. E.; Callihan, C. D.

1973-01-01

The recycle, reuse, or reclamation of single cell protein from liquid and solid agricultural waste fibers by a fermentation process is reported. It is shown that cellulose comprises the bulk of the fibers at 50% to 55% of the dry weight of the refuse and that its biodegradability is of prime importance in the choice of a substrate. The application of sodium hydroxide followed by heat and pressure serves to de-polymerize and disrupt lignin structure while swelling the cellulose to increase water uptake and pore volume. Some of the lignin, hemi-celluloses, ash, and cellulose of the material is hydrolized and solubilized. Introduction of microorganisms to the substrate fibers mixed with nutrients produces continuous fermentation of cellulose for further protein extraction and purification.

Compact representation of continuous energy surfaces for more efficient protein design

PubMed Central

Hallen, Mark A.; Gainza, Pablo; Donald, Bruce R.

2015-01-01

In macromolecular design, conformational energies are sensitive to small changes in atom coordinates, so modeling the small, continuous motions of atoms around low-energy wells confers a substantial advantage in structural accuracy; however, modeling these motions comes at the cost of a very large number of energy function calls, which form the bottleneck in the design calculation. In this work, we remove this bottleneck by consolidating all conformational energy evaluations into the precomputation of a local polynomial expansion of the energy about the “ideal” conformation for each low-energy, “rotameric” state of each residue pair. This expansion is called Energy as Polynomials in Internal Coordinates (EPIC), where the internal coordinates can be sidechain dihedrals, backrub angles, and/or any other continuous degrees of freedom of a macromolecule, and any energy function can be used without adding any asymptotic complexity to the design. We demonstrate that EPIC efficiently represents the energy surface for both molecular-mechanics and quantum-mechanical energy functions, and apply it specifically to protein design to model both sidechain and backbone degrees of freedom. PMID:26089744

Plasmonic nanohole arrays on Si-Ge heterostructures: an approach for integrated biosensors

NASA Astrophysics Data System (ADS)

Augel, L.; Fischer, I. A.; Dunbar, L. A.; Bechler, S.; Berrier, A.; Etezadi, D.; Hornung, F.; Kostecki, K.; Ozdemir, C. I.; Soler, M.; Altug, H.; Schulze, J.

2016-03-01

Nanohole array surface plasmon resonance (SPR) sensors offer a promising platform for high-throughput label-free biosensing. Integrating nanohole arrays with group-IV semiconductor photodetectors could enable low-cost and disposable biosensors compatible to Si-based complementary metal oxide semiconductor (CMOS) technology that can be combined with integrated circuitry for continuous monitoring of biosamples and fast sensor data processing. Such an integrated biosensor could be realized by structuring a nanohole array in the contact metal layer of a photodetector. We used Fouriertransform infrared spectroscopy to investigate nanohole arrays in a 100 nm Al film deposited on top of a vertical Si-Ge photodiode structure grown by molecular beam epitaxy (MBE). We find that the presence of a protein bilayer, constitute of protein AG and Immunoglobulin G (IgG), leads to a wavelength-dependent absorptance enhancement of ~ 8 %.

Driving force behind adsorption-induced protein unfolding: a time-resolved X-ray reflectivity study on lysozyme adsorbed at an air/water interface.

PubMed

Yano, Yohko F; Uruga, Tomoya; Tanida, Hajime; Toyokawa, Hidenori; Terada, Yasuko; Takagaki, Masafumi; Yamada, Hironari

2009-01-06

Time-resolved X-ray reflectivity measurements for lysozyme (LSZ) adsorbed at an air/water interface were performed to study the mechanism of adsorption-induced protein unfolding. The time dependence of the density profile at the air/water interface revealed that the molecular conformation changed significantly during adsorption. Taking into account previous work using Fourier transform infrared (FTIR) spectroscopy, we propose that the LSZ molecules initially adsorbed on the air/water interface have a flat unfolded structure, forming antiparallel beta-sheets as a result of hydrophobic interactions with the gas phase. In contrast, as adsorption continues, a second layer forms in which the molecules have a very loose structure having random coils as a result of hydrophilic interactions with the hydrophilic groups that protrude from the first layer.

[Inventive activity of the Departments of Chemistry and Biochemistry of Enzymes, and Protein Structure and Function of the Palladin Institute of Biochemistry of NAS of Ukraine. Part III. Diagnostic test-systems for analysis of fibrinolysis blood system and novel approaches to thrombosis treatment].

PubMed

Danilova, V M; Vynogradova, R P; Chernysh, I Yu

2016-01-01

This article continues analysis of scientific achievements of the Institute of Biochemistry in the study of hemostasis system. Two previous articles were focused on the studies of blood coagulation proteins and development of the immune-enzyme test-systems for evaluation of the risk of thrombosis upon various pathologies. This article highlights the research on the blood fibrinolysis system and new approaches to thrombosis treatment, which were developed (and are under development) in the Palladin Institute of Biochemistry of the NAS of Ukraine, in particular, in the Department of Chemistry and Biochemistry of Enzymes headed previously by Dr.Sci.(Biol.) S. O. Kudinov and now by Dr.Sci.(Biol.) T .V. Grinenko, and also in the Department of Protein Structure and Function headed by Dr.Biol.Sci. E. M. Makogonenko. The fundamental knowledge of protein molecule functions and mechanisms of regulation of blood coagulation and fibrinolysis opens up new opportunities to diagnose hemostasis disorders and control the effectiveness of the cardiovascular disease treatment and also contributes to development of new techniques for isolation of new proteins – promising therapeutic agents.

Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays.

PubMed

Fortes-Dias, Consuelo Latorre; Santos, Roberta Márcia Marques dos; Magro, Angelo José; Fontes, Marcos Roberto de Mattos; Chávez-Olórtegui, Carlos; Granier, Claude

2009-01-01

Crotoxin (CA.CB) is a beta-neurotoxin from Crotalus durissus terrificus snake venom that is responsible for main envenomation effects upon biting by this snake. It is a heterodimer of an acidic protein (CA) devoid of any biological activity per se and a basic, enzymatically active, PLA(2) counterpart (CB). Both lethal and enzymatic activities of crotoxin have been shown to be inhibited by CNF, a protein from the blood of C. d. terrificus snakes. CNF replaces CA in the CA.CB complex, forming a stable, non-toxic complex CNF.CB. The molecular sites involved in the tight interfacial protein-protein interactions in these PLA(2)-based complexes have not been clearly determined. To help address this question, we used the peptide arrays approach to map possible interfacial interaction sites in CA.CB and CNF.CB. Amino acid stretches putatively involved in these interactions were firstly identified in the primary structure of CB. Further analysis of the interfacial availability of these stretches in the presumed biologically active structure of CB, suggested two interaction main sites, located at the amino-terminus and beta-wing regions. Peptide segments at the carboxyl-terminus of CB were also suggested to play a secondary role in the binding of both CA and CNF.

From Protein Engineering to Immobilization: Promising Strategies for the Upgrade of Industrial Enzymes

PubMed Central

Singh, Raushan Kumar; Tiwari, Manish Kumar; Singh, Ranjitha; Lee, Jung-Kul

2013-01-01

Enzymes found in nature have been exploited in industry due to their inherent catalytic properties in complex chemical processes under mild experimental and environmental conditions. The desired industrial goal is often difficult to achieve using the native form of the enzyme. Recent developments in protein engineering have revolutionized the development of commercially available enzymes into better industrial catalysts. Protein engineering aims at modifying the sequence of a protein, and hence its structure, to create enzymes with improved functional properties such as stability, specific activity, inhibition by reaction products, and selectivity towards non-natural substrates. Soluble enzymes are often immobilized onto solid insoluble supports to be reused in continuous processes and to facilitate the economical recovery of the enzyme after the reaction without any significant loss to its biochemical properties. Immobilization confers considerable stability towards temperature variations and organic solvents. Multipoint and multisubunit covalent attachments of enzymes on appropriately functionalized supports via linkers provide rigidity to the immobilized enzyme structure, ultimately resulting in improved enzyme stability. Protein engineering and immobilization techniques are sequential and compatible approaches for the improvement of enzyme properties. The present review highlights and summarizes various studies that have aimed to improve the biochemical properties of industrially significant enzymes. PMID:23306150

Multiscale multiphysics and multidomain models—Flexibility and rigidity

PubMed Central

Xia, Kelin; Opron, Kristopher; Wei, Guo-Wei

2013-01-01

The emerging complexity of large macromolecules has led to challenges in their full scale theoretical description and computer simulation. Multiscale multiphysics and multidomain models have been introduced to reduce the number of degrees of freedom while maintaining modeling accuracy and achieving computational efficiency. A total energy functional is constructed to put energies for polar and nonpolar solvation, chemical potential, fluid flow, molecular mechanics, and elastic dynamics on an equal footing. The variational principle is utilized to derive coupled governing equations for the above mentioned multiphysical descriptions. Among these governing equations is the Poisson-Boltzmann equation which describes continuum electrostatics with atomic charges. The present work introduces the theory of continuum elasticity with atomic rigidity (CEWAR). The essence of CEWAR is to formulate the shear modulus as a continuous function of atomic rigidity. As a result, the dynamics complexity of a macromolecular system is separated from its static complexity so that the more time-consuming dynamics is handled with continuum elasticity theory, while the less time-consuming static analysis is pursued with atomic approaches. We propose a simple method, flexibility-rigidity index (FRI), to analyze macromolecular flexibility and rigidity in atomic detail. The construction of FRI relies on the fundamental assumption that protein functions, such as flexibility, rigidity, and energy, are entirely determined by the structure of the protein and its environment, although the structure is in turn determined by all the interactions. As such, the FRI measures the topological connectivity of protein atoms or residues and characterizes the geometric compactness of the protein structure. As a consequence, the FRI does not resort to the interaction Hamiltonian and bypasses matrix diagonalization, which underpins most other flexibility analysis methods. FRI's computational complexity is of \\documentclass[12pt]{minimal}\\begin{document}${\\cal O}(N^2)$\\end{document}O(N2) at most, where N is the number of atoms or residues, in contrast to \\documentclass[12pt]{minimal}\\begin{document}${\\cal O}(N^3)$\\end{document}O(N3) for Hamiltonian based methods. We demonstrate that the proposed FRI gives rise to accurate prediction of protein B-Factor for a set of 263 proteins. We show that a parameter free FRI is able to achieve about 95% accuracy of the parameter optimized FRI. An interpolation algorithm is developed to construct continuous atomic flexibility functions for visualization and use with CEWAR. PMID:24320318

Multiscale multiphysics and multidomain models—Flexibility and rigidity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xia, Kelin; Opron, Kristopher; Wei, Guo-Wei, E-mail: wei@math.msu.edu

The emerging complexity of large macromolecules has led to challenges in their full scale theoretical description and computer simulation. Multiscale multiphysics and multidomain models have been introduced to reduce the number of degrees of freedom while maintaining modeling accuracy and achieving computational efficiency. A total energy functional is constructed to put energies for polar and nonpolar solvation, chemical potential, fluid flow, molecular mechanics, and elastic dynamics on an equal footing. The variational principle is utilized to derive coupled governing equations for the above mentioned multiphysical descriptions. Among these governing equations is the Poisson-Boltzmann equation which describes continuum electrostatics with atomicmore » charges. The present work introduces the theory of continuum elasticity with atomic rigidity (CEWAR). The essence of CEWAR is to formulate the shear modulus as a continuous function of atomic rigidity. As a result, the dynamics complexity of a macromolecular system is separated from its static complexity so that the more time-consuming dynamics is handled with continuum elasticity theory, while the less time-consuming static analysis is pursued with atomic approaches. We propose a simple method, flexibility-rigidity index (FRI), to analyze macromolecular flexibility and rigidity in atomic detail. The construction of FRI relies on the fundamental assumption that protein functions, such as flexibility, rigidity, and energy, are entirely determined by the structure of the protein and its environment, although the structure is in turn determined by all the interactions. As such, the FRI measures the topological connectivity of protein atoms or residues and characterizes the geometric compactness of the protein structure. As a consequence, the FRI does not resort to the interaction Hamiltonian and bypasses matrix diagonalization, which underpins most other flexibility analysis methods. FRI's computational complexity is of O(N{sup 2}) at most, where N is the number of atoms or residues, in contrast to O(N{sup 3}) for Hamiltonian based methods. We demonstrate that the proposed FRI gives rise to accurate prediction of protein B-Factor for a set of 263 proteins. We show that a parameter free FRI is able to achieve about 95% accuracy of the parameter optimized FRI. An interpolation algorithm is developed to construct continuous atomic flexibility functions for visualization and use with CEWAR.« less

Comparing Molecular Dynamics Force Fields in the Essential Subspace

PubMed Central

Gomez-Puertas, Paulino; Boomsma, Wouter; Lindorff-Larsen, Kresten

2015-01-01

The continued development and utility of molecular dynamics simulations requires improvements in both the physical models used (force fields) and in our ability to sample the Boltzmann distribution of these models. Recent developments in both areas have made available multi-microsecond simulations of two proteins, ubiquitin and Protein G, using a number of different force fields. Although these force fields mostly share a common mathematical form, they differ in their parameters and in the philosophy by which these were derived, and previous analyses showed varying levels of agreement with experimental NMR data. To complement the comparison to experiments, we have performed a structural analysis of and comparison between these simulations, thereby providing insight into the relationship between force-field parameterization, the resulting ensemble of conformations and the agreement with experiments. In particular, our results show that, at a coarse level, many of the motional properties are preserved across several, though not all, force fields. At a finer level of detail, however, there are distinct differences in both the structure and dynamics of the two proteins, which can, together with comparison with experimental data, help to select force fields for simulations of proteins. A noteworthy observation is that force fields that have been reparameterized and improved to provide a more accurate energetic description of the balance between helical and coil structures are difficult to distinguish from their “unbalanced” counterparts in these simulations. This observation implies that simulations of stable, folded proteins, even those reaching 10 microseconds in length, may provide relatively little information that can be used to modify torsion parameters to achieve an accurate balance between different secondary structural elements. PMID:25811178

Continuous mutual improvement of macromolecular structure models in the PDB and of X-ray crystallographic software: the dual role of deposited experimental data.

PubMed

Terwilliger, Thomas C; Bricogne, Gerard

2014-10-01

Accurate crystal structures of macromolecules are of high importance in the biological and biomedical fields. Models of crystal structures in the Protein Data Bank (PDB) are in general of very high quality as deposited. However, methods for obtaining the best model of a macromolecular structure from a given set of experimental X-ray data continue to progress at a rapid pace, making it possible to improve most PDB entries after their deposition by re-analyzing the original deposited data with more recent software. This possibility represents a very significant departure from the situation that prevailed when the PDB was created, when it was envisioned as a cumulative repository of static contents. A radical paradigm shift for the PDB is therefore proposed, away from the static archive model towards a much more dynamic body of continuously improving results in symbiosis with continuously improving methods and software. These simultaneous improvements in methods and final results are made possible by the current deposition of processed crystallographic data (structure-factor amplitudes) and will be supported further by the deposition of raw data (diffraction images). It is argued that it is both desirable and feasible to carry out small-scale and large-scale efforts to make this paradigm shift a reality. Small-scale efforts would focus on optimizing structures that are of interest to specific investigators. Large-scale efforts would undertake a systematic re-optimization of all of the structures in the PDB, or alternatively the redetermination of groups of structures that are either related to or focused on specific questions. All of the resulting structures should be made generally available, along with the precursor entries, with various views of the structures being made available depending on the types of questions that users are interested in answering.

Continuous mutual improvement of macromolecular structure models in the PDB and of X-ray crystallographic software: The dual role of deposited experimental data

DOE PAGES

Terwilliger, Thomas C.; Bricogne, Gerard

2014-09-30

Accurate crystal structures of macromolecules are of high importance in the biological and biomedical fields. Models of crystal structures in the Protein Data Bank (PDB) are in general of very high quality as deposited. However, methods for obtaining the best model of a macromolecular structure from a given set of experimental X-ray data continue to progress at a rapid pace, making it possible to improve most PDB entries after their deposition by re-analyzing the original deposited data with more recent software. This possibility represents a very significant departure from the situation that prevailed when the PDB was created, when itmore » was envisioned as a cumulative repository of static contents. A radical paradigm shift for the PDB is therefore proposed, away from the static archive model towards a much more dynamic body of continuously improving results in symbiosis with continuously improving methods and software. These simultaneous improvements in methods and final results are made possible by the current deposition of processed crystallographic data (structure-factor amplitudes) and will be supported further by the deposition of raw data (diffraction images). It is argued that it is both desirable and feasible to carry out small-scale and large-scale efforts to make this paradigm shift a reality. Small-scale efforts would focus on optimizing structures that are of interest to specific investigators. Large-scale efforts would undertake a systematic re-optimization of all of the structures in the PDB, or alternatively the redetermination of groups of structures that are either related to or focused on specific questions. All of the resulting structures should be made generally available, along with the precursor entries, with various views of the structures being made available depending on the types of questions that users are interested in answering.« less

Continuous mutual improvement of macromolecular structure models in the PDB and of X-ray crystallographic software: the dual role of deposited experimental data

PubMed Central

Terwilliger, Thomas C.; Bricogne, Gerard

2014-01-01

Accurate crystal structures of macromolecules are of high importance in the biological and biomedical fields. Models of crystal structures in the Protein Data Bank (PDB) are in general of very high quality as deposited. However, methods for obtaining the best model of a macromolecular structure from a given set of experimental X-ray data continue to progress at a rapid pace, making it possible to improve most PDB entries after their deposition by re-analyzing the original deposited data with more recent software. This possibility represents a very significant departure from the situation that prevailed when the PDB was created, when it was envisioned as a cumulative repository of static contents. A radical paradigm shift for the PDB is therefore proposed, away from the static archive model towards a much more dynamic body of continuously improving results in symbiosis with continuously improving methods and software. These simultaneous improvements in methods and final results are made possible by the current deposition of processed crystallographic data (structure-factor amplitudes) and will be supported further by the deposition of raw data (diffraction images). It is argued that it is both desirable and feasible to carry out small-scale and large-scale efforts to make this paradigm shift a reality. Small-scale efforts would focus on optimizing structures that are of interest to specific investigators. Large-scale efforts would undertake a systematic re-optimization of all of the structures in the PDB, or alternatively the redetermination of groups of structures that are either related to or focused on specific questions. All of the resulting structures should be made generally available, along with the precursor entries, with various views of the structures being made available depending on the types of questions that users are interested in answering. PMID:25286839

Continuous mutual improvement of macromolecular structure models in the PDB and of X-ray crystallographic software: The dual role of deposited experimental data

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terwilliger, Thomas C.; Bricogne, Gerard

Accurate crystal structures of macromolecules are of high importance in the biological and biomedical fields. Models of crystal structures in the Protein Data Bank (PDB) are in general of very high quality as deposited. However, methods for obtaining the best model of a macromolecular structure from a given set of experimental X-ray data continue to progress at a rapid pace, making it possible to improve most PDB entries after their deposition by re-analyzing the original deposited data with more recent software. This possibility represents a very significant departure from the situation that prevailed when the PDB was created, when itmore » was envisioned as a cumulative repository of static contents. A radical paradigm shift for the PDB is therefore proposed, away from the static archive model towards a much more dynamic body of continuously improving results in symbiosis with continuously improving methods and software. These simultaneous improvements in methods and final results are made possible by the current deposition of processed crystallographic data (structure-factor amplitudes) and will be supported further by the deposition of raw data (diffraction images). It is argued that it is both desirable and feasible to carry out small-scale and large-scale efforts to make this paradigm shift a reality. Small-scale efforts would focus on optimizing structures that are of interest to specific investigators. Large-scale efforts would undertake a systematic re-optimization of all of the structures in the PDB, or alternatively the redetermination of groups of structures that are either related to or focused on specific questions. All of the resulting structures should be made generally available, along with the precursor entries, with various views of the structures being made available depending on the types of questions that users are interested in answering.« less

Response of Soft Continuous Structures and Topological Defects to a Temperature Gradient.

PubMed

Kurita, Rei; Mitsui, Shun; Tanaka, Hajime

2017-09-08

Thermophoresis, which is mass transport induced by a temperature gradient, has recently attracted considerable attention as a new way to transport materials. So far the study has been focused on the transport of discrete structures such as colloidal particles, proteins, and polymers in solutions. However, the response of soft continuous structures such as membranes and gels to a temperature gradient has been largely unexplored. Here we study the behavior of a lamellar phase made of stacked surfactant bilayer membranes under a temperature gradient. We find the migration of membranes towards a low-temperature region, causing the increase in the degree of membrane undulation fluctuations towards that direction. This is contrary to our intuition that the fluctuations are weaker at a lower temperature. We show that this can be explained by temperature-gradient-induced migration of membranes under the topological constraint coming from the connectivity of each membrane. We also reveal that the pattern of an edge dislocation array formed in a wedge-shaped cell can be controlled by a temperature gradient. These findings suggest that application of a temperature gradient provides a novel way to control the organization of soft continuous structures such as membranes, gels, and foams, in a manner essentially different from the other types of fields, and to manipulate topological defects.

Structure of a prokaryotic virtual proton pump at 3.2 Å resolution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Yiling; Jayaram, Hariharan; Shane, Tania

2009-09-15

To reach the mammalian gut, enteric bacteria must pass through the stomach. Many such organisms survive exposure to the harsh gastric environment (pH 1.5-4) by mounting extreme acid-resistance responses, one of which, the arginine-dependent system of Escherichia coli, has been studied at levels of cellular physiology, molecular genetics and protein biochemistry. This multiprotein system keeps the cytoplasm above pH 5 during acid challenge by continually pumping protons out of the cell using the free energy of arginine decarboxylation. At the heart of the process is a 'virtual proton pump' in the inner membrane, called AdiC, that imports L-arginine from themore » gastric juice and exports its decarboxylation product agmatine. AdiC belongs to the APC superfamily of membrane proteins, which transports amino acids, polyamines and organic cations in a multitude of biological roles, including delivery of arginine for nitric oxide synthesis, facilitation of insulin release from pancreatic {beta}-cells, and, when inappropriately overexpressed, provisioning of certain fast-growing neoplastic cells with amino acids. High-resolution structures and detailed transport mechanisms of APC transporters are currently unknown. Here we describe a crystal structure of AdiC at 3.2 {angstrom} resolution. The protein is captured in an outward-open, substrate-free conformation with transmembrane architecture remarkably similar to that seen in four other families of apparently unrelated transport proteins.« less

The principle of minimal episteric distortion of the water matrix and its steering role in protein folding

NASA Astrophysics Data System (ADS)

Fernández, Ariel

2013-08-01

A significant episteric ("around a solid") distortion of the hydrogen-bond structure of water is promoted by solutes with nanoscale surface detail and physico-chemical complexity, such as soluble natural proteins. These structural distortions defy analysis because the discrete nature of the solvent at the interface is not upheld by the continuous laws of electrostatics. This work derives and validates an electrostatic equation that governs the episteric distortions of the hydrogen-bond matrix. The equation correlates distortions from bulk-like structural patterns with anomalous polarization components that do not align with the electrostatic field of the solute. The result implies that the interfacial energy stored in the orthogonal polarization correlates with the distortion of the water hydrogen-bond network. The result is validated vis-à-vis experimental data on protein interfacial thermodynamics and is interpreted in terms of the interaction energy between the electrostatic field of the solute and the dipole moment induced by the anomalous polarization of interfacial water. Finally, we consider solutes capable of changing their interface through conformational transitions and introduce a principle of minimal episteric distortion (MED) of the water matrix. We assess the importance of the MED principle in the context of protein folding, concluding that the native fold may be identified topologically with the conformation that minimizes the interfacial tension or disruption of the water matrix.

Choosing non-redundant representative subsets of protein sequence data sets using submodular optimization.

PubMed

Libbrecht, Maxwell W; Bilmes, Jeffrey A; Noble, William Stafford

2018-04-01

Selecting a non-redundant representative subset of sequences is a common step in many bioinformatics workflows, such as the creation of non-redundant training sets for sequence and structural models or selection of "operational taxonomic units" from metagenomics data. Previous methods for this task, such as CD-HIT, PISCES, and UCLUST, apply a heuristic threshold-based algorithm that has no theoretical guarantees. We propose a new approach based on submodular optimization. Submodular optimization, a discrete analogue to continuous convex optimization, has been used with great success for other representative set selection problems. We demonstrate that the submodular optimization approach results in representative protein sequence subsets with greater structural diversity than sets chosen by existing methods, using as a gold standard the SCOPe library of protein domain structures. In this setting, submodular optimization consistently yields protein sequence subsets that include more SCOPe domain families than sets of the same size selected by competing approaches. We also show how the optimization framework allows us to design a mixture objective function that performs well for both large and small representative sets. The framework we describe is the best possible in polynomial time (under some assumptions), and it is flexible and intuitive because it applies a suite of generic methods to optimize one of a variety of objective functions. © 2018 Wiley Periodicals, Inc.

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

NASA Astrophysics Data System (ADS)

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

2017-05-01

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

Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

PubMed

Shynlova, Oksana; Chow, Michelle; Lye, Stephen J

2009-10-01

The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions.

Visualizing breathing motion of internal cavities in concert with ligand migration in myoglobin

PubMed Central

Tomita, Ayana; Sato, Tokushi; Ichiyanagi, Kouhei; Nozawa, Shunsuke; Ichikawa, Hirohiko; Chollet, Matthieu; Kawai, Fumihiro; Park, Sam-Yong; Tsuduki, Takayuki; Yamato, Takahisa; Koshihara, Shin-ya; Adachi, Shin-ichi

2009-01-01

Proteins harbor a number of cavities of relatively small volume. Although these packing defects are associated with the thermodynamic instability of the proteins, the cavities also play specific roles in controlling protein functions, e.g., ligand migration and binding. This issue has been extensively studied in a well-known protein, myoglobin (Mb). Mb reversibly binds gas ligands at the heme site buried in the protein matrix and possesses several internal cavities in which ligand molecules can reside. It is still an open question as to how a ligand finds its migration pathways between the internal cavities. Here, we report on the dynamic and sequential structural deformation of internal cavities during the ligand migration process in Mb. Our method, the continuous illumination of native carbonmonoxy Mb crystals with pulsed laser at cryogenic temperatures, has revealed that the migration of the CO molecule into each cavity induces structural changes of the amino acid residues around the cavity, which results in the expansion of the cavity with a breathing motion. The sequential motion of the ligand and the cavity suggests a self-opening mechanism of the ligand migration channel arising by induced fit, which is further supported by computational geometry analysis by the Delaunay tessellation method. This result suggests a crucial role of the breathing motion of internal cavities as a general mechanism of ligand migration in a protein matrix. PMID:19204297

Automation of large scale transient protein expression in mammalian cells

PubMed Central

Zhao, Yuguang; Bishop, Benjamin; Clay, Jordan E.; Lu, Weixian; Jones, Margaret; Daenke, Susan; Siebold, Christian; Stuart, David I.; Yvonne Jones, E.; Radu Aricescu, A.

2011-01-01

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative. PMID:21571074

Synthesis and Properties of Dodecyl Trehaloside Detergents for Membrane Protein Studies

PubMed Central

Tao, Houchao; Fu, Yu; Thompson, Aaron; Lee, Sung Chang; Mahoney, Nicholas; Stevens, Raymond C.; Zhang, Qinghai

2012-01-01

Sugar-based detergents, mostly derived from maltose or glucose, prevail in the extraction, solubilization, stabilization and crystallization of membrane proteins. Inspired by the broad use of trehalose for protecting biological macromolecules and lipid bilayer structures, we synthesized new trehaloside detergents for potential applications in membrane protein research. We devised an efficient synthesis of four dodecyl trehalosides, each with the 12-carboned alkyl chain attached to different hydroxyl groups of trehalose, thus presenting a structurally diverse but related family of detergents. The detergent physical properties, including solubility, hydrophobicity, critical micelle concentration (CMC) and size of micelles, were evaluated and compared with the most popular maltoside analog, β- D-dodecylmaltoside (DDM), which varied from each other due to distinct molecular geometries and possible polar group interactions in resulting micelles. Crystals of 2-dodecyl trehaloside (2-DDTre) were also obtained in methanol, and the crystal packing revealed multiple H-bonded interactions among adjacent trehalose groups. The few trehaloside detergents were tested for the solubilization and stabilization of the nociceptin/orphanin FQ peptide receptor (ORL1) and MsbA, which belong to the G-protein coupled receptor (GPCR) and ATP-binding cassette transporter families, respectively. Our results demonstrated the utility of trehaloside detergents as membrane protein solubilization reagents with the optimal detergents being protein dependent. Continuing development and investigations of trehaloside detergents are attractive given their interesting and unique chemical-physical properties and potential interactions with membrane lipids. PMID:22780816

Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guu, Tom S.Y.; Liu, Zheng; Ye, Qiaozhen

Hepatitis E virus (HEV), a small, non-enveloped RNA virus in the family Hepeviridae, is associated with endemic and epidemic acute viral hepatitis in developing countries. Our 3.5-{angstrom} structure of a HEV-like particle (VLP) shows that each capsid protein contains 3 linear domains that form distinct structural elements: S, the continuous capsid; P1, 3-fold protrusions; and P2, 2-fold spikes. The S domain adopts a jelly-roll fold commonly observed in small RNA viruses. The P1 and P2 domains both adopt {beta}-barrel folds. Each domain possesses a potential polysaccharide-binding site that may function in cell-receptor binding. Sugar binding to P1 at the capsidmore » protein interface may lead to capsid disassembly and cell entry. Structural modeling indicates that native T = 3 capsid contains flat dimers, with less curvature than those of T = 1 VLP. Our findings significantly advance the understanding of HEV molecular biology and have application to the development of vaccines and antiviral medications.« less

E-MSD: an integrated data resource for bioinformatics.

PubMed

Golovin, A; Oldfield, T J; Tate, J G; Velankar, S; Barton, G J; Boutselakis, H; Dimitropoulos, D; Fillon, J; Hussain, A; Ionides, J M C; John, M; Keller, P A; Krissinel, E; McNeil, P; Naim, A; Newman, R; Pajon, A; Pineda, J; Rachedi, A; Copeland, J; Sitnov, A; Sobhany, S; Suarez-Uruena, A; Swaminathan, G J; Tagari, M; Tromm, S; Vranken, W; Henrick, K

2004-01-01

The Macromolecular Structure Database (MSD) group (http://www.ebi.ac.uk/msd/) continues to enhance the quality and consistency of macromolecular structure data in the Protein Data Bank (PDB) and to work towards the integration of various bioinformatics data resources. We have implemented a simple form-based interface that allows users to query the MSD directly. The MSD 'atlas pages' show all of the information in the MSD for a particular PDB entry. The group has designed new search interfaces aimed at specific areas of interest, such as the environment of ligands and the secondary structures of proteins. We have also implemented a novel search interface that begins to integrate separate MSD search services in a single graphical tool. We have worked closely with collaborators to build a new visualization tool that can present both structure and sequence data in a unified interface, and this data viewer is now used throughout the MSD services for the visualization and presentation of search results. Examples showcasing the functionality and power of these tools are available from tutorial webpages (http://www. ebi.ac.uk/msd-srv/docs/roadshow_tutorial/).

E-MSD: an integrated data resource for bioinformatics

PubMed Central

Golovin, A.; Oldfield, T. J.; Tate, J. G.; Velankar, S.; Barton, G. J.; Boutselakis, H.; Dimitropoulos, D.; Fillon, J.; Hussain, A.; Ionides, J. M. C.; John, M.; Keller, P. A.; Krissinel, E.; McNeil, P.; Naim, A.; Newman, R.; Pajon, A.; Pineda, J.; Rachedi, A.; Copeland, J.; Sitnov, A.; Sobhany, S.; Suarez-Uruena, A.; Swaminathan, G. J.; Tagari, M.; Tromm, S.; Vranken, W.; Henrick, K.

2004-01-01

The Macromolecular Structure Database (MSD) group (http://www.ebi.ac.uk/msd/) continues to enhance the quality and consistency of macromolecular structure data in the Protein Data Bank (PDB) and to work towards the integration of various bioinformatics data resources. We have implemented a simple form-based interface that allows users to query the MSD directly. The MSD ‘atlas pages’ show all of the information in the MSD for a particular PDB entry. The group has designed new search interfaces aimed at specific areas of interest, such as the environment of ligands and the secondary structures of proteins. We have also implemented a novel search interface that begins to integrate separate MSD search services in a single graphical tool. We have worked closely with collaborators to build a new visualization tool that can present both structure and sequence data in a unified interface, and this data viewer is now used throughout the MSD services for the visualization and presentation of search results. Examples showcasing the functionality and power of these tools are available from tutorial webpages (http://www.ebi.ac.uk/msd-srv/docs/roadshow_tutorial/). PMID:14681397

Structural and mechanistic studies of measles virus illuminate paramyxovirus entry.

PubMed

Plemper, Richard K; Brindley, Melinda A; Iorio, Ronald M

2011-06-01

Measles virus (MeV), a member of the paramyxovirus family of enveloped RNA viruses and one of the most infectious viral pathogens identified, accounts for major pediatric morbidity and mortality worldwide although coordinated efforts to achieve global measles control are in place. Target cell entry is mediated by two viral envelope glycoproteins, the attachment (H) and fusion (F) proteins, which form a complex that achieves merger of the envelope with target cell membranes. Despite continually expanding knowledge of the entry strategies employed by enveloped viruses, our molecular insight into the organization of functional paramyxovirus fusion complexes and the mechanisms by which the receptor binding by the attachment protein triggers the required conformational rearrangements of the fusion protein remain incomplete. Recently reported crystal structures of the MeV attachment protein in complex with its cellular receptors CD46 or SLAM and newly developed functional assays have now illuminated some of the fundamental principles that govern cell entry by this archetype member of the paramyxovirus family. Here, we review these advances in our molecular understanding of MeV entry in the context of diverse entry strategies employed by other members of the paramyxovirus family.

Membrane proteins, detergents and crystals: what is the state of the art?

PubMed Central

Loll, Patrick J.

2014-01-01

At the time when the first membrane-protein crystal structure was determined, crystallization of these molecules was widely perceived as extremely arduous. Today, that perception has changed drastically, and the process is regarded as routine (or nearly so). On the occasion of the International Year of Crystallography 2014, this review presents a snapshot of the current state of the art, with an emphasis on the role of detergents in this process. A survey of membrane-protein crystal structures published since 2012 reveals that the direct crystallization of protein–detergent complexes remains the dominant method­ology; in addition, lipidic mesophases have proven immensely useful, particularly in specific niches, and bicelles, while perhaps undervalued, have provided important contributions as well. Evolving trends include the addition of lipids to protein–detergent complexes and the gradual incorporation of new detergents into the standard repertoire. Stability has emerged as a critical parameter controlling how a membrane protein behaves in the presence of detergent, and efforts to enhance stability are discussed. Finally, although discovery-based screening approaches continue to dwarf mechanistic efforts to unravel crystallization, recent technical advances offer hope that future experiments might incorporate the rational manipulation of crystallization behaviors. PMID:25484203

Development of a microsecond X-ray protein footprinting facility at the Advanced Light Source.

PubMed

Gupta, Sayan; Celestre, Richard; Petzold, Christopher J; Chance, Mark R; Ralston, Corie

2014-07-01

X-ray footprinting (XF) is an important structural biology tool used to determine macromolecular conformations and dynamics of both nucleic acids and proteins in solution on a wide range of timescales. With the impending shut-down of the National Synchrotron Light Source, it is ever more important that this tool continues to be developed at other synchrotron facilities to accommodate XF users. Toward this end, a collaborative XF program has been initiated at the Advanced Light Source using the white-light bending-magnet beamlines 5.3.1 and 3.2.1. Accessibility of the microsecond time regime for protein footprinting is demonstrated at beamline 5.3.1 using the high flux density provided by a focusing mirror in combination with a micro-capillary flow cell. It is further reported that, by saturating samples with nitrous oxide, the radiolytic labeling efficiency is increased and the imprints of bound versus bulk water can be distinguished. These results both demonstrate the suitability of the Advanced Light Source as a second home for the XF experiment, and pave the way for obtaining high-quality structural data on complex protein samples and dynamics information on the microsecond timescale.

The Importance of Being Tyrosine: Lessons in Molecular Recognition from Minimalist Synthetic Binding Proteins

PubMed Central

Koide, Shohei; Sidhu, Sachdev S.

2010-01-01

Summary Combinatorial libraries built with severely restricted chemical diversity have yielded highly functional synthetic binding proteins. Structural analyses of these minimalist binding sites have revealed the dominant role of large tyrosine residues for mediating molecular contacts and of small serine/glycine residues for providing space and flexibility. The concept of using limited residue types to construct optimized binding proteins mirrors findings in the field of small molecule drug development, where it has been proposed that most drugs are built from a limited set of side chains presented by diverse frameworks. The physicochemical properties of tyrosine make it the amino acid that is most effective for mediating molecular recognition, and protein engineers have taken advantage of these characteristics to build tyrosine-rich protein binding sites that outperform natural proteins in terms of affinity and specificity. Knowledge from preceding studies can be used to improve current designs, and thus, synthetic protein libraries will continue to evolve and improve. In the near future, it seems likely that synthetic binding proteins will supersede natural antibodies for most purposes, and moreover, synthetic proteins will enable many new applications beyond the scope of natural proteins. PMID:19298050

Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells

NASA Technical Reports Server (NTRS)

Maniotis, A. J.; Bojanowski, K.; Ingber, D. E.

1997-01-01

Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin-sensitive microfilaments. Furthermore, when ion concentrations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion-induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre-existing size, shape, number, and position by adding anti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally-unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis.

Overview of Sweeteners

NASA Astrophysics Data System (ADS)

Ellis, Jerry W.

1995-08-01

The techniques for assessing the relative sweetness of different compounds are discussed. The search for new, sweet compounds continues to be of interest to the food industry. In addition to sugars, sweet compounds with a variety of structures are surveyed and range from small inorganic molecules to large proteins. Emphasis is placed on artificial sweeteners and their current status in the marketplace. The recent theories of sweetness are briefly covered.

Electron microscope studies

NASA Astrophysics Data System (ADS)

Crewe, A. V.; Kapp, O. H.

1991-06-01

This year our laboratory has continued to make progress in the design of electron-optical systems, in the study of structure-function relationships of large multi-subunit proteins, in the development of new image processing software and in achieving a workable sub-angstrom STEM. We present an algebraic approach to the symmetrical Einzel (unipotential) lens wherein we simplify the analysis by specifying a field shape that meets some preferred set of boundary or other conditions and then calculate the fields. In a second study we generalize this approach to study of three element electrostatic lenses of which the symmetrical Einzel lens is a particular form. The purpose is to develop a method for assisting in the design of a lens for a particular purpose. In our biological work we study a stable and functional dodecameric complex of globin chains from the hemoglobin of Lumbricus terrestris. This is a complex lacking the 'linker' subunit first imaged in this lab and required for maintenance of the native structure. In addition, we do a complete work-up on the hemoglobin of the marine polychaete Eudistylia vancouverii, demonstrating the presence of a hierarchy of globin complexes. We demonstrate stable field-emission in the sub-angstrom STEM and the preliminary alignment of the beam. We continue our exploration of a algorithms for alignment of sequences of protein and DNA. Our computer facilities now include four second generation RISC workstations and we continue to take increasing advantage of the floating-point and graphical performance of these devices.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crewe, A.V.; Kapp, O.H.

This year our laboratory has continued to make progress in the design of electron-optical systems, in the study of structure-function relationships of large multi-subunit proteins, in the development of new image processing software and in achieving a workable sub-angstrom STEM. We present an algebraic approach to the symmetrical Einzel (unipotential) lens wherein we simplify the analysis by specifying a field shape that meets some preferred set of boundary or other conditions and then calculate the fields. In a second study we generalize this approach to study of three element electrostatic lenses of which the symmetrical Einzel lens is a particularmore » form. The purpose is to develop a method for assisting in the design of a lens for a particular purpose. In our biological work we study a stable and functional dodecameric complex of globin chains from the hemoglobin of Lumbricus terrestris. This is a complex lacking the linker'' subunit first imaged in this lab and required for maintenance of the native structure. In addition, we do a complete work-up on the hemoglobin of the marine polychaete Eudistylia vancouverii demonstrating the presence of a hierarchy of globin complexes. We demonstrate stable field-emission in the sub-angstrom STEM and the preliminary alignment of the beam. We continue our exploration of a algorithms for alignment of sequences of protein and DNA. Our computer facilities now include four second generation RISC workstations and we continue to take increasing advantage of the floating-point and graphical performance of these devices.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crewe, A.V.; Kapp, O.H.

This year our laboratory has continued to make progress in the design of electron-optical systems, in the study of structure-function relationships of large multi-subunit proteins, in the development of new image processing software and in achieving a workable sub-angstrom STEM. We present an algebraic approach to the symmetrical Einzel (unipotential) lens wherein we simplify the analysis by specifying a field shape that meets some preferred set of boundary or other conditions and then calculate the fields. In a second study we generalize this approach to study of three element electrostatic lenses of which the symmetrical Einzel lens is a particularmore » form. The purpose is to develop a method for assisting in the design of a lens for a particular purpose. In our biological work we study a stable and functional dodecameric complex of globin chains from the hemoglobin of Lumbricus terrestris. This is a complex lacking the ``linker`` subunit first imaged in this lab and required for maintenance of the native structure. In addition, we do a complete work-up on the hemoglobin of the marine polychaete Eudistylia vancouverii demonstrating the presence of a hierarchy of globin complexes. We demonstrate stable field-emission in the sub-angstrom STEM and the preliminary alignment of the beam. We continue our exploration of a algorithms for alignment of sequences of protein and DNA. Our computer facilities now include four second generation RISC workstations and we continue to take increasing advantage of the floating-point and graphical performance of these devices.« less

Recommendations of the wwPDB NMR Validation Task Force

PubMed Central

Montelione, Gaetano T.; Nilges, Michael; Bax, Ad; Güntert, Peter; Herrmann, Torsten; Richardson, Jane S.; Schwieters, Charles; Vranken, Wim F.; Vuister, Geerten W.; Wishart, David S.; Berman, Helen M.; Kleywegt, Gerard J.; Markley, John L.

2013-01-01

As methods for analysis of biomolecular structure and dynamics using nuclear magnetic resonance spectroscopy (NMR) continue to advance, the resulting 3D structures, chemical shifts, and other NMR data are broadly impacting biology, chemistry, and medicine. Structure model assessment is a critical area of NMR methods development, and is an essential component of the process of making these structures accessible and useful to the wider scientific community. For these reasons, the Worldwide Protein Data Bank (wwPDB) has convened an NMR Validation Task Force (NMR-VTF) to work with the wwPDB partners in developing metrics and policies for biomolecular NMR data harvesting, structure representation, and structure quality assessment. This paper summarizes the recommendations of the NMR-VTF, and lays the groundwork for future work in developing standards and metrics for biomolecular NMR structure quality assessment. PMID:24010715

The Development of Target-Specific Pose Filter Ensembles To Boost Ligand Enrichment for Structure-Based Virtual Screening.

PubMed

Xia, Jie; Hsieh, Jui-Hua; Hu, Huabin; Wu, Song; Wang, Xiang Simon

2017-06-26

Structure-based virtual screening (SBVS) has become an indispensable technique for hit identification at the early stage of drug discovery. However, the accuracy of current scoring functions is not high enough to confer success to every target and thus remains to be improved. Previously, we had developed binary pose filters (PFs) using knowledge derived from the protein-ligand interface of a single X-ray structure of a specific target. This novel approach had been validated as an effective way to improve ligand enrichment. Continuing from it, in the present work we attempted to incorporate knowledge collected from diverse protein-ligand interfaces of multiple crystal structures of the same target to build PF ensembles (PFEs). Toward this end, we first constructed a comprehensive data set to meet the requirements of ensemble modeling and validation. This set contains 10 diverse targets, 118 well-prepared X-ray structures of protein-ligand complexes, and large benchmarking actives/decoys sets. Notably, we designed a unique workflow of two-layer classifiers based on the concept of ensemble learning and applied it to the construction of PFEs for all of the targets. Through extensive benchmarking studies, we demonstrated that (1) coupling PFE with Chemgauss4 significantly improves the early enrichment of Chemgauss4 itself and (2) PFEs show greater consistency in boosting early enrichment and larger overall enrichment than our prior PFs. In addition, we analyzed the pairwise topological similarities among cognate ligands used to construct PFEs and found that it is the higher chemical diversity of the cognate ligands that leads to the improved performance of PFEs. Taken together, the results so far prove that the incorporation of knowledge from diverse protein-ligand interfaces by ensemble modeling is able to enhance the screening competence of SBVS scoring functions.

Structural rearrangement of β-lactoglobulin at different oil-water interfaces and its effect on emulsion stability.

PubMed

Zhai, Jiali; Wooster, Tim J; Hoffmann, Søren V; Lee, Tzong-Hsien; Augustin, Mary Ann; Aguilar, Marie-Isabel

2011-08-02

Understanding the factors that control protein structure and stability at the oil-water interface continues to be a major focus to optimize the formulation of protein-stabilized emulsions. In this study, a combination of synchrotron radiation circular dichroism spectroscopy, front-face fluorescence spectroscopy, and dual polarization interferometry (DPI) was used to characterize the conformation and geometric structure of β-lactoglobulin (β-Lg) upon adsorption to two oil-water interfaces: a hexadecane-water interface and a tricaprylin-water interface. The results show that, upon adsorption to both oil-water interfaces, β-Lg went through a β-sheet to α-helix transition with a corresponding loss of its globular tertiary structure. The degree of conformational change was also a function of the oil phase polarity. The hexadecane oil induced a much higher degree of non-native α-helix compared to the tricaprylin oil. In contrast to the β-Lg conformation in solution, the non-native α-helical-rich conformation of β-Lg at the interface was resistant to further conformational change upon heating. DPI measurements suggest that β-Lg formed a thin dense layer at emulsion droplet surfaces. The effects of high temperature and the presence of salt on these β-Lg emulsions were then investigated by monitoring changes in the ζ-potential and particle size. In the absence of salt, high electrostatic repulsion meant β-Lg-stabilized emulsions were resistant to heating to 90 °C. Adding salt (120 mM NaCl) before or after heating led to emulsion flocculation due to the screening of the electrostatic repulsion between colloidal particles. This study has provided insight into the structural properties of proteins adsorbed at the oil-water interface and has implications in the formulation and production of emulsions stabilized by globular proteins.

Advances in free-energy-based simulations of protein folding and ligand binding.

PubMed

Perez, Alberto; Morrone, Joseph A; Simmerling, Carlos; Dill, Ken A

2016-02-01

Free-energy-based simulations are increasingly providing the narratives about the structures, dynamics and biological mechanisms that constitute the fabric of protein science. Here, we review two recent successes. It is becoming practical: first, to fold small proteins with free-energy methods without knowing substructures and second, to compute ligand-protein binding affinities, not just their binding poses. Over the past 40 years, the timescales that can be simulated by atomistic MD are doubling every 1.3 years--which is faster than Moore's law. Thus, these advances are not simply due to the availability of faster computers. Force fields, solvation models and simulation methodology have kept pace with computing advancements, and are now quite good. At the tip of the spear recently are GPU-based computing, improved fast-solvation methods, continued advances in force fields, and conformational sampling methods that harness external information. Copyright © 2015 Elsevier Ltd. All rights reserved.

Properties of Bread Dough with Added Fiber Polysaccharides and Phenolic Antioxidants: A Review

PubMed Central

Sivam, Anusooya S; Sun-Waterhouse, Dongxiao; Quek, SiewYoung; Perera, Conrad O

2010-01-01

During breadmaking, different ingredients are used to ensure the development of a continuous protein network that is essential for bread quality. Interests in incorporating bioactive ingredients such as dietary fiber (DF) and phenolic antioxidants into popular foods such as bread have grown rapidly, due to the increased consumer health awareness. The added bioactive ingredients may or may not promote the protein cross-links. Appropriate cross-links among wheat proteins, fiber polysaccharides, and phenolic antioxidants could be the most critical factor for bread dough enhanced with DF and phenolic antioxidants. Such cross-links may influence the structure and properties of a bread system during baking. This article presents a brief overview of our current knowledge of the fate of the key components (wheat proteins, fibers, and phenolic antioxidants) and how they might interact during bread dough development and baking. PMID:21535512

The MORPHEUS II protein crystallization screen

PubMed Central

Gorrec, Fabrice

2015-01-01

High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions. PMID:26144227

The MORPHEUS II protein crystallization screen.

PubMed

Gorrec, Fabrice

2015-07-01

High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected from the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.

The film tells the story: Physical-chemical characteristics of IgG at the liquid-air interface.

PubMed

Koepf, Ellen; Schroeder, Rudolf; Brezesinski, Gerald; Friess, Wolfgang

2017-10-01

The presence of liquid-air interfaces in protein pharmaceuticals is known to negatively impact product stability. Nevertheless, the mechanisms behind interface-related protein aggregation are not yet fully understood. Little is known about the physical-chemical behavior of proteins adsorbed to the interface. Therefore, the combinatorial use of appropriate surface-sensitive analytical methods such as Langmuir trough experiments, Infrared Reflection-Absorption Spectroscopy (IRRAS), Brewster Angle Microscopy (BAM), and Atomic Force Microscopy (AFM) is highly expedient to uncover structures and events at the liquid-air interface directly. Concentration-dependent adsorption of a human immunoglobulin G (IgG) and characteristic surface-pressure/area isotherms substantiated the amphiphilic nature of the protein molecules as well as the formation of a compressible protein film at the liquid-air interface. Upon compression, the IgG molecules do not readily desorb but form a highly compressible interfacial film. IRRA spectra proved not only the presence of the protein at the interface, but also showed that the secondary structure does not change considerably during adsorption or compression. IRRAS experiments at different angles of incidence indicated that the film thickness and/or packing density increases upon compression. Furthermore, BAM images exposed the presence of a coherent but heterogeneous distribution of the protein at the interface. Topographical differences within the protein film after adsorption, compression and decompression were revealed using underwater AFM. The combinatorial use of physical-chemical, spectroscopic and microscopic methods provided useful insights into the liquid-air interfacial protein behavior and revealed the formation of a continuous but inhomogeneous film of native-like protein molecules whose topographical appearance is affected by compressive forces. Copyright © 2017 Elsevier B.V. All rights reserved.

The movement of proteins across the insect and tick digestive system.

PubMed

Jeffers, Laura A; Michael Roe, R

2008-02-01

The movement of intact proteins across the digestive system was shown in a number of different blood-feeding and non-blood-feeding insects in the orders Blattaria, Coleoptera, Diptera, Hemiptera, Lepidoptera, Orthoptera, Neuroptera and Siphonaptera, as well as in two tick families Ixodidae and Argasidae. Protein movement was observed for both normal dietary and xenobiotic proteins, which suggest that the mechanism for transfer is not substrate specific. The number of studies on the mechanism of movement is limited. The research so far suggests that movement can occur by either a transcellular or an intercellular pathway in the ventriculus with most of the research describing the former. Transfer is by continuous diffusion with no evidence of pinocytosis or vesicular transport common in mammalian systems. Proteins can move across the digestive system without modification of their primary or multimeric structure and with retention of their functional characteristics. Accumulation in the hemolymph is the result of the protein degradation rate in the gut and hemolymph and transfer rate across the digestive system and can be highly variable depending on species. Research on the development of delivery systems to enhance protein movement across the insect digestive system is in its infancy. The approaches so far considered with some success include the use of lipophilic-polyethylene glycol (PEG) polymers, the development of fusion proteins with lectins, reduced gut protease activity and the development of amphiphilic peptidic analogs. Additional research on understanding the basic mechanisms of protein delivery across the insect digestive system, the importance of structure activity in this transfer and the development of technology to improve movement across the gut could be highly significant to the future of protein and nucleic acid-based insecticide development as well as traditional chemical insecticidal technologies.

Combination of acoustic levitation with small angle scattering techniques and synchrotron radiation circular dichroism. Application to the study of protein solutions.

PubMed

Cristiglio, Viviana; Grillo, Isabelle; Fomina, Margarita; Wien, Frank; Shalaev, Evgenyi; Novikov, Alexey; Brassamin, Séverine; Réfrégiers, Matthieu; Pérez, Javier; Hennet, Louis

2017-01-01

The acoustic levitation technique is a useful sample handling method for small solid and liquids samples, suspended in air by means of an ultrasonic field. This method was previously used at synchrotron sources for studying pharmaceutical liquids and protein solutions using x-ray diffraction and small angle x-ray scattering (SAXS). In this work we combined for the first time this containerless method with small angle neutron scattering (SANS) and synchrotron radiation circular dichroism (SRCD) to study the structural behavior of proteins in solutions during the water evaporation. SANS results are also compared with SAXS experiments. The aggregation behavior of 45μl droplets of lysozyme protein diluted in water was followed during the continuous increase of the sample concentration by evaporating the solvent. The evaporation kinetics was followed at different drying stage by SANS and SAXS with a good data quality. In a prospective work using SRCD, we also studied the evolution of the secondary structure of the myoglobin protein in water solution in the same evaporation conditions. Acoustic levitation was applied for the first time with SANS and the high performances of the used neutron instruments made it possible to monitor fast container-less reactions in situ. A preliminary work using SRCD shows the potentiality of its combination with acoustic levitation for studying the evolution of the protein structure with time. This multi-techniques approach could give novel insights into crystallization and self-assembly phenomena of biological compound with promising potential applications in pharmaceutical, food and cosmetics industry. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo. Copyright © 2016 Elsevier B.V. All rights reserved.

Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications.

PubMed

Hiruma-Shimizu, Kazumi; Shimizu, Hiroki; Thompson, Gary S; Kalverda, Arnout P; Patching, Simon G

2015-01-01

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halavaty, Andrei S.; Northwestern University, Chicago, IL 60611; Kim, Youngchang

The structural characterization of acyl-carrier-protein synthase (AcpS) from three different pathogenic microorganisms is reported. One interesting finding of the present work is a crystal artifact related to the activity of the enzyme, which fortuitously represents an opportunity for a strategy to design a potential inhibitor of a pathogenic AcpS. Some bacterial type II fatty-acid synthesis (FAS II) enzymes have been shown to be important candidates for drug discovery. The scientific and medical quest for new FAS II protein targets continues to stimulate research in this field. One of the possible additional candidates is the acyl-carrier-protein synthase (AcpS) enzyme. Its holomore » form post-translationally modifies the apo form of an acyl carrier protein (ACP), which assures the constant delivery of thioester intermediates to the discrete enzymes of FAS II. At the Center for Structural Genomics of Infectious Diseases (CSGID), AcpSs from Staphylococcus aureus (AcpS{sub SA}), Vibrio cholerae (AcpS{sub VC}) and Bacillus anthracis (AcpS{sub BA}) have been structurally characterized in their apo, holo and product-bound forms, respectively. The structure of AcpS{sub BA} is emphasized because of the two 3′, 5′-adenosine diphosphate (3′, 5′-ADP) product molecules that are found in each of the three coenzyme A (CoA) binding sites of the trimeric protein. One 3′, 5′-ADP is bound as the 3′, 5′-ADP part of CoA in the known structures of the CoA–AcpS and 3′, 5′-ADP–AcpS binary complexes. The position of the second 3′, 5′-ADP has never been described before. It is in close proximity to the first 3′, 5′-ADP and the ACP-binding site. The coordination of two ADPs in AcpS{sub BA} may possibly be exploited for the design of AcpS inhibitors that can block binding of both CoA and ACP.« less

Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation.

PubMed

Wybenga-Groot, Leanne E; McGlade, C Jane

2013-12-01

The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3-SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3-SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3-SH2 connector, and SH2 domain of SLAP2 (SLAP2-32). While both domains adopt typical folds, the short SH3-SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding. © 2013.

Solution structure of dimeric Mnt repressor (1-76).

PubMed

Burgering, M J; Boelens, R; Gilbert, D E; Breg, J N; Knight, K L; Sauer, R T; Kaptein, R

1994-12-20

Wild-type Mnt repressor of Salmonella bacteriophage P22 is a tetrameric protein of 82 residues per monomer. A C-terminal deletion mutant of the repressor denoted Mnt (1-76) is a dimer in solution. The structure of this dimer has been determined using NMR. The NMR assignments of the majority of the 1H, 15N, and 13C resonances were obtained using 2D and triple-resonance 3D techniques. Elements of secondary structure were identified on the basis of characteristic sequential and medium range NOEs. For the structure determination more than 1000 NOEs per monomer were obtained, and structures were generated using distance geometry and restrained simulated annealing calculations. The discrimination of intra- vs intermonomer NOEs was based upon the observation of intersubunit NOEs in [15N,13C] double half-filtered NOESY experiments. The N-terminal part of Mnt (residues 1-44), which shows a 40% sequence homology with the Arc repressor, has a similar secondary and tertiary structure. Mnt (1-76) continues with a loop region of irregular structure, a third alpha-helix, and a random coil C-terminal peptide. Analysis of the secondary structure NOEs, the exchange rates, and the backbone chemical shifts suggests that the carboxy-terminal third helix is less stable than the remainder of the protein, but the observation of intersubunit NOEs for this part of the protein enables the positioning of this helix. The rsmd's between the backbone atoms of the N-terminal part of the Mnt repressor (residues 5-43, 5'-43') and the Arc repressor is 1.58 A, and between this region and the corresponding part of the MetJ repressor 1.43 A.

Cooperativity and modularity in protein folding

PubMed Central

Sasai, Masaki; Chikenji, George; Terada, Tomoki P.

2016-01-01

A simple statistical mechanical model proposed by Wako and Saitô has explained the aspects of protein folding surprisingly well. This model was systematically applied to multiple proteins by Muñoz and Eaton and has since been referred to as the Wako-Saitô-Muñoz-Eaton (WSME) model. The success of the WSME model in explaining the folding of many proteins has verified the hypothesis that the folding is dominated by native interactions, which makes the energy landscape globally biased toward native conformation. Using the WSME and other related models, Saitô emphasized the importance of the hierarchical pathway in protein folding; folding starts with the creation of contiguous segments having a native-like configuration and proceeds as growth and coalescence of these segments. The Φ-values calculated for barnase with the WSME model suggested that segments contributing to the folding nucleus are similar to the structural modules defined by the pattern of native atomic contacts. The WSME model was extended to explain folding of multi-domain proteins having a complex topology, which opened the way to comprehensively understanding the folding process of multi-domain proteins. The WSME model was also extended to describe allosteric transitions, indicating that the allosteric structural movement does not occur as a deterministic sequential change between two conformations but as a stochastic diffusive motion over the dynamically changing energy landscape. Statistical mechanical viewpoint on folding, as highlighted by the WSME model, has been renovated in the context of modern methods and ideas, and will continue to provide insights on equilibrium and dynamical features of proteins. PMID:28409080

Proteome complexity and the forces that drive proteome imbalance

PubMed Central

Harper, J. Wade; Bennett, Eric J.

2016-01-01

Summary The cellular proteome is a complex microcosm of structural and regulatory networks that requires continuous surveillance and modification to meet the dynamic needs of the cell. It is therefore crucial that the protein flux of the cell remains in balance to ensure proper cell function. Genetic alterations that range from chromosome imbalance to oncogene activation can affect the speed, fidelity and capacity of protein biogenesis and degradation systems, which often results in proteome imbalance. An improved understanding of the causes and consequences of proteome imbalance is helping to reveal how these systems can be targeted to treat diseases such as cancer. PMID:27629639

Multiple Simulated Annealing-Molecular Dynamics (MSA-MD) for Conformational Space Search of Peptide and Miniprotein

PubMed Central

Hao, Ge-Fei; Xu, Wei-Fang; Yang, Sheng-Gang; Yang, Guang-Fu

2015-01-01

Protein and peptide structure predictions are of paramount importance for understanding their functions, as well as the interactions with other molecules. However, the use of molecular simulation techniques to directly predict the peptide structure from the primary amino acid sequence is always hindered by the rough topology of the conformational space and the limited simulation time scale. We developed here a new strategy, named Multiple Simulated Annealing-Molecular Dynamics (MSA-MD) to identify the native states of a peptide and miniprotein. A cluster of near native structures could be obtained by using the MSA-MD method, which turned out to be significantly more efficient in reaching the native structure compared to continuous MD and conventional SA-MD simulation. PMID:26492886

How Do Rab Proteins Determine Golgi Structure?

PubMed Central

Liu, Shijie; Storrie, Brian

2015-01-01

Rab proteins, small GTPases, are key regulators of mammalian Golgi apparatus organization. Based on the effect of Rab activation state, Rab proteins fall into two functional classes. In Class1, inactivation induces Golgi ribbon fragmentation and/or redistribution of Golgi enzymes to the ER, while overexpression of wild type or activation has little, if any, effect on Golgi ribbon organization. In Class 2, the reverse is true. We give emphasis to Rab6, the most abundant Golgi-associated Rab protein. Rab6 depletion in HeLa cells causes an increase in Golgi cisternal number, longer, more continuous cisternae, and a pronounced accumulation of vesicles; the effect of Rab6 on Golgi ribbon organization is probably through regulation of vesicle transport. In effector studies, motor proteins and their regulators are found to be key Rab6 effectors. A related Rab, Rab41, affects Golgi ribbon organization in a contrasting manner. The balance between minus- and plus-end directed motor recruitment may well be the major Rab-dependent factor in Golgi ribbon organization. PMID:25708460

The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

DOE PAGES

Hu, Xiaohu; Hong, Liang; Smith, Micholas Dean; ...

2015-11-23

Here, internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behavior with effective relaxation times existing over many decades in time, from ps up to ~10 2s (refs 1-4). Here, using molecular dynamics simulations, we show that, on timescales from 10 –12 to 10 –5s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of themore » energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behavior persists up to timescales approaching the in vivo lifespan of individual protein molecules.« less

Simulating the bio nanoelectronic interface

NASA Astrophysics Data System (ADS)

Millar, Campbell; Roy, Scott; Brown, Andrew R.; Asenov, Asen

2007-05-01

As the size of conventional nano-CMOS devices continues to shrink, they are beginning to approach the size of biologically relevant macromolecules such as ion channels. This, in concert with the increasing understanding of the behaviour of proteins in vivo, creates the potential for a revolution in the sensing, measurement and interaction with biological systems. In this paper we will demonstrate the theoretical possibility of directly coupling a nanoscale MOSFET with a model ion channel protein. This will potentially allow a much better understanding of the behaviour of biologically relevant molecules, since the measurement of the motion of charged particles can reveal a substantial amount of information about protein structure-function relationships. We can use the MOSFET's innate sensitivity to stray charge to detect the positions of single ions and, thus, better explore the dynamics of ion conduction in channel proteins. In addition, we also demonstrate that the MOSFET can be 'tuned' to sense current flow through channel proteins, thus providing, for the first time, a direct solid state/biological interface at the atomic level.

Gels prepared from egg yolk and its fractions for tissue engineering.

PubMed

Rodil, Andrea; Laca, Amanda; Paredes, Benjamín; Rendueles, Manuel; Meana, Álvaro; Díaz, Mario

2016-11-01

New biomaterials prepared from egg yolk and its main fractions (plasma and granules) have been developed for use in tissue engineering. Protein gels obtained via transglutaminase cross-linking were characterized by rheometry, texturometry and scanning electron microscopy. All the gels exhibited suitable physical and mechanical characteristics for use as potential biomaterials in skin regeneration. Specifically, results showed that these materials presented a compact, uniform structure, with granular gel being found to be the most resistant as well as the most elastic material. Accordingly, these gels were subsequently evaluated as scaffolds for murine fibroblast growth. The best results were obtained with granule gels. Not only adhesion and cell growth were detected when using these gels, but also continuous coatings of cells growing on their surface. These findings can be attributed to the higher protein content of this fraction and to the particular structure of its proteins. Thus, granules have proved to be an interesting potential raw material for scaffold development. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1577-1583, 2016. © 2016 American Institute of Chemical Engineers.

The Nucleation and Growth of Protein Crystals

NASA Technical Reports Server (NTRS)

Pusey, Marc

2004-01-01

Obtaining crystals of suitable size and high quality continues to be a major bottleneck in macromolecular crystallography. Currently, structural genomics efforts are achieving on average about a 10% success rate in going from purified protein to a deposited crystal structure. Growth of crystals in microgravity was proposed as a means of overcoming size and quality problems, which subsequently led to a major NASA effort in microgravity crystal growth, with the agency also funding research into understanding the process. Studies of the macromolecule crystal nucleation and growth process were carried out in a number of labs in an effort to understand what affected the resultant crystal quality on Earth, and how microgravity improved the process. Based upon experimental evidence, as well as simple starting assumptions, we have proposed that crystal nucleation occurs by a series of discrete self assembly steps, which 'set' the underlying crystal symmetry. This talk will review the model developed, and its origins, in our laboratory for how crystals nucleate and grow, and will then present, along with preliminary data, how we propose to use this model to improve the success rate for obtaining crystals from a given protein.

Directed evolution of a model primordial enzyme provides insights into the development of the genetic code.

PubMed

Müller, Manuel M; Allison, Jane R; Hongdilokkul, Narupat; Gaillon, Laurent; Kast, Peter; van Gunsteren, Wilfred F; Marlière, Philippe; Hilvert, Donald

2013-01-01

The contemporary proteinogenic repertoire contains 20 amino acids with diverse functional groups and side chain geometries. Primordial proteins, in contrast, were presumably constructed from a subset of these building blocks. Subsequent expansion of the proteinogenic alphabet would have enhanced their capabilities, fostering the metabolic prowess and organismal fitness of early living systems. While the addition of amino acids bearing innovative functional groups directly enhances the chemical repertoire of proteomes, the inclusion of chemically redundant monomers is difficult to rationalize. Here, we studied how a simplified chorismate mutase evolves upon expanding its amino acid alphabet from nine to potentially 20 letters. Continuous evolution provided an enhanced enzyme variant that has only two point mutations, both of which extend the alphabet and jointly improve protein stability by >4 kcal/mol and catalytic activity tenfold. The same, seemingly innocuous substitutions (Ile→Thr, Leu→Val) occurred in several independent evolutionary trajectories. The increase in fitness they confer indicates that building blocks with very similar side chain structures are highly beneficial for fine-tuning protein structure and function.

Expression of the type VI intermediate filament proteins CP49 and filensin in the mouse lens epithelium.

PubMed

FitzGerald, Paul; Sun, Ning; Shibata, Brad; Hess, John F

2016-01-01

The differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age. Localization of CP49 was achieved with immunocytochemical probing of wild-type, CP49 knockout, filensin knockout, and vimentin knockout mice, in sections and in the explanted lens epithelium, at the light microscope and electron microscope levels. The relationship between CP49 and other cytoskeletal elements was probed using fluorescent phalloidin, as well as with antibodies to vimentin, GFAP, and α-tubulin. The relationship between CP49 and the aggresome was probed with antibodies to γ-tubulin, ubiquitin, and HDAC6. CP49 and filensin were expressed in the mouse lens epithelium, but only after 5 weeks of age. At the light microscope level, these two proteins colocalize to a large tubular structure, approximately 7 × 1 μm, which was typically present at one to two copies per cell. This structure is found in the anterior and anterolateral lens epithelium, including the zone where mitosis occurs. The structure becomes smaller and largely undetectable closer to the equator where the cell exits the cell cycle and commits to fiber cell differentiation. This structure bears some resemblance to the aggresome and is reactive with antibodies to HDAC6, a marker for the aggresome. However, the structure does not colocalize with antibodies to γ-tubulin or ubiquitin, also markers for the aggresome. The structure also colocalizes with actin but appears to largely exclude vimentin and α-tubulin. In the CP49 and filensin knockouts, this structure is absent, confirming the identity of CP49 and filensin in this structure, and suggesting a requirement for the physiologic coassembly of CP49 and filensin. CP49 and filensin have been considered robust markers for mouse lens fiber cell differentiation. The data reported here, however, document both proteins in the mouse lens epithelium, but only after 5 weeks of age, when lens epithelial growth and mitotic activity have slowed. Because of this, CP49 and filensin must be considered markers of differentiation for both fiber cells and the lens epithelium in the mouse. In addition, to our knowledge, no other protein has been shown to emerge so late in the development of the mouse lens epithelium, suggesting that lens epithelial differentiation may continue well into post-natal life. If this structure is related to the aggresome, it is a rare, or perhaps unique example of a large, stable aggresome in wild-type tissue.

Structure of catalase determined by MicroED

PubMed Central

Nannenga, Brent L; Shi, Dan; Hattne, Johan; Reyes, Francis E; Gonen, Tamir

2014-01-01

MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination. DOI: http://dx.doi.org/10.7554/eLife.03600.001 PMID:25303172

Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

PubMed Central

Zhao, Hua

2015-01-01

There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule. PMID:26949281

Defining scaffold geometries for interacting with proteins: geometrical classification of secondary structure linking regions.

PubMed

Tran, Tran T; Kulis, Christina; Long, Steven M; Bryant, Darryn; Adams, Peter; Smythe, Mark L

2010-11-01

Medicinal chemists synthesize arrays of molecules by attaching functional groups to scaffolds. There is evidence suggesting that some scaffolds yield biologically active molecules more than others, these are termed privileged substructures. One role of the scaffold is to present its side-chains for molecular recognition, and biologically relevant scaffolds may present side-chains in biologically relevant geometries or shapes. Since drug discovery is primarily focused on the discovery of compounds that bind to proteinaceous targets, we have been deciphering the scaffold shapes that are used for binding proteins as they reflect biologically relevant shapes. To decipher the scaffold architecture that is important for binding protein surfaces, we have analyzed the scaffold architecture of protein loops, which are defined in this context as continuous four residue segments of a protein chain that are not part of an α-helix or β-strand secondary structure. Loops are an important molecular recognition motif of proteins. We have found that 39 clusters reflect the scaffold architecture of 89% of the 23,331 loops in the dataset, with average intra-cluster and inter-cluster RMSD of 0.47 and 1.91, respectively. These protein loop scaffolds all have distinct shapes. We have used these 39 clusters that reflect the scaffold architecture of protein loops as biological descriptors. This involved generation of a small dataset of scaffold-based peptidomimetics. We found that peptidomimetic scaffolds with reported biological activities matched loop scaffold geometries and those peptidomimetic scaffolds with no reported biologically activities did not. This preliminary evidence suggests that organic scaffolds with tight matches to the preferred loop scaffolds of proteins, implies the likelihood of the scaffold to be biologically relevant.

Defining scaffold geometries for interacting with proteins: geometrical classification of secondary structure linking regions

NASA Astrophysics Data System (ADS)

Tran, Tran T.; Kulis, Christina; Long, Steven M.; Bryant, Darryn; Adams, Peter; Smythe, Mark L.

2010-11-01

Medicinal chemists synthesize arrays of molecules by attaching functional groups to scaffolds. There is evidence suggesting that some scaffolds yield biologically active molecules more than others, these are termed privileged substructures. One role of the scaffold is to present its side-chains for molecular recognition, and biologically relevant scaffolds may present side-chains in biologically relevant geometries or shapes. Since drug discovery is primarily focused on the discovery of compounds that bind to proteinaceous targets, we have been deciphering the scaffold shapes that are used for binding proteins as they reflect biologically relevant shapes. To decipher the scaffold architecture that is important for binding protein surfaces, we have analyzed the scaffold architecture of protein loops, which are defined in this context as continuous four residue segments of a protein chain that are not part of an α-helix or β-strand secondary structure. Loops are an important molecular recognition motif of proteins. We have found that 39 clusters reflect the scaffold architecture of 89% of the 23,331 loops in the dataset, with average intra-cluster and inter-cluster RMSD of 0.47 and 1.91, respectively. These protein loop scaffolds all have distinct shapes. We have used these 39 clusters that reflect the scaffold architecture of protein loops as biological descriptors. This involved generation of a small dataset of scaffold-based peptidomimetics. We found that peptidomimetic scaffolds with reported biological activities matched loop scaffold geometries and those peptidomimetic scaffolds with no reported biologically activities did not. This preliminary evidence suggests that organic scaffolds with tight matches to the preferred loop scaffolds of proteins, implies the likelihood of the scaffold to be biologically relevant.

Parallel Computational Protein Design.

PubMed

Zhou, Yichao; Donald, Bruce R; Zeng, Jianyang

2017-01-01

Computational structure-based protein design (CSPD) is an important problem in computational biology, which aims to design or improve a prescribed protein function based on a protein structure template. It provides a practical tool for real-world protein engineering applications. A popular CSPD method that guarantees to find the global minimum energy solution (GMEC) is to combine both dead-end elimination (DEE) and A* tree search algorithms. However, in this framework, the A* search algorithm can run in exponential time in the worst case, which may become the computation bottleneck of large-scale computational protein design process. To address this issue, we extend and add a new module to the OSPREY program that was previously developed in the Donald lab (Gainza et al., Methods Enzymol 523:87, 2013) to implement a GPU-based massively parallel A* algorithm for improving protein design pipeline. By exploiting the modern GPU computational framework and optimizing the computation of the heuristic function for A* search, our new program, called gOSPREY, can provide up to four orders of magnitude speedups in large protein design cases with a small memory overhead comparing to the traditional A* search algorithm implementation, while still guaranteeing the optimality. In addition, gOSPREY can be configured to run in a bounded-memory mode to tackle the problems in which the conformation space is too large and the global optimal solution cannot be computed previously. Furthermore, the GPU-based A* algorithm implemented in the gOSPREY program can be combined with the state-of-the-art rotamer pruning algorithms such as iMinDEE (Gainza et al., PLoS Comput Biol 8:e1002335, 2012) and DEEPer (Hallen et al., Proteins 81:18-39, 2013) to also consider continuous backbone and side-chain flexibility.

LCS-TA to identify similar fragments in RNA 3D structures.

PubMed

Wiedemann, Jakub; Zok, Tomasz; Milostan, Maciej; Szachniuk, Marta

2017-10-23

In modern structural bioinformatics, comparison of molecular structures aimed to identify and assess similarities and differences between them is one of the most commonly performed procedures. It gives the basis for evaluation of in silico predicted models. It constitutes the preliminary step in searching for structural motifs. In particular, it supports tracing the molecular evolution. Faced with an ever-increasing amount of available structural data, researchers need a range of methods enabling comparative analysis of the structures from either global or local perspective. Herein, we present a new, superposition-independent method which processes pairs of RNA 3D structures to identify their local similarities. The similarity is considered in the context of structure bending and bonds' rotation which are described by torsion angles. In the analyzed RNA structures, the method finds the longest continuous segments that show similar torsion within a user-defined threshold. The length of the segment is provided as local similarity measure. The method has been implemented as LCS-TA algorithm (Longest Continuous Segments in Torsion Angle space) and is incorporated into our MCQ4Structures application, freely available for download from http://www.cs.put.poznan.pl/tzok/mcq/ . The presented approach ties torsion-angle-based method of structure analysis with the idea of local similarity identification by handling continuous 3D structure segments. The first method, implemented in MCQ4Structures, has been successfully utilized in RNA-Puzzles initiative. The second one, originally applied in Euclidean space, is a component of LGA (Local-Global Alignment) algorithm commonly used in assessing protein models submitted to CASP. This unique combination of concepts implemented in LCS-TA provides a new perspective on structure quality assessment in local and quantitative aspect. A series of computational experiments show the first results of applying our method to comparison of RNA 3D models. LCS-TA can be used for identifying strengths and weaknesses in the prediction of RNA tertiary structures.

Membrane Assembly during the Infection Cycle of the Giant Mimivirus

PubMed Central

Mutsafi, Yael; Shimoni, Eyal; Shimon, Amir; Minsky, Abraham

2013-01-01

Although extensively studied, the structure, cellular origin and assembly mechanism of internal membranes during viral infection remain unclear. By combining diverse imaging techniques, including the novel Scanning-Transmission Electron Microscopy tomography, we elucidate the structural stages of membrane biogenesis during the assembly of the giant DNA virus Mimivirus. We show that this elaborate multistage process occurs at a well-defined zone localized at the periphery of large viral factories that are generated in the host cytoplasm. Membrane biogenesis is initiated by fusion of multiple vesicles, ∼70 nm in diameter, that apparently derive from the host ER network and enable continuous supply of lipid components to the membrane-assembly zone. The resulting multivesicular bodies subsequently rupture to form large open single-layered membrane sheets from which viral membranes are generated. Membrane generation is accompanied by the assembly of icosahedral viral capsids in a process involving the hypothetical major capsid protein L425 that acts as a scaffolding protein. The assembly model proposed here reveals how multiple Mimivirus progeny can be continuously and efficiently generated and underscores the similarity between the infection cycles of Mimivirus and Vaccinia virus. Moreover, the membrane biogenesis process indicated by our findings provides new insights into the pathways that might mediate assembly of internal viral membranes in general. PMID:23737745

BlockLogo: visualization of peptide and sequence motif conservation

PubMed Central

Olsen, Lars Rønn; Kudahl, Ulrich Johan; Simon, Christian; Sun, Jing; Schönbach, Christian; Reinherz, Ellis L.; Zhang, Guang Lan; Brusic, Vladimir

2013-01-01

BlockLogo is a web-server application for visualization of protein and nucleotide fragments, continuous protein sequence motifs, and discontinuous sequence motifs using calculation of block entropy from multiple sequence alignments. The user input consists of a multiple sequence alignment, selection of motif positions, type of sequence, and output format definition. The output has BlockLogo along with the sequence logo, and a table of motif frequencies. We deployed BlockLogo as an online application and have demonstrated its utility through examples that show visualization of T-cell epitopes and B-cell epitopes (both continuous and discontinuous). Our additional example shows a visualization and analysis of structural motifs that determine specificity of peptide binding to HLA-DR molecules. The BlockLogo server also employs selected experimentally validated prediction algorithms to enable on-the-fly prediction of MHC binding affinity to 15 common HLA class I and class II alleles as well as visual analysis of discontinuous epitopes from multiple sequence alignments. It enables the visualization and analysis of structural and functional motifs that are usually described as regular expressions. It provides a compact view of discontinuous motifs composed of distant positions within biological sequences. BlockLogo is available at: http://research4.dfci.harvard.edu/cvc/blocklogo/ and http://methilab.bu.edu/blocklogo/ PMID:24001880

[The true story and advantages of the famous Hepatitis B virus core particles: Outlook 2016].

PubMed

Pumpens, P; Grens, E

2016-01-01

This review article is a continuation of the paper "Hepatitis B core particles as a universal display model: a structure-function basis for development" written by Pumpens P. and Grens E., ordered by Professor Lev Kisselev and published in FEBS Letters, 1999, 442, 1-6. The past 17 years have strengthened the paper's finding that the human hepatitis B virus core protein, along with other Hepadnaviridae family member core proteins, is a mysterious, multifunctional protein. The core gene of the Hepadnaviridae genome encodes five partially collinear proteins. The most important of these is the HBV core protein p21, or HBc. It can self-assemble by forming viral HBc particles, but also plays a crucial role in the regulation of viral replication. Since 1986, the HBc protein has been one of the first and the most successful tools of the virus-like particle (VLP) technology. Later, the woodchuck hepatitis virus core protein (WHc) was also used as a VLP carrier. The Hepadnaviridae core proteins remain favourite VLP candidates for the knowledge-based design of future vaccines, gene therapy vectors, specifically targeted nanocontainers, and other modern nanotechnological tools for prospective medical use.

Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement.

PubMed

Khang, Chang Hyun; Berruyer, Romain; Giraldo, Martha C; Kankanala, Prasanna; Park, Sook-Young; Czymmek, Kirk; Kang, Seogchan; Valent, Barbara

2010-04-01

Knowledge remains limited about how fungal pathogens that colonize living plant cells translocate effector proteins inside host cells to regulate cellular processes and neutralize defense responses. To cause the globally important rice blast disease, specialized invasive hyphae (IH) invade successive living rice (Oryza sativa) cells while enclosed in host-derived extrainvasive hyphal membrane. Using live-cell imaging, we identified a highly localized structure, the biotrophic interfacial complex (BIC), which accumulates fluorescently labeled effectors secreted by IH. In each newly entered rice cell, effectors were first secreted into BICs at the tips of the initially filamentous hyphae in the cell. These tip BICs were left behind beside the first-differentiated bulbous IH cells as the fungus continued to colonize the host cell. Fluorescence recovery after photobleaching experiments showed that the effector protein PWL2 (for prevents pathogenicity toward weeping lovegrass [Eragrostis curvula]) continued to accumulate in BICs after IH were growing elsewhere. PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion. We report robust assays for elucidating the molecular mechanisms that underpin effector secretion into BICs, translocation to the rice cytoplasm, and cell-to-cell movement in rice.

Study on the Mechanisms of Active Compounds in Traditional Chinese Medicine for the Treatment of Influenza Virus by Virtual Screening.

PubMed

Ai, Haixin; Wu, Xuewei; Qi, Mengyuan; Zhang, Li; Hu, Huan; Zhao, Qi; Zhao, Jian; Liu, Hongsheng

2018-06-01

In recent years, new strains of influenza virus such as H7N9, H10N8, H5N6 and H5N8 had continued to emerge. There was an urgent need for discovery of new anti-influenza virus drugs as well as accurate and efficient large-scale inhibitor screening methods. In this study, we focused on six influenza virus proteins that could be anti-influenza drug targets, including neuraminidase (NA), hemagglutinin (HA), matrix protein 1 (M1), M2 proton channel (M2), nucleoprotein (NP) and non-structural protein 1 (NS1). Structure-based molecular docking was utilized to identify potential inhibitors for these drug targets from 13144 compounds in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform. The results showed that 56 compounds could inhibit more than two drug targets simultaneously. Further, we utilized reverse docking to study the interaction of these compounds with host targets. Finally, the 22 compound inhibitors could stably bind to host targets with high binding free energy. The results showed that the Chinese herbal medicines had a multi-target effect, which could directly inhibit influenza virus by the target viral protein and indirectly inhibit virus by the human target protein. This method was of great value for large-scale virtual screening of new anti-influenza virus compounds.

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

PubMed Central

Lugari, Adrien; Betzi, Stephane; Decroly, Etienne; Bonnaud, Emmanuel; Hermant, Aurélie; Guillemot, Jean-Claude; Debarnot, Claire; Borg, Jean-Paul; Bouvet, Mickaël; Canard, Bruno; Morelli, Xavier; Lécine, Patrick

2010-01-01

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses. PMID:20699222

Ambient ionisation mass spectrometry for in situ analysis of intact proteins

PubMed Central

Kocurek, Klaudia I.; Griffiths, Rian L.

2018-01-01

Abstract Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein‐protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed. PMID:29607564

Measurements of Attractive Forces between Proteins and End-Grafted Poly(Ethylene Glycol) Chains

NASA Astrophysics Data System (ADS)

Sheth, S. R.; Leckband, D.

1997-08-01

The surface force apparatus was used to measure directly the molecular forces between streptavidin and lipid bilayers displaying grafted Mr 2,000 poly(ethylene glycol) (PEG). These measurements provide direct evidence for the formation of relatively strong attractive forces between PEG and protein. At low compressive loads, the forces were repulsive, but they became attractive when the proteins were pressed into the polymer layer at higher loads. The adhesion was sufficiently robust that separation of the streptavidin and PEG uprooted anchored polymer from the supporting membrane. These interactions altered the properties of the grafted chains. After the onset of the attraction, the polymer continued to bind protein for several hours. The changes were not due to protein denaturation. These data demonstrate directly that the biological activity of PEG is not due solely to properties of simple polymers such as the excluded volume. It is also coupled to the competitive interactions between solvent and other materials such as proteins for the chain segments and to the ability of this material to adopt higher order intrachain structures.

A dielectric barrier discharge terminally inactivates RNase A by oxidizing sulfur-containing amino acids and breaking structural disulfide bonds

NASA Astrophysics Data System (ADS)

Lackmann, J.-W.; Baldus, S.; Steinborn, E.; Edengeiser, E.; Kogelheide, F.; Langklotz, S.; Schneider, S.; Leichert, L. I. O.; Benedikt, J.; Awakowicz, P.; Bandow, J. E.

2015-12-01

RNases are among the most stable proteins in nature. They even refold spontaneously after heat inactivation, regaining full activity. Due to their stability and universal presence, they often pose a problem when experimenting with RNA. We investigated the capabilities of nonthermal atmospheric-pressure plasmas to inactivate RNase A and studied the inactivation mechanism on a molecular level. While prolonged heating above 90 °C is required for heat inactivating RNase A, direct plasma treatment with a dielectric barrier discharge (DBD) source caused permanent inactivation within minutes. Circular dichroism spectroscopy showed that DBD-treated RNase A unfolds rapidly. Raman spectroscopy indicated methionine modifications and formation of sulfonic acid. A mass spectrometry-based analysis of the protein modifications that occur during plasma treatment over time revealed that methionine sulfoxide formation coincides with protein inactivation. Chemical reduction of methionine sulfoxides partially restored RNase A activity confirming that sulfoxidation is causal and sufficient for RNase A inactivation. Continued plasma exposure led to over-oxidation of structural disulfide bonds. Using antibodies, disulfide bond over-oxidation was shown to be a general protein inactivation mechanism of the DBD. The antibody’s heavy and light chains linked by disulfide bonds dissociated after plasma exposure. Based on their ability to inactivate proteins by oxidation of sulfur-containing amino acids and over-oxidation of disulfide bonds, DBD devices present a viable option for inactivating undesired or hazardous proteins on heat or solvent-sensitive surfaces.

In Silico Analysis for the Study of Botulinum Toxin Structure

NASA Astrophysics Data System (ADS)

Suzuki, Tomonori; Miyazaki, Satoru

2010-01-01

Protein-protein interactions play many important roles in biological function. Knowledge of protein-protein complex structure is required for understanding the function. The determination of protein-protein complex structure by experimental studies remains difficult, therefore computational prediction of protein structures by structure modeling and docking studies is valuable method. In addition, MD simulation is also one of the most popular methods for protein structure modeling and characteristics. Here, we attempt to predict protein-protein complex structure and property using some of bioinformatic methods, and we focus botulinum toxin complex as target structure.

In Situ formation of microstructures near live cells using spatially structured near-infrared laser microbeam

NASA Astrophysics Data System (ADS)

Ingle, Ninad; Gu, Ling; Mohanty, Samarendra K.

2011-03-01

Here, we report in situ formation of microstructures from the regular constituents of culture media near live cells using spatially-structured near infrared (NIR) laser beam. Irradiation with the continuous wave (cw) NIR laser microbeam for few seconds onto the regular cell culture media containing fetal bovine serum resulted in accumulation of dense material inside the media as evidenced by phase contrast microscopy. The time to form the phase dense material was found to depend on the laser beam power. Switching off the laser beam led to diffusion of phase dark material. However, the proteins could be stitched together by use of carbon nanoparticles and continuous wave (cw) Ti: Sapphire laser beam. Further, by use of spatially-structured beam profiles different structures near live cells could be formed. The microfabricated structure could be held by the Gravito-optical trap and repositioned by movement of the sample stage. Orientation of these microstructures was achieved by rotating the elliptical laser beam profile. Thus, multiple microstructures were formed and organized near live cells. This method would enable study of response of cells/axons to the immediate physical hindrance provided by such structure formation and also eliminate the biocompatibility requirement posed on artificial microstructure materials.

Investigations on the effect of forage source, grinding, and urea supplementation on ruminal fermentation and microbial protein flow in a semi-continuous rumen simulation system.

PubMed

Hildebrand, Bastian; Boguhn, Jeannette; Rodehutscord, Markus

2011-10-01

The objective of the present study was to compare the effect of maize silage and grass silage on microbial fermentation and protein flow in a semi-continuous rumen simulation system (Rusitec) when milling screen size (MSS) during grinding was varied. Oven-dried silages were milled through screens of 1, 4 or 9 mm pore size and incubated for 48 h in a Rusitec system. Furthermore, the effect of N supplementation to maize silage (MSS: 4 mm) was investigated and single dose vs. continuous infusion of urea-N were compared. Degradation of organic matter (OM), crude protein (CP), fibre fractions and non-structural carbohydrates (NSC) as well as short-chain fatty acid production differed significantly between forage sources. Urea-N supplementation improved the degradation of NSC, but not that of fibre fractions in maize silage. The way of urea supply had only marginal effects on fermentation characteristics. An increase in MSS, and consequently in mean feed particle size, led to an improvement in the degradation of OM, CP and NSC, but efficiency of microbial net protein synthesis (EMPS; mg microbial N flow/g degraded OM) and the microbial amino acid profile were less affected. EMPS was higher in grass silage than in maize silage and was improved by urea-N supplementation in maize silage. This study indicates that fermentation of NSC as well as EMPS during incubation of maize silage was limited by availability of NH3-N. Furthermore, an increase in MSS above 1 mm seems to improve fermentation of silages in the Rusitec system.

Efficiently explore the energy landscape of proteins in molecular dynamics simulations by amplifying collective motions

NASA Astrophysics Data System (ADS)

He, Jianbin; Zhang, Zhiyong; Shi, Yunyu; Liu, Haiyan

2003-08-01

We describe a method for efficient sampling of the energy landscape of a protein in atomic molecular dynamics simulations. A simulation is divided into alternatively occurring relaxation phases and excitation phases. In the relaxation phase (conventional simulation), we use a frequently updated reference structure and deviations from this reference structure to mark whether the system has been trapped in a local minimum. In that case, the simulation enters the excitation phase, during which a few slow collective modes of the system are coupled to a higher temperature bath. After the system has escaped from the minimum (also judged by deviations from the reference structure) the simulation reenters the relaxation phase. The collective modes are obtained from a coarse-grained Gaussian elastic network model. The scheme, which we call ACM-AME (amplified collective motion-assisted minimum escaping), is compared with conventional simulations as well as an alternative scheme that elevates the temperature of all degrees of freedom during the excitation phase (amplified overall motion-assisted minimum escaping, or AOM-AME). Comparison is made using simulations on four peptides starting from non-native extended or all helical structures. In terms of sampling low energy conformations and continuously sampling new conformations throughout a simulation, the ACM-AME scheme demonstrates very good performance while the AOM-AME scheme shows little improvement upon conventional simulations. Limited success is achieved in producing structures close to the native structures of the peptides: for an S-peptide analog, the ACM-AME approach is able to reproduce its native helical structure, and starting from an all-helical structure of the villin headpiece subdomain (HP-36) in implicit solvent, two out of three 150 ns ACM-AME runs are able to sample structures with 3-4 Å backbone root-mean-square deviations from the nuclear magnetic resonance structure of the protein.

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a 'function follows form' model of differentiation.

PubMed

Kankeu, Cynthia; Clarke, Kylie; Van Haver, Delphi; Gevaert, Kris; Impens, Francis; Dittrich, Anna; Roderick, H Llewelyn; Passante, Egle; Huber, Heinrich J

2018-05-17

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes.

Resolving protein structure-function-binding site relationships from a binding site similarity network perspective.

PubMed

Mudgal, Richa; Srinivasan, Narayanaswamy; Chandra, Nagasuma

2017-07-01

Functional annotation is seldom straightforward with complexities arising due to functional divergence in protein families or functional convergence between non-homologous protein families, leading to mis-annotations. An enzyme may contain multiple domains and not all domains may be involved in a given function, adding to the complexity in function annotation. To address this, we use binding site information from bound cognate ligands and catalytic residues, since it can help in resolving fold-function relationships at a finer level and with higher confidence. A comprehensive database of 2,020 fold-function-binding site relationships has been systematically generated. A network-based approach is employed to capture the complexity in these relationships, from which different types of associations are deciphered, that identify versatile protein folds performing diverse functions, same function associated with multiple folds and one-to-one relationships. Binding site similarity networks integrated with fold, function, and ligand similarity information are generated to understand the depth of these relationships. Apart from the observed continuity in the functional site space, network properties of these revealed versatile families with topologically different or dissimilar binding sites and structural families that perform very similar functions. As a case study, subtle changes in the active site of a set of evolutionarily related superfamilies are studied using these networks. Tracing of such similarities in evolutionarily related proteins provide clues into the transition and evolution of protein functions. Insights from this study will be helpful in accurate and reliable functional annotations of uncharacterized proteins, poly-pharmacology, and designing enzymes with new functional capabilities. Proteins 2017; 85:1319-1335. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

New developments in high field electron paramagnetic resonance with applications in structural biology

NASA Astrophysics Data System (ADS)

Bennati, Marina; Prisner, Thomas F.

2005-02-01

Recent developments in microwave technologies have led to a renaissance of electron paramagnetic resonance (EPR) due to the implementation of new spectrometers operating at frequencies >=90 GHz. EPR at high fields and high frequencies (HF-EPR) has been established up to THz (very high frequency (VHF) EPR) in continuous wave (cw) operation and up to about 300 GHz in pulsed operation. To date, its most prominent application field is structural biology. This review article first gives an overview of the theoretical basics and the technical aspects of HF-EPR methodologies, such as cw and pulsed HF-EPR, as well as electron nuclear double resonance at high fields (HF-ENDOR). In the second part, the article illustrates different application areas of HF-EPR in studies of protein structure and function. In particular, HF-EPR has delivered essential contributions to disentangling complex spectra of radical cofactors or reaction intermediates in photosynthetic reaction centres, radical enzymes (such as ribonucleotide reductase) and in metalloproteins. Furthermore, HF-EPR combined with site-directed spin labelling in membranes and soluble proteins provides new methods of investigating complex molecular dynamics and intermolecular distances.

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures.

PubMed

Riquelme, Meritxell; Aguirre, Jesús; Bartnicki-García, Salomon; Braus, Gerhard H; Feldbrügge, Michael; Fleig, Ursula; Hansberg, Wilhelm; Herrera-Estrella, Alfredo; Kämper, Jörg; Kück, Ulrich; Mouriño-Pérez, Rosa R; Takeshita, Norio; Fischer, Reinhard

2018-06-01

Filamentous fungi constitute a large group of eukaryotic microorganisms that grow by forming simple tube-like hyphae that are capable of differentiating into more-complex morphological structures and distinct cell types. Hyphae form filamentous networks by extending at their tips while branching in subapical regions. Rapid tip elongation requires massive membrane insertion and extension of the rigid chitin-containing cell wall. This process is sustained by a continuous flow of secretory vesicles that depends on the coordinated action of the microtubule and actin cytoskeletons and the corresponding motors and associated proteins. Vesicles transport cell wall-synthesizing enzymes and accumulate in a special structure, the Spitzenkörper, before traveling further and fusing with the tip membrane. The place of vesicle fusion and growth direction are enabled and defined by the position of the Spitzenkörper, the so-called cell end markers, and other proteins involved in the exocytic process. Also important for tip extension is membrane recycling by endocytosis via early endosomes, which function as multipurpose transport vehicles for mRNA, septins, ribosomes, and peroxisomes. Cell integrity, hyphal branching, and morphogenesis are all processes that are largely dependent on vesicle and cytoskeleton dynamics. When hyphae differentiate structures for asexual or sexual reproduction or to mediate interspecies interactions, the hyphal basic cellular machinery may be reprogrammed through the synthesis of new proteins and/or the modification of protein activity. Although some transcriptional networks involved in such reprogramming of hyphae are well studied in several model filamentous fungi, clear connections between these networks and known determinants of hyphal morphogenesis are yet to be established. Copyright © 2018 American Society for Microbiology.

Single-Molecule Encoders for Tracking Motor Proteins on DNA

NASA Astrophysics Data System (ADS)

Lipman, Everett A.

2012-02-01

Devices such as inkjet printers and disk drives track position and velocity using optical encoders, which produce periodic signals precisely synchronized with linear or rotational motion. We have implemented this technique at the nanometer scale by labeling DNA with regularly spaced fluorescent dyes. The resulting molecular encoders can be used in several ways for high-resolution continuous tracking of individual motor proteins. These measurements do not require mechanical coupling to macroscopic instrumentation, are automatically calibrated by the underlying structure of DNA, and depend on signal periodicity rather than absolute level. I will describe the synthesis of single-molecule encoders, data from and modeling of experiments on a helicase and a DNA polymerase, and some ideas for future work.

Protein Engineering Towards Natural Product Synthesis and Diversification

PubMed Central

Zabala, Angelica O.; Cacho, Ralph A.; Tang, Yi

2014-01-01

A dazzling array of enzymes is used by nature in making structurally complex natural products. These enzymes constitute a molecular toolbox that may be used in the construction and fine-tuning of pharmaceutically active molecules. Aided by technological advancements in protein engineering, it is now possible to tailor the activities and specificities of these enzymes as biocatalysts in the production of both natural products and their unnatural derivatives. These efforts are crucial in drug discovery and development, where there is a continuous quest for more potent agents. Both rational and random evolution techniques have been utilized in engineering these enzymes. This review will highlight some examples from several large families of natural products. PMID:22006344

Cell adhesion in zebrafish myogenesis: distribution of intermediate filaments, microfilaments, intracellular adhesion structures and extracellular matrix.

PubMed

Costa, Manoel L; Escaleira, Roberta C; Jazenko, Fernanda; Mermelstein, Claudia S

2008-10-01

To overcome the limitations of in vitro studies, we have been studying myogenesis in situ in zebrafish embryos, at a sub-cellular level. While in previous works we focused on myofibrillogenesis and some aspects of adhesion structures, here we describe in more detail cell adhesion structures and interactions among cytoskeletal components, membrane and extracellular matrix during zebrafish muscle development. We studied the intermediate filaments, and we describe the full range of desmin distribution in zebrafish development, from perinuclear to striated, until its deposition around the intersomite septa of older somites. This adhesion structure, positive for desmin and actin, has not been previously observed in myogenesis in vitro. We also show that actin is initially located in the intersomite septum region whereas it is confined to the myofibrils later on. While actin localization changes during development, the adhesion complex proteins vinculin, paxillin, talin, dystrophin, laminin and fibronectin always appear exclusively at the intersomite septa, and appear to be co-distributed, even though the extracellular proteins accumulates before the intracellular ones. Contrary to the adhesion proteins, that are continuously distributed, desmin and sarcomeric actin form triangular aggregates among the septa and the cytoskeleton. We studied the cytoskeletal linker plectin as well, and we show that it has a distribution similar to desmin and not to actin. We conclude that the in situ adhesion structures differ from their in vitro counterparts, and that the actual zebrafish embryo myogenesis is quite different than that which occurs in in vitro systems. Copyright 2008 Wiley-Liss, Inc.

Predicting protein-protein interactions on a proteome scale by matching evolutionary and structural similarities at interfaces using PRISM.

PubMed

Tuncbag, Nurcan; Gursoy, Attila; Nussinov, Ruth; Keskin, Ozlem

2011-08-11

Prediction of protein-protein interactions at the structural level on the proteome scale is important because it allows prediction of protein function, helps drug discovery and takes steps toward genome-wide structural systems biology. We provide a protocol (termed PRISM, protein interactions by structural matching) for large-scale prediction of protein-protein interactions and assembly of protein complex structures. The method consists of two components: rigid-body structural comparisons of target proteins to known template protein-protein interfaces and flexible refinement using a docking energy function. The PRISM rationale follows our observation that globally different protein structures can interact via similar architectural motifs. PRISM predicts binding residues by using structural similarity and evolutionary conservation of putative binding residue 'hot spots'. Ultimately, PRISM could help to construct cellular pathways and functional, proteome-scale annotation. PRISM is implemented in Python and runs in a UNIX environment. The program accepts Protein Data Bank-formatted protein structures and is available at http://prism.ccbb.ku.edu.tr/prism_protocol/.

Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

PubMed

Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

2016-12-13

Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

GPCRM: a homology modeling web service with triple membrane-fitted quality assessment of GPCR models.

PubMed

Miszta, Przemyslaw; Pasznik, Pawel; Jakowiecki, Jakub; Sztyler, Agnieszka; Latek, Dorota; Filipek, Slawomir

2018-05-21

Due to the involvement of G protein-coupled receptors (GPCRs) in most of the physiological and pathological processes in humans they have been attracting a lot of attention from pharmaceutical industry as well as from scientific community. Therefore, the need for new, high quality structures of GPCRs is enormous. The updated homology modeling service GPCRM (http://gpcrm.biomodellab.eu/) meets those expectations by greatly reducing the execution time of submissions (from days to hours/minutes) with nearly the same average quality of obtained models. Additionally, due to three different scoring functions (Rosetta, Rosetta-MP, BCL::Score) it is possible to select accurate models for the required purposes: the structure of the binding site, the transmembrane domain or the overall shape of the receptor. Currently, no other web service for GPCR modeling provides this possibility. GPCRM is continually upgraded in a semi-automatic way and the number of template structures has increased from 20 in 2013 to over 90 including structures the same receptor with different ligands which can influence the structure not only in the on/off manner. Two types of protein viewers can be used for visual inspection of obtained models. The extended sortable tables with available templates provide links to external databases and display ligand-receptor interactions in visual form.

Next generation calmodulin affinity purification: Clickable calmodulin facilitates improved protein purification

PubMed Central

Kinzer-Ursem, Tamara L.

2018-01-01

As the proteomics field continues to expand, scientists are looking to integrate cross-disciplinary tools for studying protein structure, function, and interactions. Protein purification remains a key tool for many characterization studies. Calmodulin (CaM) is a calcium-binding messenger protein with over a hundred downstream binding partners, and is involved in a host of physiological processes, from learning and memory to immune and cardiac function. To facilitate biophysical studies of calmodulin, researchers have designed a site-specific labeling process for use in bioconjugation applications while maintaining high levels of protein activity. Here, we present a platform for selective conjugation of calmodulin directly from clarified cell lysates under bioorthogonal reaction conditions. Using a chemoenzymatically modified calmodulin, we employ popular click chemistry reactions for the conjugation of calmodulin to Sepharose resin, thereby streamlining a previously multi-step purification and conjugation process. We show that this “next-generation” calmodulin-Sepharose resin is not only easy to produce, but is also able to purify more calmodulin-binding proteins per volume of resin than traditional calmodulin-Sepharose resins. We expect these methods to be translatable to other proteins of interest and to other conjugation applications such as surface-based assays for the characterization of protein-protein interaction dynamics. PMID:29864125

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer.

PubMed

Yan, Jing; Zhou, Mowei; Gilbert, Joshua D; Wolff, Jeremy J; Somogyi, Árpád; Pedder, Randall E; Quintyn, Royston S; Morrison, Lindsay J; Easterling, Michael L; Paša-Tolić, Ljiljana; Wysocki, Vicki H

2017-01-03

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.

Microdomains of endoplasmic reticulum within the sarcoplasmic reticulum of skeletal myofibers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaakinen, Mika; Papponen, Hinni; Metsikkoe, Kalervo

2008-01-15

The relationship between the endoplasmic reticulum (ER) and the sarcoplasmic reticulum (SR) of skeletal muscle cells has remained obscure. In this study, we found that ER- and SR-specific membrane proteins exhibited diverse solubility properties when extracted with mild detergents. Accordingly, the major SR-specific protein Ca{sup 2+}-ATPase (SERCA) remained insoluble in Brij 58 and floated in sucrose gradients while typical ER proteins were partially or fully soluble. Sphingomyelinase treatment rendered SERCA soluble in Brij 58. Immunofluorescence staining for resident ER proteins revealed dispersed dots over I bands contrasting the continuous staining pattern of SERCA. Infection of isolated myofibers with enveloped virusesmore » indicated that interfibrillar protein synthesis occurred. Furthermore, we found that GFP-tagged Dad1, able to incorporate into the oligosaccharyltransferase complex, showed the dot-like structures but the fusion protein was also present in membranes over the Z lines. This behaviour mimics that of cargo proteins that accumulated over the Z lines when blocked in the ER. Taken together, the results suggest that resident ER proteins comprised Brij 58-soluble microdomains within the insoluble SR membrane. After synthesis and folding in the ER-microdomains, cargo proteins and non-incorporated GFP-Dad1 diffused into the Z line-flanking compartment which likely represents the ER exit sites.« less

Outside-in assembly pathway of the type IV pilus system in Myxococcus xanthus.

PubMed

Friedrich, Carmen; Bulyha, Iryna; Søgaard-Andersen, Lotte

2014-01-01

Type IV pili (T4P) are ubiquitous bacterial cell surface structures that undergo cycles of extension, adhesion, and retraction. T4P function depends on a highly conserved envelope-spanning macromolecular machinery consisting of 10 proteins that localizes polarly in Myxococcus xanthus. Using this localization, we investigated the entire T4P machinery assembly pathway by systematically profiling the stability of all and the localization of eight of these proteins in the absence of other T4P machinery proteins as well as by mapping direct protein-protein interactions. Our experiments uncovered a sequential, outside-in pathway starting with the outer membrane (OM) PilQ secretin ring. PilQ recruits a subcomplex consisting of the inner membrane (IM) lipoprotein PilP and the integral IM proteins PilN and PilO by direct interaction with the periplasmic domain of PilP. The PilP/PilN/PilO subcomplex recruits the cytoplasmic PilM protein, by direct interaction between PilN and PilM, and the integral IM protein PilC. The PilB/PilT ATPases that power extension/retraction localize independently of other T4P machinery proteins. Thus, assembly of the T4P machinery initiates with formation of the OM secretin ring and continues inwards over the periplasm and IM to the cytoplasm.

Subnuclear organization and trafficking of regulatory proteins: implications for biological control and cancer.

PubMed

Stein, G S; van Wijnen, A J; Stein, J L; Lian, J B; Montecino, M; Zaidi, K; Javed, A

2000-01-01

The regulated and regulatory components that interrelate nuclear structure and function must be experimentally established. A formidable challenge is to define further the control of transcription factor targeting to acceptor sites associated with the nuclear matrix. It will be important to determine whether acceptor proteins are associated with a pre-existing core-filament structural lattice or whether a compositely organized scaffold of regulatory factors is dynamically assembled. An inclusive model for all steps in the targeting of proteins to subnuclear sites cannot yet be proposed. However, this model must account for the apparent diversity of intranuclear targeting signals. It is also important to assess the extent to which regulatory discrimination is mediated by subnuclear domain-specific trafficking signals. Furthermore, the checkpoints that monitor subnuclear distribution of regulatory factors and the sorting steps that ensure both structural and functional fidelity of nuclear domains in which replication and expression of genes occur must be biochemically and mechanistically defined. There is emerging recognition that placement of regulatory components of gene expression must be temporally and spatially coordinated to facilitate biological control. The consequences of breaches in nuclear structure-function relationships are observed in an expanding series of diseases that include cancer [Weis et al., 1994; Rogaia et al., 1997; Yano et al., 1997; Rowley, 1998; Zeng et al., 1998; McNeil et al., 1999; Tao and Levine, 1999a] and neurological disorders [Skinner et al., 1997]. As the repertoire of architecture-associated regulatory factors and cofactors expands, workers in the field are becoming increasingly confident that nuclear organization contributes significantly to control of transcription. To gain increased appreciation for the complexities of subnuclear organization and gene regulation, we must continue to characterize mechanisms that direct regulatory proteins to specific transcription sites within the nucleus so that these proteins are in the right place at the right time. J. Cell. Biochem. Suppl. 35:84-92, 2000. Copyright 2001 Wiley-Liss, Inc.

Expression of the type VI intermediate filament proteins CP49 and filensin in the mouse lens epithelium

PubMed Central

Sun, Ning; Shibata, Brad; Hess, John F.

2016-01-01

Purpose The differentiated lens fiber cell assembles a filamentous cytoskeletal structure referred to as the beaded filament (BF). The BF requires CP49 (bfsp2) and filensin (bfsp1) for assembly, both of which are highly divergent members of the large intermediate filament (IF) family of proteins. Thus far, these two proteins have been reported only in the differentiated lens fiber cell. For this reason, both proteins have been considered robust markers of fiber cell differentiation. We report here that both proteins are also expressed in the mouse lens epithelium, but only after 5 weeks of age. Methods Localization of CP49 was achieved with immunocytochemical probing of wild-type, CP49 knockout, filensin knockout, and vimentin knockout mice, in sections and in the explanted lens epithelium, at the light microscope and electron microscope levels. The relationship between CP49 and other cytoskeletal elements was probed using fluorescent phalloidin, as well as with antibodies to vimentin, GFAP, and α-tubulin. The relationship between CP49 and the aggresome was probed with antibodies to γ-tubulin, ubiquitin, and HDAC6. Results CP49 and filensin were expressed in the mouse lens epithelium, but only after 5 weeks of age. At the light microscope level, these two proteins colocalize to a large tubular structure, approximately 7 × 1 μm, which was typically present at one to two copies per cell. This structure is found in the anterior and anterolateral lens epithelium, including the zone where mitosis occurs. The structure becomes smaller and largely undetectable closer to the equator where the cell exits the cell cycle and commits to fiber cell differentiation. This structure bears some resemblance to the aggresome and is reactive with antibodies to HDAC6, a marker for the aggresome. However, the structure does not colocalize with antibodies to γ-tubulin or ubiquitin, also markers for the aggresome. The structure also colocalizes with actin but appears to largely exclude vimentin and α-tubulin. In the CP49 and filensin knockouts, this structure is absent, confirming the identity of CP49 and filensin in this structure, and suggesting a requirement for the physiologic coassembly of CP49 and filensin. Conclusions CP49 and filensin have been considered robust markers for mouse lens fiber cell differentiation. The data reported here, however, document both proteins in the mouse lens epithelium, but only after 5 weeks of age, when lens epithelial growth and mitotic activity have slowed. Because of this, CP49 and filensin must be considered markers of differentiation for both fiber cells and the lens epithelium in the mouse. In addition, to our knowledge, no other protein has been shown to emerge so late in the development of the mouse lens epithelium, suggesting that lens epithelial differentiation may continue well into post-natal life. If this structure is related to the aggresome, it is a rare, or perhaps unique example of a large, stable aggresome in wild-type tissue. PMID:27559293

Characterization of extended channel bioreactors for continuous-flow protein production

DOE PAGES

Timm, Andrea C.; Shankles, Peter G.; Foster, Carmen M.; ...

2015-10-02

In this paper, protein based therapeutics are an important class of drugs, used to treat a variety of medical conditions including cancer and autoimmune diseases. Requiring continuous cold storage, and having a limited shelf life, the ability to produce such therapeutics at the point-of-care would open up new opportunities in distributing medicines and treating patients in more remote locations. Here, the authors describe the first steps in the development of a microfluidic platform that can be used for point-of-care protein synthesis. While biologic medicines, including therapeutic proteins, are commonly produced using recombinant deoxyribonucleic acid (DNA) technology in large batch cellmore » cultures, the system developed here utilizes cell-free protein synthesis (CFPS) technology. CFPS is a scalable technology that uses cell extracts containing the biological machinery required for transcription and translation and combines those extracts with DNA, encoding a specific gene, and the additional metabolites required to produce proteins in vitro. While CFPS reactions are typically performed in batch or fed-batch reactions, a well-engineered reaction scheme may improve both the rate of protein production and the economic efficiency of protein synthesis reactions, as well as enable a more streamlined method for subsequent purification of the protein product—all necessary requirements for point-of-care protein synthesis. In this work, the authors describe a new bioreactor design capable of continuous production of protein using cell-free protein synthesis. The bioreactors were designed with three inlets to separate reactive components prior to on-chip mixing, which lead into a long, narrow, serpentine channel. These multiscale, serpentine channel bioreactors were designed to take advantage of microscale diffusion distances across narrow channels in reactors containing enough volume to produce a therapeutic dose of protein, and open the possibility of performing these reactions continuously and in line with downstream purification modules. Here, the authors demonstrate the capability to produce protein over time with continuous-flow reactions and examine basic design features and operation specifications fundamental to continuous microfluidic protein synthesis.« less

Utilization of protein intrinsic disorder knowledge in structural proteomics

PubMed Central

Oldfield, Christopher J.; Xue, Bin; Van, Ya-Yue; Ulrich, Eldon L.; Markley, John L.; Dunker, A. Keith; Uversky, Vladimir N.

2014-01-01

Intrinsically disordered proteins (IDPs) and proteins with long disordered regions are highly abundant in various proteomes. Despite their lack of well-defined ordered structure, these proteins and regions are frequently involved in crucial biological processes. Although in recent years these proteins have attracted the attention of many researchers, IDPs represent a significant challenge for structural characterization since these proteins can impact many of the processes in the structure determination pipeline. Here we investigate the effects of IDPs on the structure determination process and the utility of disorder prediction in selecting and improving proteins for structural characterization. Examination of the extent of intrinsic disorder in existing crystal structures found that relatively few protein crystal structures contain extensive regions of intrinsic disorder. Although intrinsic disorder is not the only cause of crystallization failures and many structured proteins cannot be crystallized, filtering out highly disordered proteins from structure-determination target lists is still likely to be cost effective. Therefore it is desirable to avoid highly disordered proteins from structure-determination target lists and we show that disorder prediction can be applied effectively to enrich structure determination pipelines with proteins more likely to yield crystal structures. For structural investigation of specific proteins, disorder prediction can be used to improve targets for structure determination. Finally, a framework for considering intrinsic disorder in the structure determination pipeline is proposed. PMID:23232152

Structure based alignment and clustering of proteins (STRALCP)

DOEpatents

Zemla, Adam T.; Zhou, Carol E.; Smith, Jason R.; Lam, Marisa W.

2013-06-18

Disclosed are computational methods of clustering a set of protein structures based on local and pair-wise global similarity values. Pair-wise local and global similarity values are generated based on pair-wise structural alignments for each protein in the set of protein structures. Initially, the protein structures are clustered based on pair-wise local similarity values. The protein structures are then clustered based on pair-wise global similarity values. For each given cluster both a representative structure and spans of conserved residues are identified. The representative protein structure is used to assign newly-solved protein structures to a group. The spans are used to characterize conservation and assign a "structural footprint" to the cluster.

Dynamics of the microbial community during continuous methane fermentation in continuously stirred tank reactors.

PubMed

Tang, Yue-Qin; Shigematsu, Toru; Morimura, Shigeru; Kida, Kenji

2015-04-01

Methane fermentation is an attractive technology for the treatment of organic wastes and wastewaters. However, the process is difficult to control, and treatment rates and digestion efficiency require further optimization. Understanding the microbiology mechanisms of methane fermentation is of fundamental importance to improving this process. In this review, we summarize the dynamics of microbial communities in methane fermentation chemostats that are operated using completely stirred tank reactors (CSTRs). Each chemostat was supplied with one substrate as the sole carbon source. The substrates include acetate, propionate, butyrate, long-chain fatty acids, glycerol, protein, glucose, and starch. These carbon sources are general substrates and intermediates of methane fermentation. The factors that affect the structure of the microbial community are discussed. The carbon source, the final product, and the operation conditions appear to be the main factors that affect methane fermentation and determine the structure of the microbial community. Understanding the structure of the microbial community during methane fermentation will guide the design and operation of practical wastewater treatments. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Collective Dynamics of Periplasmic Glutamine Binding Protein upon Domain Closure

PubMed Central

Loeffler, Hannes H.; Kitao, Akio

2009-01-01

The glutamine binding protein is a vital component of the associated ATP binding cassette transport systems responsible for the uptake of glutamine into the cell. We have investigated the global movements of this protein by molecular dynamics simulations and principal component analysis (PCA). We confirm that the most dominant mode corresponds to the biological function of the protein, i.e., a hinge-type motion upon ligand binding. The closure itself was directly observed from two independent trajectories whereby PCA was used to elucidate the nature of this closing reaction. Two intermediary states are identified and described in detail. The ligand binding induces the structural change of the hinge regions from a discontinuous β-sheet to a continuous one, which also enhances softness of the hinge and modifies the direction of hinge motion to enable closing. We also investigated the convergence behavior of PCA modes, which were found to converge rather quickly when the associated magnitudes of the eigenvalues are well separated. PMID:19883597

On the formation and functions of high and very high magnesium calcites in the continuously growing teeth of the echinoderm Lytechinus variegatus: development of crystallinity and protein involvement.

PubMed

Veis, Arthur; Stock, Stuart R; Alvares, Keith; Lux, Elizabeth

2011-01-01

Sea urchin teeth grow continuously and develop a complex mineralized structure consisting of spatially separate but crystallographically aligned first stage calcitic elements of high Mg content (5-15 mol% mineral). These become cemented together by epitaxially oriented second stage very high Mg calcite (30-40 mol% mineral). In the tooth plumula, ingressing preodontoblasts create layered cellular syncytia. Mineral deposits develop within membrane-bound compartments between cellular syncytial layers. We seek to understand how this complex tooth architecture is developed, how individual crystalline calcitic elements become crystallographically aligned, and how their Mg composition is regulated. Synchrotron microbeam X-ray scattering was performed on live, freshly dissected teeth. We observed that the initial diffracting crystals lie within independent syncytial spaces in the plumula. These diffraction patterns match those of mature tooth calcite. Thus, the spatially separate crystallites grow with the same crystallographic orientation seen in the mature tooth. Mineral-related proteins from regions with differing Mg contents were isolated, sequenced, and characterized. A tooth cDNA library was constructed, and selected matrix-related proteins were cloned. Antibodies were prepared and used for immunolocaliztion. Matrix-related proteins are acidic, phosphorylated, and associated with the syncytial membranes. Time-of-flight secondary ion mass spectroscopy of various crystal elements shows unique amino acid, Mg, and Ca ion distributions. High and very high Mg calcites differ in Asp content. Matrix-related proteins are phosphorylated. Very high Mg calcite is associated with Asp-rich protein, and it is restricted to the second stage mineral. Thus, the composition at each part of the tooth is related to architecture and function. Copyright © 2011 S. Karger AG, Basel.

Characteristics and EGFP expression of goat mammary gland epithelial cells.

PubMed

Zheng, Y-M; He, X-Y; Zhang, Y

2010-12-01

The aims of this study were (i) to establish a goat mammary gland epithelial (GMGE) cell line, and (ii) to determine if these GMGE cells could be maintained long-term in culture by continuous subculturing following transfection with a reporter gene, enhanced green fluorescence protein (EGFP). Primary culture of GMGE cells was achieved by outgrowth of migrating cells from the fragments of the mammary gland tissue of a lactating goat. The passage 16 GMGE cells were transfected with EGFP gene using lipofection. The expression of Cell keratins of epithelial cells in GMGE cells was test by immunofluorescence. Βeta-Casein gene mRNA was test for GMGE cells by RT-PCR. The results showed that when grown at low density on a plastic substratum, the GMGE cells formed islands, and when grown to confluency, the cells formed a monolayer and aggregated with the characteristic cobble-stone morphology of epithelial cells. GMGE cells could form dome-like structure which looked like nipple, and the lumen-like structures formed among the cells. Several blister-like structures appeared in the appearance of the cells. The GMGE cells contained different cell types, majority of the cells were short shuttle-like or polygon which were beehive-like. A part of cells were round and flat, a small number of cells were elongated. Some of the GMGE cells contained milk drops. The cell nuclei were round which had 2-4 obvious cores. The expression of Cell keratins demonstrated the property of epithelial cells in GMGE cells by immunofluorescence. The GMGE cells could express transcript encoding a Βeta-Casein protein. EGFP gene was successfully transferred into the GMGE cells, and the transfected cells could be maintained long-term in culture by continuous subculturing. In conclusion, we have established a EGFP gene transfected GMGE (ET-GMGE) cell line and maintained it long-term in culture by continuous subculturing. © 2010 Blackwell Verlag GmbH.

Bioinformatic prediction and in vivo validation of residue-residue interactions in human proteins

NASA Astrophysics Data System (ADS)

Jordan, Daniel; Davis, Erica; Katsanis, Nicholas; Sunyaev, Shamil

2014-03-01

Identifying residue-residue interactions in protein molecules is important for understanding both protein structure and function in the context of evolutionary dynamics and medical genetics. Such interactions can be difficult to predict using existing empirical or physical potentials, especially when residues are far from each other in sequence space. Using a multiple sequence alignment of 46 diverse vertebrate species we explore the space of allowed sequences for orthologous protein families. Amino acid changes that are known to damage protein function allow us to identify specific changes that are likely to have interacting partners. We fit the parameters of the continuous-time Markov process used in the alignment to conclude that these interactions are primarily pairwise, rather than higher order. Candidates for sites under pairwise epistasis are predicted, which can then be tested by experiment. We report the results of an initial round of in vivo experiments in a zebrafish model that verify the presence of multiple pairwise interactions predicted by our model. These experimentally validated interactions are novel, distant in sequence, and are not readily explained by known biochemical or biophysical features.

Protein crystal growth in microgravity review of large scale temperature induction method: Bovine insulin, human insulin and human α-interferon

NASA Astrophysics Data System (ADS)

Long, Marianna M.; Bishop, John Bradford; Delucas, Lawrence J.; Nagabhushan, Tattanhalli L.; Reichert, Paul; Smith, G. David

1997-01-01

The Protein Crystal Growth Facility (PCF) is space-flight hardware that accommodates large scale protein crystal growth experiments using temperature change as the inductive step. Recent modifications include specialized instrumentation for monitoring crystal nucleation with laser light scattering. This paper reviews results from its first seven flights on the Space Shuttle, the last with laser light scattering instrumentation in place. The PCF's objective is twofold: (1) the production of high quality protein crystals for x-ray analysis and subsequent structure-based drug design and (2) preparation of a large quantity of relatively contaminant free crystals for use as time-release protein pharmaceuticals. The first three Shuttle flights with bovine insulin constituted the PCF's proof of concept, demonstrating that the space-grown crystals were larger and diffracted to higher resolution than their earth-grown counterparts. The later four PCF missions were used to grow recombinant human insulin crystals for x-ray analysis and continue productions trials aimed at the development of a processing facility for crystalline recombinant a-interferon.

SLLE for predicting membrane protein types.

PubMed

Wang, Meng; Yang, Jie; Xu, Zhi-Jie; Chou, Kuo-Chen

2005-01-07

Introduction of the concept of pseudo amino acid composition (PROTEINS: Structure, Function, and Genetics 43 (2001) 246; Erratum: ibid. 44 (2001) 60) has made it possible to incorporate a considerable amount of sequence-order effects by representing a protein sample in terms of a set of discrete numbers, and hence can significantly enhance the prediction quality of membrane protein type. As a continuous effort along such a line, the Supervised Locally Linear Embedding (SLLE) technique for nonlinear dimensionality reduction is introduced (Science 22 (2000) 2323). The advantage of using SLLE is that it can reduce the operational space by extracting the essential features from the high-dimensional pseudo amino acid composition space, and that the cluster-tolerant capacity can be increased accordingly. As a consequence by combining these two approaches, high success rates have been observed during the tests of self-consistency, jackknife and independent data set, respectively, by using the simplest nearest neighbour classifier. The current approach represents a new strategy to deal with the problems of protein attribute prediction, and hence may become a useful vehicle in the area of bioinformatics and proteomics.

Interactions of Ras proteins with the plasma membrane and their roles in signaling.

PubMed

Eisenberg, Sharon; Henis, Yoav I

2008-01-01

The complex dynamic structure of the plasma membrane plays critical roles in cellular signaling; interactions with the membrane lipid milieu, spatial segregation within and between cellular membranes and/or targeting to specific membrane-associated scaffolds are intimately involved in many signal transduction pathways. In this review, we focus on the membrane interactions of Ras proteins. These small GTPases play central roles in the regulation of cell growth and proliferation, and their excessive activation is commonly encountered in human tumors. Ras proteins associate with the membrane continuously via C-terminal lipidation and additional interactions in both their inactive and active forms; this association, as well as the targeting of specific Ras isoforms to plasma membrane microdomains and to intracellular organelles, have recently been implicated in Ras signaling and oncogenic potential. We discuss biochemical and biophysical evidence for the roles of specific domains of Ras proteins in mediating their association with the plasma membrane, and consider the potential effects of lateral segregation and interactions with membrane-associated protein assemblies on the signaling outcomes.

Native Mass Spectrometry Characterizes the Photosynthetic Reaction Center Complex from the Purple Bacterium Rhodobacter sphaeroides

NASA Astrophysics Data System (ADS)

Zhang, Hao; Harrington, Lucas B.; Lu, Yue; Prado, Mindy; Saer, Rafael; Rempel, Don; Blankenship, Robert E.; Gross, Michael L.

2017-01-01

Native mass spectrometry (MS) is an emerging approach to study protein complexes in their near-native states and to elucidate their stoichiometry and topology. Here, we report a native MS study of the membrane-embedded reaction center (RC) protein complex from the purple photosynthetic bacterium Rhodobacter sphaeroides. The membrane-embedded RC protein complex is stabilized by detergent micelles in aqueous solution, directly introduced into a mass spectrometer by nano-electrospray (nESI), and freed of detergents and dissociated in the gas phase by collisional activation. As the collision energy is increased, the chlorophyll pigments are gradually released from the RC complex, suggesting that native MS introduces a near-native structure that continues to bind pigments. Two bacteriochlorophyll a pigments remain tightly bound to the RC protein at the highest collision energy. The order of pigment release and their resistance to release by gas-phase activation indicates the strength of pigment interaction in the RC complex. This investigation sets the stage for future native MS studies of membrane-embedded photosynthetic pigment-protein and related complexes.

A Q-switched Ho:YAG laser assisted nanosecond time-resolved T-jump transient mid-IR absorbance spectroscopy with high sensitivity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Deyong; Li, Yunliang; Li, Hao

2015-05-15

Knowledge of dynamical structure of protein is an important clue to understand its biological function in vivo. Temperature-jump (T-jump) time-resolved transient mid-IR absorbance spectroscopy is a powerful tool in elucidating the protein dynamical structures and the folding/unfolding kinetics of proteins in solution. A home-built setup of T-jump time-resolved transient mid-IR absorbance spectroscopy with high sensitivity is developed, which is composed of a Q-switched Cr, Tm, Ho:YAG laser with an output wavelength at 2.09 μm as the T-jump heating source, and a continuous working CO laser tunable from 1580 to 1980 cm{sup −1} as the IR probe. The results demonstrate thatmore » this system has a sensitivity of 1 × 10{sup −4} ΔOD for a single wavelength detection, and 2 × 10{sup −4} ΔOD for spectral detection in amide I′ region, as well as a temporal resolution of 20 ns. Moreover, the data quality coming from the CO laser is comparable to the one using the commercial quantum cascade laser.« less

Validation of Molecular Dynamics Simulations for Prediction of Three-Dimensional Structures of Small Proteins.

PubMed

Kato, Koichi; Nakayoshi, Tomoki; Fukuyoshi, Shuichi; Kurimoto, Eiji; Oda, Akifumi

2017-10-12

Although various higher-order protein structure prediction methods have been developed, almost all of them were developed based on the three-dimensional (3D) structure information of known proteins. Here we predicted the short protein structures by molecular dynamics (MD) simulations in which only Newton's equations of motion were used and 3D structural information of known proteins was not required. To evaluate the ability of MD simulationto predict protein structures, we calculated seven short test protein (10-46 residues) in the denatured state and compared their predicted and experimental structures. The predicted structure for Trp-cage (20 residues) was close to the experimental structure by 200-ns MD simulation. For proteins shorter or longer than Trp-cage, root-mean square deviation values were larger than those for Trp-cage. However, secondary structures could be reproduced by MD simulations for proteins with 10-34 residues. Simulations by replica exchange MD were performed, but the results were similar to those from normal MD simulations. These results suggest that normal MD simulations can roughly predict short protein structures and 200-ns simulations are frequently sufficient for estimating the secondary structures of protein (approximately 20 residues). Structural prediction method using only fundamental physical laws are useful for investigating non-natural proteins, such as primitive proteins and artificial proteins for peptide-based drug delivery systems.

Improved protein-protein interactions prediction via weighted sparse representation model combining continuous wavelet descriptor and PseAA composition.

PubMed

Huang, Yu-An; You, Zhu-Hong; Chen, Xing; Yan, Gui-Ying

2016-12-23

Protein-protein interactions (PPIs) are essential to most biological processes. Since bioscience has entered into the era of genome and proteome, there is a growing demand for the knowledge about PPI network. High-throughput biological technologies can be used to identify new PPIs, but they are expensive, time-consuming, and tedious. Therefore, computational methods for predicting PPIs have an important role. For the past years, an increasing number of computational methods such as protein structure-based approaches have been proposed for predicting PPIs. The major limitation in principle of these methods lies in the prior information of the protein to infer PPIs. Therefore, it is of much significance to develop computational methods which only use the information of protein amino acids sequence. Here, we report a highly efficient approach for predicting PPIs. The main improvements come from the use of a novel protein sequence representation by combining continuous wavelet descriptor and Chou's pseudo amino acid composition (PseAAC), and from adopting weighted sparse representation based classifier (WSRC). This method, cross-validated on the PPIs datasets of Saccharomyces cerevisiae, Human and H. pylori, achieves an excellent results with accuracies as high as 92.50%, 95.54% and 84.28% respectively, significantly better than previously proposed methods. Extensive experiments are performed to compare the proposed method with state-of-the-art Support Vector Machine (SVM) classifier. The outstanding results yield by our model that the proposed feature extraction method combing two kinds of descriptors have strong expression ability and are expected to provide comprehensive and effective information for machine learning-based classification models. In addition, the prediction performance in the comparison experiments shows the well cooperation between the combined feature and WSRC. Thus, the proposed method is a very efficient method to predict PPIs and may be a useful supplementary tool for future proteomics studies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stepanyuk, Galina A.; Serrano, Pedro; Peralta, Eigen

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein–protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39–U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemicalmore » shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35–U2AF65 and RBM39–SF3b155, the RBM39-UHM–U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM–ULM binding interface, providing a rationale for the known specificity of UHM–ULM interactions. This study therefore establishes a structural basis for specific UHM–ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.« less

Origin of sphinx, a young chimeric RNA gene in Drosophila melanogaster

PubMed Central

Wang, Wen; Brunet, Frédéric G.; Nevo, Eviatar; Long, Manyuan

2002-01-01

Non-protein-coding RNA genes play an important role in various biological processes. How new RNA genes originated and whether this process is controlled by similar evolutionary mechanisms for the origin of protein-coding genes remains unclear. A young chimeric RNA gene that we term sphinx (spx) provides the first insight into the early stage of evolution of RNA genes. spx originated as an insertion of a retroposed sequence of the ATP synthase chain F gene at the cytological region 60DB since the divergence of Drosophila melanogaster from its sibling species 2–3 million years ago. This retrosequence, which is located at 102F on the fourth chromosome, recruited a nearby exon and intron, thereby evolving a chimeric gene structure. This molecular process suggests that the mechanism of exon shuffling, which can generate protein-coding genes, also plays a role in the origin of RNA genes. The subsequent evolutionary process of spx has been associated with a high nucleotide substitution rate, possibly driven by a continuous positive Darwinian selection for a novel function, as is shown in its sex- and development-specific alternative splicing. To test whether spx has adapted to different environments, we investigated its population genetic structure in the unique “Evolution Canyon” in Israel, revealing a similar haplotype structure in spx, and thus similar evolutionary forces operating on spx between environments. PMID:11904380

Target specific proteochemometric model development for BACE1 - protein flexibility and structural water are critical in virtual screening.

PubMed

Manoharan, Prabu; Chennoju, Kiranmai; Ghoshal, Nanda

2015-07-01

BACE1 is an attractive target in Alzheimer's disease (AD) treatment. A rational drug design effort for the inhibition of BACE1 is actively pursued by researchers in both academic and pharmaceutical industries. This continued effort led to the steady accumulation of BACE1 crystal structures, co-complexed with different classes of inhibitors. This wealth of information is used in this study to develop target specific proteochemometric models and these models are exploited for predicting the prospective BACE1 inhibitors. The models developed in this study have performed excellently in predicting the computationally generated poses, separately obtained from single and ensemble docking approaches. The simple protein-ligand contact (SPLC) model outperforms other sophisticated high end models, in virtual screening performance, developed during this study. In an attempt to account for BACE1 protein active site flexibility information in predictive models, we included the change in the area of solvent accessible surface and the change in the volume of solvent accessible surface in our models. The ensemble and single receptor docking results obtained from this study indicate that the structural water mediated interactions improve the virtual screening results. Also, these waters are essential for recapitulating bioactive conformation during docking study. The proteochemometric models developed in this study can be used for the prediction of BACE1 inhibitors, during the early stage of AD drug discovery.

The Role of Severe Acute Respiratory Syndrome (SARS)-Coronavirus Accessory Proteins in Virus Pathogenesis

PubMed Central

McBride, Ruth; Fielding, Burtram C.

2012-01-01

A respiratory disease caused by a novel coronavirus, termed the severe acute respiratory syndrome coronavirus (SARS-CoV), was first reported in China in late 2002. The subsequent efficient human-to-human transmission of this virus eventually affected more than 30 countries worldwide, resulting in a mortality rate of ~10% of infected individuals. The spread of the virus was ultimately controlled by isolation of infected individuals and there has been no infections reported since April 2004. However, the natural reservoir of the virus was never identified and it is not known if this virus will re-emerge and, therefore, research on this virus continues. The SARS-CoV genome is about 30 kb in length and is predicted to contain 14 functional open reading frames (ORFs). The genome encodes for proteins that are homologous to known coronavirus proteins, such as the replicase proteins (ORFs 1a and 1b) and the four major structural proteins: nucleocapsid (N), spike (S), membrane (M) and envelope (E). SARS-CoV also encodes for eight unique proteins, called accessory proteins, with no known homologues. This review will summarize the current knowledge on SARS-CoV accessory proteins and will include: (i) expression and processing; (ii) the effects on cellular processes; and (iii) functional studies. PMID:23202509

An exploration of alternative visualisations of the basic helix-loop-helix protein interaction network

PubMed Central

Holden, Brian J; Pinney, John W; Lovell, Simon C; Amoutzias, Grigoris D; Robertson, David L

2007-01-01

Background Alternative representations of biochemical networks emphasise different aspects of the data and contribute to the understanding of complex biological systems. In this study we present a variety of automated methods for visualisation of a protein-protein interaction network, using the basic helix-loop-helix (bHLH) family of transcription factors as an example. Results Network representations that arrange nodes (proteins) according to either continuous or discrete information are investigated, revealing the existence of protein sub-families and the retention of interactions following gene duplication events. Methods of network visualisation in conjunction with a phylogenetic tree are presented, highlighting the evolutionary relationships between proteins, and clarifying the context of network hubs and interaction clusters. Finally, an optimisation technique is used to create a three-dimensional layout of the phylogenetic tree upon which the protein-protein interactions may be projected. Conclusion We show that by incorporating secondary genomic, functional or phylogenetic information into network visualisation, it is possible to move beyond simple layout algorithms based on network topology towards more biologically meaningful representations. These new visualisations can give structure to complex networks and will greatly help in interpreting their evolutionary origins and functional implications. Three open source software packages (InterView, TVi and OptiMage) implementing our methods are available. PMID:17683601

Advanced Computational Methods for High-accuracy Refinement of Protein Low-quality Models

NASA Astrophysics Data System (ADS)

Zang, Tianwu

Predicting the 3-dimentional structure of protein has been a major interest in the modern computational biology. While lots of successful methods can generate models with 3˜5A root-mean-square deviation (RMSD) from the solution, the progress of refining these models is quite slow. It is therefore urgently needed to develop effective methods to bring low-quality models to higher-accuracy ranges (e.g., less than 2 A RMSD). In this thesis, I present several novel computational methods to address the high-accuracy refinement problem. First, an enhanced sampling method, named parallel continuous simulated tempering (PCST), is developed to accelerate the molecular dynamics (MD) simulation. Second, two energy biasing methods, Structure-Based Model (SBM) and Ensemble-Based Model (EBM), are introduced to perform targeted sampling around important conformations. Third, a three-step method is developed to blindly select high-quality models along the MD simulation. These methods work together to make significant refinement of low-quality models without any knowledge of the solution. The effectiveness of these methods is examined in different applications. Using the PCST-SBM method, models with higher global distance test scores (GDT_TS) are generated and selected in the MD simulation of 18 targets from the refinement category of the 10th Critical Assessment of Structure Prediction (CASP10). In addition, in the refinement test of two CASP10 targets using the PCST-EBM method, it is indicated that EBM may bring the initial model to even higher-quality levels. Furthermore, a multi-round refinement protocol of PCST-SBM improves the model quality of a protein to the level that is sufficient high for the molecular replacement in X-ray crystallography. Our results justify the crucial position of enhanced sampling in the protein structure prediction and demonstrate that a considerable improvement of low-accuracy structures is still achievable with current force fields.

Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

PubMed

Brylinski, Michal

2013-11-25

A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. In this communication, we describe eSimDock, a new approach to ligand docking and binding affinity prediction. eSimDock employs nonlinear machine learning-based scoring functions to improve the accuracy of ligand ranking and similarity-based binding pose prediction, and to increase the tolerance to structural imperfections in the target structures. In large-scale benchmarking using the Astex/CCDC data set, we show that 53.9% (67.9%) of the predicted ligand poses have RMSD of <2 Å (<3 Å). Moreover, using binding sites predicted by recently developed eFindSite, eSimDock models ligand binding poses with an RMSD of 4 Å for 50.0-39.7% of the complexes at the protein homology level limited to 80-40%. Simulations against non-native receptor structures, whose mean backbone rearrangements vary from 0.5 to 5.0 Å Cα-RMSD, show that the ratio of docking accuracy and the estimated upper bound is at a constant level of ∼0.65. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. Finally, two case studies demonstrate that eSimDock can be customized to specific applications as well. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models. eSimDock is freely available to the academic community as a Web server at http://www.brylinski.org/esimdock .

DNASU plasmid and PSI:Biology-Materials repositories: resources to accelerate biological research

PubMed Central

Seiler, Catherine Y.; Park, Jin G.; Sharma, Amit; Hunter, Preston; Surapaneni, Padmini; Sedillo, Casey; Field, James; Algar, Rhys; Price, Andrea; Steel, Jason; Throop, Andrea; Fiacco, Michael; LaBaer, Joshua

2014-01-01

The mission of the DNASU Plasmid Repository is to accelerate research by providing high-quality, annotated plasmid samples and online plasmid resources to the research community through the curated DNASU database, website and repository (http://dnasu.asu.edu or http://dnasu.org). The collection includes plasmids from grant-funded, high-throughput cloning projects performed in our laboratory, plasmids from external researchers, and large collections from consortia such as the ORFeome Collaboration and the NIGMS-funded Protein Structure Initiative: Biology (PSI:Biology). Through DNASU, researchers can search for and access detailed information about each plasmid such as the full length gene insert sequence, vector information, associated publications, and links to external resources that provide additional protein annotations and experimental protocols. Plasmids can be requested directly through the DNASU website. DNASU and the PSI:Biology-Materials Repositories were previously described in the 2010 NAR Database Issue (Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G. et al. (2010) Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res., 38, D743–D749.). In this update we will describe the plasmid collection and highlight the new features in the website redesign, including new browse/search options, plasmid annotations and a dynamic vector mapping feature that was developed in collaboration with LabGenius. Overall, these plasmid resources continue to enable research with the goal of elucidating the role of proteins in both normal biological processes and disease. PMID:24225319

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA [Structural and Biophysical Characterization of Rv0577, a Protein Associated with Neutral Red Staining of Virulent Strains of Mycobacterium tuberculosis and homolog of the Streptomyces coelicolor protein KbpA

DOE PAGES

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan; ...

2017-07-10

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å,more » binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA [Structural and Biophysical Characterization of Rv0577, a Protein Associated with Neutral Red Staining of Virulent Strains of Mycobacterium tuberculosis and homolog of the Streptomyces coelicolor protein KbpA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchko, Garry W.; Echols, Nathaniel; Flynn, E. Megan

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å,more » binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a βαβββ topology arranged radially in consecutive pairs to form two continuous eight-strand β-sheets capped on both ends with an α-helix. The two β-sheets intersect in the center at roughly a right angle and form two asymmetric deep “saddles” that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state { 1H}– 15N nuclear Overhauser effect, T 1, and T 2 values were generally uniform throughout the sequence with only a few modest pockets of differences. As a result, circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.« less

Proteome-wide Structural Analysis of PTM Hotspots Reveals Regulatory Elements Predicted to Impact Biological Function and Disease.

PubMed

Torres, Matthew P; Dewhurst, Henry; Sundararaman, Niveda

2016-11-01

Post-translational modifications (PTMs) regulate protein behavior through modulation of protein-protein interactions, enzymatic activity, and protein stability essential in the translation of genotype to phenotype in eukaryotes. Currently, less than 4% of all eukaryotic PTMs are reported to have biological function - a statistic that continues to decrease with an increasing rate of PTM detection. Previously, we developed SAPH-ire (Structural Analysis of PTM Hotspots) - a method for the prioritization of PTM function potential that has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we apply SAPH-ire to the set of eukaryotic protein families containing experimental PTM and 3D structure data - capturing 1,325 protein families with 50,839 unique PTM sites organized into 31,747 modified alignment positions (MAPs), of which 2010 (∼6%) possess known biological function. Here, we show that using an artificial neural network model (SAPH-ire NN) trained to identify MAP hotspots with biological function results in prediction outcomes that far surpass the use of single hotspot features, including nearest neighbor PTM clustering methods. We find the greatest enhancement in prediction for positions with PTM counts of five or less, which represent 98% of all MAPs in the eukaryotic proteome and 90% of all MAPs found to have biological function. Analysis of the top 1092 MAP hotspots revealed 267 of truly unknown function (containing 5443 distinct PTMs). Of these, 165 hotspots could be mapped to human KEGG pathways for normal and/or disease physiology. Many high-ranking hotspots were also found to be disease-associated pathogenic sites of amino acid substitution despite the lack of observable PTM in the human protein family member. Taken together, these experiments demonstrate that the functional relevance of a PTM can be predicted very effectively by neural network models, revealing a large but testable body of potential regulatory elements that impact hundreds of different biological processes important in eukaryotic biology and human health. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Proteome-wide Structural Analysis of PTM Hotspots Reveals Regulatory Elements Predicted to Impact Biological Function and Disease*

PubMed Central

Dewhurst, Henry; Sundararaman, Niveda

2016-01-01

Post-translational modifications (PTMs) regulate protein behavior through modulation of protein-protein interactions, enzymatic activity, and protein stability essential in the translation of genotype to phenotype in eukaryotes. Currently, less than 4% of all eukaryotic PTMs are reported to have biological function - a statistic that continues to decrease with an increasing rate of PTM detection. Previously, we developed SAPH-ire (Structural Analysis of PTM Hotspots) - a method for the prioritization of PTM function potential that has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we apply SAPH-ire to the set of eukaryotic protein families containing experimental PTM and 3D structure data - capturing 1,325 protein families with 50,839 unique PTM sites organized into 31,747 modified alignment positions (MAPs), of which 2010 (∼6%) possess known biological function. Here, we show that using an artificial neural network model (SAPH-ire NN) trained to identify MAP hotspots with biological function results in prediction outcomes that far surpass the use of single hotspot features, including nearest neighbor PTM clustering methods. We find the greatest enhancement in prediction for positions with PTM counts of five or less, which represent 98% of all MAPs in the eukaryotic proteome and 90% of all MAPs found to have biological function. Analysis of the top 1092 MAP hotspots revealed 267 of truly unknown function (containing 5443 distinct PTMs). Of these, 165 hotspots could be mapped to human KEGG pathways for normal and/or disease physiology. Many high-ranking hotspots were also found to be disease-associated pathogenic sites of amino acid substitution despite the lack of observable PTM in the human protein family member. Taken together, these experiments demonstrate that the functional relevance of a PTM can be predicted very effectively by neural network models, revealing a large but testable body of potential regulatory elements that impact hundreds of different biological processes important in eukaryotic biology and human health. PMID:27697855

Template-based modeling and ab initio refinement of protein oligomer structures using GALAXY in CAPRI round 30.

PubMed

Lee, Hasup; Baek, Minkyung; Lee, Gyu Rie; Park, Sangwoo; Seok, Chaok

2017-03-01

Many proteins function as homo- or hetero-oligomers; therefore, attempts to understand and regulate protein functions require knowledge of protein oligomer structures. The number of available experimental protein structures is increasing, and oligomer structures can be predicted using the experimental structures of related proteins as templates. However, template-based models may have errors due to sequence differences between the target and template proteins, which can lead to functional differences. Such structural differences may be predicted by loop modeling of local regions or refinement of the overall structure. In CAPRI (Critical Assessment of PRotein Interactions) round 30, we used recently developed features of the GALAXY protein modeling package, including template-based structure prediction, loop modeling, model refinement, and protein-protein docking to predict protein complex structures from amino acid sequences. Out of the 25 CAPRI targets, medium and acceptable quality models were obtained for 14 and 1 target(s), respectively, for which proper oligomer or monomer templates could be detected. Symmetric interface loop modeling on oligomer model structures successfully improved model quality, while loop modeling on monomer model structures failed. Overall refinement of the predicted oligomer structures consistently improved the model quality, in particular in interface contacts. Proteins 2017; 85:399-407. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Protein flexibility in the light of structural alphabets

PubMed Central

Craveur, Pierrick; Joseph, Agnel P.; Esque, Jeremy; Narwani, Tarun J.; Noël, Floriane; Shinada, Nicolas; Goguet, Matthieu; Leonard, Sylvain; Poulain, Pierre; Bertrand, Olivier; Faure, Guilhem; Rebehmed, Joseph; Ghozlane, Amine; Swapna, Lakshmipuram S.; Bhaskara, Ramachandra M.; Barnoud, Jonathan; Téletchéa, Stéphane; Jallu, Vincent; Cerny, Jiri; Schneider, Bohdan; Etchebest, Catherine; Srinivasan, Narayanaswamy; Gelly, Jean-Christophe; de Brevern, Alexandre G.

2015-01-01

Protein structures are valuable tools to understand protein function. Nonetheless, proteins are often considered as rigid macromolecules while their structures exhibit specific flexibility, which is essential to complete their functions. Analyses of protein structures and dynamics are often performed with a simplified three-state description, i.e., the classical secondary structures. More precise and complete description of protein backbone conformation can be obtained using libraries of small protein fragments that are able to approximate every part of protein structures. These libraries, called structural alphabets (SAs), have been widely used in structure analysis field, from definition of ligand binding sites to superimposition of protein structures. SAs are also well suited to analyze the dynamics of protein structures. Here, we review innovative approaches that investigate protein flexibility based on SAs description. Coupled to various sources of experimental data (e.g., B-factor) and computational methodology (e.g., Molecular Dynamic simulation), SAs turn out to be powerful tools to analyze protein dynamics, e.g., to examine allosteric mechanisms in large set of structures in complexes, to identify order/disorder transition. SAs were also shown to be quite efficient to predict protein flexibility from amino-acid sequence. Finally, in this review, we exemplify the interest of SAs for studying flexibility with different cases of proteins implicated in pathologies and diseases. PMID:26075209

Development of Specific Inhibitors for Breast Cancer-Associated Variants of ErbB2

DTIC Science & Technology

2015-10-01

activity measurements (Months 9-15) Specific Aim 3: Identifying inhibitors of ErbB2 mutants.* Major Task 5: Produce ErbB2 structures for drug -lead...identified the activated cancer- associated ErbB2 mutants that will be used for drug screening, and we have established enzyme assays that will be suitable...during protein expression and purification. We measured enzyme activity using two assays: (1) a continuous spectrophotometric assay. In this assay

Hierarchical and non-hierarchical mineralisation of collagen

PubMed Central

Liu, Yan; Kim, Young-Kyung; Dai, Lin; Li, Nan; Khan, Sara; Pashley, David H.; Tay, Franklin R.

2010-01-01

Biomineralisation of collagen involves functional motifs incorporated in extracellular matrix protein molecules to accomplish the objectives of stabilising amorphous calcium phosphate into nanoprecursors and directing the nucleation and growth of apatite within collagen fibrils. Here we report the use of small inorganic polyphosphate molecules to template hierarchical intrafibrillar apatite assembly in reconstituted collagen in the presence of polyacrylic acid to sequester calcium and phosphate into transient amorphous nanophases. The use of polyphosphate without a sequestration analogue resulted only in randomly-oriented extrafibrillar precipitations along the fibrillar surface. Conversely, the use of polyacrylic acid without a templating analogue resulted only in non-hierarchical intrafibrillar mineralisation with continuous apatite strands instead of discrete crystallites. The ability of using simple non-protein molecules to recapitulate different levels of structural hierarchy in mineralised collagen signifies the ultimate simplicity in Nature’s biomineralisation design principles and challenges the need for using more complex recombinant matrix proteins in bioengineering applications. PMID:21040969

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states

PubMed Central

Linnemann, Amelia K.; Krawetz, Stephen A.

2010-01-01

Summary The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease. PMID:20948980

Maintenance of a functional higher order chromatin structure: The role of the nuclear matrix in normal and disease states.

PubMed

Linnemann, Amelia K; Krawetz, Stephen A

2009-01-01

The ordered packaging of DNA within the nucleus of somatic cells reflects a dynamic supportive structure that facilitates stable transcription interrupted by intermittent cycles of extreme condensation. This dynamic mode of packing and unpacking chromatin is intimately linked to the ability of the genome to specifically complex with both histones and non-histone proteins. Understanding the underlying mechanism that governs the formation of higher order chromatin structures is a key to understanding how local architecture modulates transcription. In part, the formation of these structures appears to be regulated through genomic looping that is dynamically mediated by attachment to the nuclear scaffold/matrix at S/MARs, i.e., Scaffold/Matrix Attachment Regions. Although the mechanism guiding the formation and use of these higher-ordered structures remains unknown, S/MARs continue to reveal a multitude of roles in development and the pathogenesis of disease.

Axonal Membrane Proteins Are Transported in Distinct Carriers: A Two-Color Video Microscopy Study in Cultured Hippocampal NeuronsV⃞

PubMed Central

Kaether, Christoph; Skehel, Paul; Dotti, Carlos G.

2000-01-01

Neurons transport newly synthesized membrane proteins along axons by microtubule-mediated fast axonal transport. Membrane proteins destined for different axonal subdomains are thought to be transported in different transport carriers. To analyze this differential transport in living neurons, we tagged the amyloid precursor protein (APP) and synaptophysin (p38) with green fluorescent protein (GFP) variants. The resulting fusion proteins, APP-yellow fluorescent protein (YFP), p38-enhanced GFP, and p38-enhanced cyan fluorescent protein, were expressed in hippocampal neurons, and the cells were imaged by video microscopy. APP-YFP was transported in elongated tubules that moved extremely fast (on average 4.5 μm/s) and over long distances. In contrast, p38-enhanced GFP-transporting structures were more vesicular and moved four times slower (0.9 μm/s) and over shorter distances only. Two-color video microscopy showed that the two proteins were sorted to different carriers that moved with different characteristics along axons of doubly transfected neurons. Antisense treatment using oligonucleotides against the kinesin heavy chain slowed down the long, continuous movement of APP-YFP tubules and increased frequency of directional changes. These results demonstrate for the first time directly the sorting and transport of two axonal membrane proteins into different carriers. Moreover, the extremely fast-moving tubules represent a previously unidentified type of axonal carrier. PMID:10749925

In silico work flow for scaffold hopping in Leishmania.

PubMed

Waugh, Barnali; Ghosh, Ambarnil; Bhattacharyya, Dhananjay; Ghoshal, Nanda; Banerjee, Rahul

2014-11-17

Leishmaniasis,a broad spectrum of diseases caused by several sister species of protozoa belonging to family trypanosomatidae and genus leishmania , generally affects poorer sections of the populace in third world countries. With the emergence of strains resistant to traditional therapies and the high cost of second line drugs which generally have severe side effects, it becomes imperative to continue the search for alternative drugs to combat the disease. In this work, the leishmanial genomes and the human genome have been compared to identify proteins unique to the parasite and whose structures (or those of close homologues) are available in the Protein Data Bank. Subsequent to the prioritization of these proteins (based on their essentiality, virulence factor etc.), inhibitors have been identified for a subset of these prospective drug targets by means of an exhaustive literature survey. A set of three dimensional protein-ligand complexes have been assembled from the list of leishmanial drug targets by culling structures from the Protein Data Bank or by means of template based homology modeling followed by ligand docking with the GOLD software. Based on these complexes several structure based pharmacophores have been designed and used to search for alternative inhibitors in the ZINC database. This process led to a list of prospective compounds which could serve as potential antileishmanials. These small molecules were also used to search the Drug Bank to identify prospective lead compounds already in use as approved drugs. Interestingly, paromomycin which is currently being used as an antileishmanial drug spontaneously appeared in the list, probably giving added confidence to the 'scaffold hopping' computational procedures adopted in this work. The report thus provides the basis to experimentally verify several lead compounds for their predicted antileishmanial activity and includes several useful data bases of prospective drug targets in leishmania, their inhibitors and protein--inhibitor three dimensional complexes.

A Template-Based Protein Structure Reconstruction Method Using Deep Autoencoder Learning.

PubMed

Li, Haiou; Lyu, Qiang; Cheng, Jianlin

2016-12-01

Protein structure prediction is an important problem in computational biology, and is widely applied to various biomedical problems such as protein function study, protein design, and drug design. In this work, we developed a novel deep learning approach based on a deeply stacked denoising autoencoder for protein structure reconstruction. We applied our approach to a template-based protein structure prediction using only the 3D structural coordinates of homologous template proteins as input. The templates were identified for a target protein by a PSI-BLAST search. 3DRobot (a program that automatically generates diverse and well-packed protein structure decoys) was used to generate initial decoy models for the target from the templates. A stacked denoising autoencoder was trained on the decoys to obtain a deep learning model for the target protein. The trained deep model was then used to reconstruct the final structural model for the target sequence. With target proteins that have highly similar template proteins as benchmarks, the GDT-TS score of the predicted structures is greater than 0.7, suggesting that the deep autoencoder is a promising method for protein structure reconstruction.

Structure and proposed mechanism of α-glycerophosphate oxidase from Mycoplasma pneumoniae

DOE PAGES

Elkhal, Callia K.; Kean, Kelsey M.; Parsonage, Derek; ...

2015-03-14

In this study, the formation of hydrogen peroxide (H₂O₂) by the FAD-dependent α-glycerophosphate oxidase (GlpO), is important for the pathogenesis of Streptococcus pneumoniae and Mycoplasma pneumoniae. The structurally known GlpO from Streptococcus sp. ( SspGlpO) is similar to the pneumococcal protein ( SpGlpO) and provides a guide for drug design against that target. However, M. pneumoniae GlpO ( MpGlpO), having <20% sequence identity with structurally known GlpOs, appears to represent a second type of GlpO we designate as Type II GlpOs. Here, the recombinant His-tagged MpGlpO structure is described at ~2.5 Å resolution, solved by molecular replacement using as amore » search model the Bordetella pertussis protein 3253 (Bp3253) a protein of unknown function solved by structural genomics efforts. Recombinant MpGlpO is an active oxidase with a turnover number of ~580 min⁻¹ while Bp3253 showed no GlpO activity. No substantial differences exist between the oxidized and dithionite-reduced MpGlpO structures. Although, no liganded structures were determined, a comparison with the tartrate-bound Bp3253 structure and consideration of residue conservation patterns guided the construction of a model for α-glycerophosphate (Glp) recognition and turnover by MpGlpO. The predicted binding mode also appears relevant for the type I GlpOs (such as SspGlpO) despite differences in substrate recognition residues, and it implicates a histidine conserved in type I and II Glp oxidases and dehydrogenases as the catalytic acid/base. This work provides a solid foundation for guiding further studies of the mitochondrial Glp dehydrogenases as well as for continued studies of M. pneumoniae and S. pneumoniae glycerol metabolism and the development of novel therapeutics targeting MpGlpO and SpGlpO.« less

Structure and proposed mechanism of α-glycerophosphate oxidase from Mycoplasma pneumoniae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elkhal, Callia K.; Kean, Kelsey M.; Parsonage, Derek

In this study, the formation of hydrogen peroxide (H₂O₂) by the FAD-dependent α-glycerophosphate oxidase (GlpO), is important for the pathogenesis of Streptococcus pneumoniae and Mycoplasma pneumoniae. The structurally known GlpO from Streptococcus sp. ( SspGlpO) is similar to the pneumococcal protein ( SpGlpO) and provides a guide for drug design against that target. However, M. pneumoniae GlpO ( MpGlpO), having <20% sequence identity with structurally known GlpOs, appears to represent a second type of GlpO we designate as Type II GlpOs. Here, the recombinant His-tagged MpGlpO structure is described at ~2.5 Å resolution, solved by molecular replacement using as amore » search model the Bordetella pertussis protein 3253 (Bp3253) a protein of unknown function solved by structural genomics efforts. Recombinant MpGlpO is an active oxidase with a turnover number of ~580 min⁻¹ while Bp3253 showed no GlpO activity. No substantial differences exist between the oxidized and dithionite-reduced MpGlpO structures. Although, no liganded structures were determined, a comparison with the tartrate-bound Bp3253 structure and consideration of residue conservation patterns guided the construction of a model for α-glycerophosphate (Glp) recognition and turnover by MpGlpO. The predicted binding mode also appears relevant for the type I GlpOs (such as SspGlpO) despite differences in substrate recognition residues, and it implicates a histidine conserved in type I and II Glp oxidases and dehydrogenases as the catalytic acid/base. This work provides a solid foundation for guiding further studies of the mitochondrial Glp dehydrogenases as well as for continued studies of M. pneumoniae and S. pneumoniae glycerol metabolism and the development of novel therapeutics targeting MpGlpO and SpGlpO.« less

Protein structure similarity from Principle Component Correlation analysis.

PubMed

Zhou, Xiaobo; Chou, James; Wong, Stephen T C

2006-01-25

Owing to rapid expansion of protein structure databases in recent years, methods of structure comparison are becoming increasingly effective and important in revealing novel information on functional properties of proteins and their roles in the grand scheme of evolutionary biology. Currently, the structural similarity between two proteins is measured by the root-mean-square-deviation (RMSD) in their best-superimposed atomic coordinates. RMSD is the golden rule of measuring structural similarity when the structures are nearly identical; it, however, fails to detect the higher order topological similarities in proteins evolved into different shapes. We propose new algorithms for extracting geometrical invariants of proteins that can be effectively used to identify homologous protein structures or topologies in order to quantify both close and remote structural similarities. We measure structural similarity between proteins by correlating the principle components of their secondary structure interaction matrix. In our approach, the Principle Component Correlation (PCC) analysis, a symmetric interaction matrix for a protein structure is constructed with relationship parameters between secondary elements that can take the form of distance, orientation, or other relevant structural invariants. When using a distance-based construction in the presence or absence of encoded N to C terminal sense, there are strong correlations between the principle components of interaction matrices of structurally or topologically similar proteins. The PCC method is extensively tested for protein structures that belong to the same topological class but are significantly different by RMSD measure. The PCC analysis can also differentiate proteins having similar shapes but different topological arrangements. Additionally, we demonstrate that when using two independently defined interaction matrices, comparison of their maximum eigenvalues can be highly effective in clustering structurally or topologically similar proteins. We believe that the PCC analysis of interaction matrix is highly flexible in adopting various structural parameters for protein structure comparison.

Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility

PubMed Central

Loving, Kathryn A.; Lin, Andy; Cheng, Alan C.

2014-01-01

Advances reported over the last few years and the increasing availability of protein crystal structure data have greatly improved structure-based druggability approaches. However, in practice, nearly all druggability estimation methods are applied to protein crystal structures as rigid proteins, with protein flexibility often not directly addressed. The inclusion of protein flexibility is important in correctly identifying the druggability of pockets that would be missed by methods based solely on the rigid crystal structure. These include cryptic pockets and flexible pockets often found at protein-protein interaction interfaces. Here, we apply an approach that uses protein modeling in concert with druggability estimation to account for light protein backbone movement and protein side-chain flexibility in protein binding sites. We assess the advantages and limitations of this approach on widely-used protein druggability sets. Applying the approach to all mammalian protein crystal structures in the PDB results in identification of 69 proteins with potential druggable cryptic pockets. PMID:25079060

On the state of crystallography at the dawn of the electron microscopy revolution.

PubMed

Higgins, Matthew K; Lea, Susan M

2017-10-01

While protein crystallography has, for many years, been the most used method for structural analysis of macromolecular complexes, remarkable recent advances in high-resolution electron cryo-microscopy led to suggestions that 'the revolution will not be crystallised'. Here we highlight the current success rate, speed and ease of modern crystallographic structure determination and some recent triumphs of both 'classical' crystallography and the use of X-ray free electron lasers. We also outline fundamental differences between structure determination using X-ray crystallography and electron microscopy. We suggest that crystallography will continue to co-exist with electron microscopy as part of an integrated array of methods, allowing structural biologists to focus on fundamental biological questions rather than being constrained by the methods available. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Identification of epitopes on nonstructural protein 7 of porcine reproductive and respiratory syndrome virus recognized by monoclonal antibodies using phage-display technology.

PubMed

Wang, Heng; Liu, Rongchang; Zhang, Weidong; Sun, Lingshuang; Ning, Zhangyong; Ji, Fangxiao; Cui, Jin; Zhang, Guihong

2017-08-01

Nonstructural protein 7 (nsp7) of porcine reproductive and respiratory syndrome virus (PRRSV) is considered to be a suitable reagent for the development of serological diagnostic assays. It can be expressed as a soluble recombinant protein in Escherichia coli, and its antibody response may continue up to 202 days post-infection. Furthermore, the region encoded by nsp7 is highly homologous among various strains within the genotype, and the results of nsp7-based enzyme-linked immunosorbent assay (ELISA) showed high agreement with previous Idexx ELISA results. All these evidences suggest the existence of important epitopes on nsp7, though the characteristics of these epitopes remain unclear. In the present study, we prepared three monoclonal antibodies against nsp7 protein and used them to screen the epitope-distribution characteristics of PRRSV nsp7 protein by phage-display technology. We identified a linear epitope NAWGDEDRLN at amino acids 153-162 type II PRRSV nsp7β subunit. This newly defined epitope showed excellent reactivity with PRSSV-positive serum samples. These results further our understanding of the antigenic structure of nsp7 protein, and provide efficient reagents for PRRSV serological tests.

Domain fusion analysis by applying relational algebra to protein sequence and domain databases

PubMed Central

Truong, Kevin; Ikura, Mitsuhiko

2003-01-01

Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

What are the structural features that drive partitioning of proteins in aqueous two-phase systems?

PubMed

Wu, Zhonghua; Hu, Gang; Wang, Kui; Zaslavsky, Boris Yu; Kurgan, Lukasz; Uversky, Vladimir N

2017-01-01

Protein partitioning in aqueous two-phase systems (ATPSs) represents a convenient, inexpensive, and easy to scale-up protein separation technique. Since partition behavior of a protein dramatically depends on an ATPS composition, it would be highly beneficial to have reliable means for (even qualitative) prediction of partitioning of a target protein under different conditions. Our aim was to understand which structural features of proteins contribute to partitioning of a query protein in a given ATPS. We undertook a systematic empirical analysis of relations between 57 numerical structural descriptors derived from the corresponding amino acid sequences and crystal structures of 10 well-characterized proteins and the partition behavior of these proteins in 29 different ATPSs. This analysis revealed that just a few structural characteristics of proteins can accurately determine behavior of these proteins in a given ATPS. However, partition behavior of proteins in different ATPSs relies on different structural features. In other words, we could not find a unique set of protein structural features derived from their crystal structures that could be used for the description of the protein partition behavior of all proteins in all ATPSs analyzed in this study. We likely need to gain better insight into relationships between protein-solvent interactions and protein structure peculiarities, in particular given limitations of the used here crystal structures, to be able to construct a model that accurately predicts protein partition behavior across all ATPSs. Copyright © 2016 Elsevier B.V. All rights reserved.

Investigating Molecular Structures of Bio-Fuel and Bio-Oil Seeds as Predictors To Estimate Protein Bioavailability for Ruminants by Advanced Nondestructive Vibrational Molecular Spectroscopy.

PubMed

Ban, Yajing; L Prates, Luciana; Yu, Peiqiang

2017-10-18

This study was conducted to (1) determine protein and carbohydrate molecular structure profiles and (2) quantify the relationship between structural features and protein bioavailability of newly developed carinata and canola seeds for dairy cows by using Fourier transform infrared molecular spectroscopy. Results showed similarity in protein structural makeup within the entire protein structural region between carinata and canola seeds. The highest area ratios related to structural CHO, total CHO, and cellulosic compounds were obtained for carinata seeds. Carinata and canola seeds showed similar carbohydrate and protein molecular structures by multivariate analyses. Carbohydrate molecular structure profiles were highly correlated to protein rumen degradation and intestinal digestion characteristics. In conclusion, the molecular spectroscopy can detect inherent structural characteristics in carinata and canola seeds in which carbohydrate-relative structural features are related to protein metabolism and utilization. Protein and carbohydrate spectral profiles could be used as predictors of rumen protein bioavailability in cows.

Exploring Human Diseases and Biological Mechanisms by Protein Structure Prediction and Modeling.

PubMed

Wang, Juexin; Luttrell, Joseph; Zhang, Ning; Khan, Saad; Shi, NianQing; Wang, Michael X; Kang, Jing-Qiong; Wang, Zheng; Xu, Dong

2016-01-01

Protein structure prediction and modeling provide a tool for understanding protein functions by computationally constructing protein structures from amino acid sequences and analyzing them. With help from protein prediction tools and web servers, users can obtain the three-dimensional protein structure models and gain knowledge of functions from the proteins. In this chapter, we will provide several examples of such studies. As an example, structure modeling methods were used to investigate the relation between mutation-caused misfolding of protein and human diseases including epilepsy and leukemia. Protein structure prediction and modeling were also applied in nucleotide-gated channels and their interaction interfaces to investigate their roles in brain and heart cells. In molecular mechanism studies of plants, rice salinity tolerance mechanism was studied via structure modeling on crucial proteins identified by systems biology analysis; trait-associated protein-protein interactions were modeled, which sheds some light on the roles of mutations in soybean oil/protein content. In the age of precision medicine, we believe protein structure prediction and modeling will play more and more important roles in investigating biomedical mechanism of diseases and drug design.

Self-Assembly of Large Amyloid Fibers

NASA Astrophysics Data System (ADS)

Ridgley, Devin M.

Functional amyloids found throughout nature have demonstrated that amyloid fibers are potential industrial biomaterials. This work introduces a new "template plus adder" cooperative mechanism for the spontaneous self-assembly of micrometer sized amyloid fibers. A short hydrophobic template peptide induces a conformation change within a highly alpha-helical adder protein to form beta-sheets that continue to assemble into micrometer sized amyloid fibers. This study utilizes a variety of proteins that have template or adder characteristics which suggests that this mechanism may be employed throughout nature. Depending on the amino acid composition of the proteins used the mixtures form amyloid fibers of a cylindrical ( 10 mum diameter, 2 GPa Young's modulus) or tape (5- 10 mum height, 10-20 mum width and 100-200 MPa Young's modulus) morphology. Processing conditions are altered to manipulate the morphology and structural characteristics of the fibers. Spectroscopy is utilized to identify certain amino acid groups that contribute to the self-assembly process. Aliphatic amino acids (A, I, V and L) are responsible for initiating conformation change of the adder proteins to assemble into amyloid tapes. Additional polyglutamine segments (Q-blocks) within the protein mixtures will form Q hydrogen bonds to reinforce the amyloid structure and form a cylindrical fiber of higher modulus. Atomic force microscopy is utilized to delineate the self-assembly of amyloid tapes and cylindrical fibers from protofibrils (15-30 nm width) to fibers (10-20 mum width) spanning three orders of magnitude. The aliphatic amino acid content of the adder proteins' alpha-helices is a good predictor of high density beta-sheet formation within the protein mixture. Thus, it is possible to predict the propensity of a protein to undergo conformation change into amyloid structures. Finally, Escherichia coli is genetically engineered to express a template protein which self-assembles into large amyloid fibers when combined with extracellular myoglobin, an adder protein. The goal of this thesis is to produce, manipulate and characterize the self-assembly of large amyloid fibers for their potential industrial biomaterial applications. The techniques used throughout this study outline various methods to design and engineer amyloid fibers of a tailored modulus and morphology. Furthermore, the mechanisms described here may offer some insight into naturally occurring amyloid forming systems.

Knowledge-based computational intelligence development for predicting protein secondary structures from sequences.

PubMed

Shen, Hong-Bin; Yi, Dong-Liang; Yao, Li-Xiu; Yang, Jie; Chou, Kuo-Chen

2008-10-01

In the postgenomic age, with the avalanche of protein sequences generated and relatively slow progress in determining their structures by experiments, it is important to develop automated methods to predict the structure of a protein from its sequence. The membrane proteins are a special group in the protein family that accounts for approximately 30% of all proteins; however, solved membrane protein structures only represent less than 1% of known protein structures to date. Although a great success has been achieved for developing computational intelligence techniques to predict secondary structures in both globular and membrane proteins, there is still much challenging work in this regard. In this review article, we firstly summarize the recent progress of automation methodology development in predicting protein secondary structures, especially in membrane proteins; we will then give some future directions in this research field.

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

PubMed Central

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

2013-01-01

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

The Prediction of Botulinum Toxin Structure Based on in Silico and in Vitro Analysis

NASA Astrophysics Data System (ADS)

Suzuki, Tomonori; Miyazaki, Satoru

2011-01-01

Many of biological system mediated through protein-protein interactions. Knowledge of protein-protein complex structure is required for understanding the function. The determination of huge size and flexible protein-protein complex structure by experimental studies remains difficult, costly and five-consuming, therefore computational prediction of protein structures by homolog modeling and docking studies is valuable method. In addition, MD simulation is also one of the most powerful methods allowing to see the real dynamics of proteins. Here, we predict protein-protein complex structure of botulinum toxin to analyze its property. These bioinformatics methods are useful to report the relation between the flexibility of backbone structure and the activity.

Site-directed mutagenesis of the hinge peptide from the hemagglutinin protein: enhancement of the pH-responsive conformational change.

PubMed

Casali, Monica; Banta, Scott; Zambonelli, Carlo; Megeed, Zaki; Yarmush, Martin L

2008-06-01

Environmentally responsive proteins and peptides are increasingly finding utility in various engineered systems due to their ability to respond to the presentation of external stimuli. A classic example of this behavior is the influenza hemagglutinin (HA) fusion protein. At neutral pH, HA exists in a non-fusogenic state, but upon exposure to low pH, the conformation of the structure changes to expose a fusogenic peptide. During this structural change, massive rearrangements occur in a subunit of HA (HA2). Crystallography data has shown that a loop of 28 amino acids (residues 54-81) undergoes a dramatic transition from a random coil to an alpha-helix. This segment connects to two flanking helical regions (short and long) to form a long, continuous helix. Here, we report the results of site-directed mutagenesis study on LOOP-36 to further understand the mechanism of this important stimulus-responsive peptide. The conformational transition of a bacterially expressed LOOP-36 was found to be less dramatic than has been previously reported. The systematic mutation of glutamate and histidine residues in the peptide to glutamines (glutamine scanning) did not impact the conformational behavior of the peptide, but the substitution of the glycine residue at position 22 with alanine resulted in significant pH-responsive behavior. Therefore this mutant stimulus-responsive peptide may be more valuable for future protein engineering and bionanotechnology efforts.

Reciprocating free-flow isoelectric focusing device for preparative separation of proteins.

PubMed

Kong, Fan-Zhi; Yang, Ying; Wang, Yi; Li, Guo-Qing; Li, Shan; Xiao, Hua; Fan, Liu-Yin; Liu, Shao-Rong; Cao, Cheng-Xi

2015-11-27

The traditional recycling free-flow isoelectric focusing (RFFIEF) suffered from complex structure, tedious operations and poor extensibility as well as high cost. To address these issues, a novel reciprocating free-flow isoelectric focusing device (ReFFIEF) was developed for proteins or peptides pre-fractionation. In the new device, a reciprocating background flow was for the first time introduced into free flow electrophoresis (FFE) system. The gas cushion injector (GCI) used in the previous continuous free-flow electrophoresis (CFFE) was redesigned for the reciprocating background flow. With the GCI, the reciprocating background flow could be achieved between the GCI, separation chamber and transient self-balance collector (tSBC). In a run, process fluid flowed to and from, forming a stable reciprocating fluid flow in the separation chamber. A pH gradient was created within the separation chamber, and at the same time proteins were focused repeatedly when passing through the chamber under perpendicular electric field. The ReFFIEF procedure was optimized for fractionations of three model proteins, and the optimized method was further used for pre-fractionation of model human serum samples. As compared with the traditional RFFIEF devices developed about 25 years ago, the new ReFFIEF system showed several merits, such as simple design and structure, user-friendly operation and easy to extend as well as low cost. Copyright © 2015 Elsevier B.V. All rights reserved.

The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis.

PubMed

Music, Nedzad; Gagnon, Carl A

2010-12-01

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide. The etiological agent, PRRS virus (PRRSV), possesses a RNA viral genome with nine open reading frames (ORFs). The ORF1a and ORF1b replicase-associated genes encode the polyproteins pp1a and pp1ab, respectively. The pp1a is processed in nine non-structural proteins (nsps): nsp1α, nsp1β, and nsp2 to nsp8. Proteolytic cleavage of pp1ab generates products nsp9 to nsp12. The proteolytic pp1a cleavage products process and cleave pp1a and pp1ab into nsp products. The nsp9 to nsp12 are involved in virus genome transcription and replication. The 3' end of the viral genome encodes four minor and three major structural proteins. The GP(2a), GP₃ and GP₄ (encoded by ORF2a, 3 and 4), are glycosylated membrane associated minor structural proteins. The fourth minor structural protein, the E protein (encoded by ORF2b), is an unglycosylated membrane associated protein. The viral envelope contains two major structural proteins: a glycosylated major envelope protein GP₅ (encoded by ORF5) and an unglycosylated membrane M protein (encoded by ORF6). The third major structural protein is the nucleocapsid N protein (encoded by ORF7). All PRRSV non-structural and structural proteins are essential for virus replication, and PRRSV infectivity is relatively intolerant to subtle changes within the structural proteins. PRRSV virulence is multigenic and resides in both the non-structural and structural viral proteins. This review discusses the molecular characteristics, biological and immunological functions of the PRRSV structural and nsps and their involvement in the virus pathogenesis.

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.

An approach to functionally relevant clustering of the protein universe: Active site profile‐based clustering of protein structures and sequences

PubMed Central

Knutson, Stacy T.; Westwood, Brian M.; Leuthaeuser, Janelle B.; Turner, Brandon E.; Nguyendac, Don; Shea, Gabrielle; Kumar, Kiran; Hayden, Julia D.; Harper, Angela F.; Brown, Shoshana D.; Morris, John H.; Ferrin, Thomas E.; Babbitt, Patricia C.

2017-01-01

Abstract Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification—amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two‐Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure‐Function Linkage Database, SFLD) self‐identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self‐identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well‐curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP‐identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F‐measure and performance analysis on the enolase search results and comparison to GEMMA and SCI‐PHY demonstrate that TuLIP avoids the over‐division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results. PMID:28054422

New paradigm in ankyrin repeats: Beyond protein-protein interaction module.

PubMed

Islam, Zeyaul; Nagampalli, Raghavendra Sashi Krishna; Fatima, Munazza Tamkeen; Ashraf, Ghulam Md

2018-04-01

Classically, ankyrin repeat (ANK) proteins are built from tandems of two or more repeats and form curved solenoid structures that are associated with protein-protein interactions. These are short, widespread structural motif of around 33 amino acids repeats in tandem, having a canonical helix-loop-helix fold, found individually or in combination with other domains. The multiplicity of structural pattern enables it to form assemblies of diverse sizes, required for their abilities to confer multiple binding and structural roles of proteins. Three-dimensional structures of these repeats determined to date reveal a degree of structural variability that translates into the considerable functional versatility of this protein superfamily. Recent work on the ANK has proposed novel structural information, especially protein-lipid, protein-sugar and protein-protein interaction. Self-assembly of these repeats was also shown to prevent the associated protein in forming filaments. In this review, we summarize the latest findings and how the new structural information has increased our understanding of the structural determinants of ANK proteins. We discussed latest findings on how these proteins participate in various interactions to diversify the ANK roles in numerous biological processes, and explored the emerging and evolving field of designer ankyrins and its framework for protein engineering emphasizing on biotechnological applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Classification of proteins: available structural space for molecular modeling.

PubMed

Andreeva, Antonina

2012-01-01

The wealth of available protein structural data provides unprecedented opportunity to study and better understand the underlying principles of protein folding and protein structure evolution. A key to achieving this lies in the ability to analyse these data and to organize them in a coherent classification scheme. Over the past years several protein classifications have been developed that aim to group proteins based on their structural relationships. Some of these classification schemes explore the concept of structural neighbourhood (structural continuum), whereas other utilize the notion of protein evolution and thus provide a discrete rather than continuum view of protein structure space. This chapter presents a strategy for classification of proteins with known three-dimensional structure. Steps in the classification process along with basic definitions are introduced. Examples illustrating some fundamental concepts of protein folding and evolution with a special focus on the exceptions to them are presented.

DOE Office of Scientific and Technical Information (OSTI.GOV)

DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar

Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulatemore » the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 {angstrom} resolution and two structures of HP HA at 2.95 and 3.10 {angstrom} resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.« less

Upon Infection the Cellular WD Repeat-containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication.

PubMed

Ma, Dzwokai; George, Cyril X; Nomburg, Jason; Pfaller, Christian K; Cattaneo, Roberto; Samuel, Charles E

2017-12-13

Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5 deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus. Copyright © 2017 American Society for Microbiology.

Structural alterations in rat liver proteins due to streptozotocin-induced diabetes and the recovery effect of selenium: Fourier transform infrared microspectroscopy and neural network study

NASA Astrophysics Data System (ADS)

Bozkurt, Ozlem; Haman Bayari, Sevgi; Severcan, Mete; Krafft, Christoph; Popp, Jürgen; Severcan, Feride

2012-07-01

The relation between protein structural alterations and tissue dysfunction is a major concern as protein fibrillation and/or aggregation due to structural alterations has been reported in many disease states. In the current study, Fourier transform infrared microspectroscopic imaging has been used to investigate diabetes-induced changes on protein secondary structure and macromolecular content in streptozotocin-induced diabetic rat liver. Protein secondary structural alterations were predicted using neural network approach utilizing the amide I region. Moreover, the role of selenium in the recovery of diabetes-induced alterations on macromolecular content and protein secondary structure was also studied. The results revealed that diabetes induced a decrease in lipid to protein and glycogen to protein ratios in diabetic livers. Significant alterations in protein secondary structure were observed with a decrease in α-helical and an increase in β-sheet content. Both doses of selenium restored diabetes-induced changes in lipid to protein and glycogen to protein ratios. However, low-dose selenium supplementation was not sufficient to recover the effects of diabetes on protein secondary structure, while a higher dose of selenium fully restored diabetes-induced alterations in protein structure.

Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: implications for packaged DNA structure.

PubMed

Mullaney, J M; Black, L W

1998-11-13

The phage-derived expression, packaging, and processing (PEPP) system was used to target foreign proteins into the bacteriophage capsid to probe the intracapsid environment and the structure of packaged DNA. Small proteins with minimal requirements for activity were selected, staphylococcal nuclease (SN) and green fluorescent protein (GFP). These proteins were targeted into the T4 head by means of IPIII (internal protein III) fusions or CTS (capsid targeting sequence) fusions. Additional evidence is provided that foreign proteins are targeted into T4 by the N-terminal ten amino acid residue consensus CTS of IPIII identified in previous work. Fusion proteins were produced within host bacteria by expression from plasmids or by produc tion from recombinant phage carrying the fusion genes. Packaged fusion proteins CTS IPIII SN, CTS IPIII TSN, CTS IPIII GFP, CTS IPIII TGFP, and CTS GFP, where [symbol: see text] indicates a linkage peptide sequence Leu(Ile)-N-Glu cleaved by the T4 head morphogenetic proteinase gp21 during head maturation, are observed to exhibit intracapsid activity. SN activity within the head is demonstrated by loss of phage viability and by digested genomic DNA patterns visualized by gel electrophoresis when viable phage are incubated in Ca2+. Green fluorescent phage result immediately after packaging GFP produced at 30 degreesC and below, and continue to give green fluorescence under 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are produced in bacteria at 37 degreesC, and phage packaged with these proteins achieve a fluorescent state after incubation for several months at 4 degreesC. GFP-packaged phage and proheads analyzed by fluorescence spectroscopy show that the mature head and the DNA-empty prohead package identical numbers of GFP-fusion proteins. Encapsidated GFP and SN can be injected into bacteria and rapidly exhibit intracellular activity. In vivo SN digestion of encapsidated DNA gives an intriguing pattern of DNA fragments by gel analysis, predominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome digestion ladder, This quasi-limit DNA digestion pattern, reached >100-fold more slowly than the loss of titer, is invariant over a range

Leucine pulses enhance skeletal muscle protein synthesis during continuous feeding in neonatal pigs

USDA-ARS?s Scientific Manuscript database

Infants unable to maintain oral feeding can be nourished by orogastric tube. We have shown that orogastric continuous feeding restricts muscle protein synthesis compared with intermittent bolus feeding in neonatal pigs. To determine whether leucine leu infusion can be used to enhance protein synthes...

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Jing; Zhou, Mowei; Gilbert, Joshua D.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. Here in this study, an SID device was designed and successfully installed in amore » hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. Lastly, SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.« less

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Jing; Zhou, Mowei; Gilbert, Joshua D.

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on non-covalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybridmore » FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 kDa to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.« less

Surface-Induced Dissociation of Protein Complexes in a Hybrid Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

DOE PAGES

Yan, Jing; Zhou, Mowei; Gilbert, Joshua D.; ...

2016-12-02

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. Here in this study, an SID device was designed and successfully installed in amore » hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. Lastly, SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR.« less

The Primary Mechanism of Cellular Internalization for a Short Cell- Penetrating Peptide as a Nano-Scale Delivery System.

PubMed

Liu, Betty R; Huang, Yue-Wern; Korivi, Mallikarjuna; Lo, Shih-Yen; Aronstam, Robert S; Lee, Han-Jung

2017-01-01

Development of effective drug delivery systems (DDS) is a critical issue in health care and medicine. Advances in molecular biology and nanotechnology have allowed the introduction of nanomaterial-based drug delivery systems. Cell-penetrating peptides (CPPs) can form the basis of drug delivery systems by virtue of their ability to support the transport of cargoes into the cell. Potential cargoes include proteins, DNA, RNA, liposomes, and nanomaterials. These cargoes generally retain their bioactivities upon entering cells. In the present study, the smallest, fully-active lactoferricin-derived CPP, L5a is used to demonstrate the primary contributor of cellular internalization. The secondary helical structure of L5a encompasses symmetrical positive charges around the periphery. The contributions of cell-specificity, peptide length, concentration, zeta potential, particle size, and spatial structure of the peptides were examined, but only zeta potential and spatial structure affected protein transduction efficiency. FITC-labeled L5a appeared to enter cells via direct membrane translocation insofar as endocytic modulators did not block FITC-L5a entry. This is the same mechanism of protein transduction active in Cy5 labeled DNA delivery mediated by FITC-L5a. A significant reduction of transduction efficiency was observed with structurally incomplete FITC-L5a formed by tryptic destruction, in which case the mechanism of internalization switched to a classical energydependent endocytosis pathway. These results support the continued development of the non-cytotoxic L5a as an efficient tool for drug delivery. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Theoretical models for electrochemical impedance spectroscopy and local ζ-potential of unfolded proteins in nanopores

NASA Astrophysics Data System (ADS)

Vitarelli, Michael J.; Talaga, David S.

2013-09-01

Single solid-state nanopores find increasing use for electrical detection and/or manipulation of macromolecules. These applications exploit the changes in signals due to the geometry and electrical properties of the molecular species found within the nanopore. The sensitivity and resolution of such measurements are also influenced by the geometric and electrical properties of the nanopore. This paper continues the development of an analytical theory to predict the electrochemical impedance spectra of nanopores by including the influence of the presence of an unfolded protein using the variable topology finite Warburg impedance model previously published by the authors. The local excluded volume of, and charges present on, the segment of protein sampled by the nanopore are shown to influence the shape and peak frequency of the electrochemical impedance spectrum. An analytical theory is used to relate the capacitive response of the electrical double layer at the surface of the protein to both the charge density at the protein surface and the more commonly measured zeta potential. Illustrative examples show how the theory predicts that the varying sequential regions of surface charge density and excluded volume dictated by the protein primary structure may allow for an impedance-based approach to identifying unfolded proteins.

Control of Protein Crystal Nucleation and Growth Using Stirring Solution

NASA Astrophysics Data System (ADS)

Niino, Ai; Adachi, Hiroaki; Takano, Kazufumi; Matsumura, Hiroyoshi; Kinoshita, Takayoshi; Warizaya, Masaichi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo

2004-11-01

We have previously developed a protein crystallization technique using a stirring protein solution and revealed that (i) continuous stirring prevents excess spontaneous nucleation and accelerates the growth of protein crystals and (ii) prestirring (solution stirring in advance) promotes the crystal nucleation of hen egg-white lysozyme. In bovine adenosine deaminase (ADA) crystallization, continuous stirring improves the crystal quality but elongates the nucleation time. In this paper, in order to control both the crystal nucleation and growth of ADA using a Micro-Stirring technique, we carried out five different stirring patterns such as (i) no stirring, (ii) continuous stirring, (iii) prestirring, (iv) poststirring (stirring late in the growth period) and (v) restirring (combined pre- and poststirring). The results showed that high-quality well-shaped crystals were obtained under the continuous stirring and restirring conditions and the nucleation time under the prestirring and restirring conditions was shorter than that under the continuous stirring and poststirring conditions. Consequently, high-quality crystals were promptly obtained under the restirring condition. These results suggest that we are able to control both the nucleation and growth of protein crystals with the stirring techniques.

Multiscale design and synthesis of biomimetic gradient protein/biosilica composites for interfacial tissue engineering.

PubMed

Guo, Jin; Li, Chunmei; Ling, Shengjie; Huang, Wenwen; Chen, Ying; Kaplan, David L

2017-11-01

Continuous gradients present at tissue interfaces such as osteochondral systems, reflect complex tissue functions and involve changes in extracellular matrix compositions, cell types and mechanical properties. New and versatile biomaterial strategies are needed to create suitable biomimetic engineered grafts for interfacial tissue engineering. Silk protein-based composites, coupled with selective peptides with mineralization domains, were utilized to mimic the soft-to-hard transition in osteochondral interfaces. The gradient composites supported tunable mineralization and mechanical properties corresponding to the spatial concentration gradient of the mineralization domains (R5 peptide). The composite system exhibited continuous transitions in terms of composition, structure and mechanical properties, as well as cytocompatibility and biodegradability. The gradient silicified silk/R5 composites promoted and regulated osteogenic differentiation of human mesenchymal stem cells in an osteoinductive environment in vitro. The cells differentiated along the composites in a manner consistent with the R5-gradient profile. This novel biomimetic gradient biomaterial design offers a useful approach to meet a broad range of needs in regenerative medicine. Copyright © 2017 Elsevier Ltd. All rights reserved.

Xenon and Other Volatile Anesthetics Change Domain Structure in Model Lipid Raft Membranes

PubMed Central

Weinrich, Michael; Worcester, David L.

2014-01-01

Inhalation anesthetics have been in clinical use for over 160 years, but the molecular mechanisms of action continue to be investigated. Direct interactions with ion channels received much attention after it was found that anesthetics do not change the structure of homogeneous model membranes. However, it was recently found that halothane, a prototypical anesthetic, changes domain structure of a binary lipid membrane. The noble gas xenon is an excellent anesthetic and provides a pivotal test of the generality of this finding, extended to ternary lipid raft mixtures. We report that xenon and conventional anesthetics change the domain equilibrium in two canonical ternary lipid raft mixtures. These findings demonstrate a membrane-mediated mechanism whereby inhalation anesthetics can affect the lipid environment of trans-membrane proteins. PMID:24299622

Gaia: automated quality assessment of protein structure models.

PubMed

Kota, Pradeep; Ding, Feng; Ramachandran, Srinivas; Dokholyan, Nikolay V

2011-08-15

Increasing use of structural modeling for understanding structure-function relationships in proteins has led to the need to ensure that the protein models being used are of acceptable quality. Quality of a given protein structure can be assessed by comparing various intrinsic structural properties of the protein to those observed in high-resolution protein structures. In this study, we present tools to compare a given structure to high-resolution crystal structures. We assess packing by calculating the total void volume, the percentage of unsatisfied hydrogen bonds, the number of steric clashes and the scaling of the accessible surface area. We assess covalent geometry by determining bond lengths, angles, dihedrals and rotamers. The statistical parameters for the above measures, obtained from high-resolution crystal structures enable us to provide a quality-score that points to specific areas where a given protein structural model needs improvement. We provide these tools that appraise protein structures in the form of a web server Gaia (http://chiron.dokhlab.org). Gaia evaluates the packing and covalent geometry of a given protein structure and provides quantitative comparison of the given structure to high-resolution crystal structures. dokh@unc.edu Supplementary data are available at Bioinformatics online.

Protein Structure Prediction by Protein Threading

NASA Astrophysics Data System (ADS)

Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

3D-SURFER 2.0: web platform for real-time search and characterization of protein surfaces.

PubMed

Xiong, Yi; Esquivel-Rodriguez, Juan; Sael, Lee; Kihara, Daisuke

2014-01-01

The increasing number of uncharacterized protein structures necessitates the development of computational approaches for function annotation using the protein tertiary structures. Protein structure database search is the basis of any structure-based functional elucidation of proteins. 3D-SURFER is a web platform for real-time protein surface comparison of a given protein structure against the entire PDB using 3D Zernike descriptors. It can smoothly navigate the protein structure space in real-time from one query structure to another. A major new feature of Release 2.0 is the ability to compare the protein surface of a single chain, a single domain, or a single complex against databases of protein chains, domains, complexes, or a combination of all three in the latest PDB. Additionally, two types of protein structures can now be compared: all-atom-surface and backbone-atom-surface. The server can also accept a batch job for a large number of database searches. Pockets in protein surfaces can be identified by VisGrid and LIGSITE (csc) . The server is available at http://kiharalab.org/3d-surfer/.

Protein Structure Determination using Metagenome sequence data

PubMed Central

Ovchinnikov, Sergey; Park, Hahnbeom; Varghese, Neha; Huang, Po-Ssu; Pavlopoulos, Georgios A.; Kim, David E.; Kamisetty, Hetunandan; Kyrpides, Nikos C.; Baker, David

2017-01-01

Despite decades of work by structural biologists, there are still ~5200 protein families with unknown structure outside the range of comparative modeling. We show that Rosetta structure prediction guided by residue-residue contacts inferred from evolutionary information can accurately model proteins that belong to large families, and that metagenome sequence data more than triples the number of protein families with sufficient sequences for accurate modeling. We then integrate metagenome data, contact based structure matching and Rosetta structure calculations to generate models for 614 protein families with currently unknown structures; 206 are membrane proteins and 137 have folds not represented in the PDB. This approach provides the representative models for large protein families originally envisioned as the goal of the protein structure initiative at a fraction of the cost. PMID:28104891

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 representations to extract information about protein structures, as well as organizing and mining protein structure space with mature text mining tools. PMID:25080993

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. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution.

PubMed

Schlinkmann, Karola M; Hillenbrand, Matthias; Rittner, Alexander; Künz, Madeleine; Strohner, Ralf; Plückthun, Andreas

2012-09-21

To identify structural features in a G-protein-coupled receptor (GPCR) crucial for biosynthesis, stability in the membrane and stability in detergent micelles, we developed an evolutionary approach using expression in the inner membrane of Escherichia coli. From the analysis of 800,000 sequences of the rat neurotensin receptor 1, in which every amino acid had been varied to all 64 codons, we uncovered several "shift" positions, where the selected population focuses on a residue different from wild type. Here, we employed in vitro DNA recombination and a comprehensive synthetic binary library made by the Slonomics® technology, allowing us to uncover additive and synergistic effects in the structure that maximize both detergent stability and functional expression. We identified variants with >25,000 functional molecules per E. coli cell, a 50-fold increase over wild type, and observed strong coevolution of detergent stability. We arrived at receptor variants highly stable in short-chain detergents, much more so than those found by alanine scanning on the same receptor. These evolved GPCRs continue to be able to signal through the G-protein. We discuss the structural reasons for these improvements achieved through directed evolution. Copyright © 2012 Elsevier Ltd. All rights reserved.

Towards enamel biomimetics: Structure, mechanical properties and biomineralization of dental enamel

NASA Astrophysics Data System (ADS)

Fong, Hanson Kwok

Dental enamel is the most mineralized tissue in the human body. This bioceramic, composed largely of hydroxyapatite (HAp), is also one of the most durable tissues despite a lifetime of masticatory loading and bacterial attack. The biosynthesis of enamel, which occurs in physiological conditions is a complex orchestration of protein assembly and mineral formation. The resulting product is the hardest tissue in the vertebrate body with the longest and most organized arrangement of hydroxyapatite crystals known to biomineralizing systems. Detail understanding of the structure of enamel in relationship to its mechanical function and the biomineralization process will provide a framework for enamel regeneration as well as potential lessons in the design of engineering materials. The objective of this study, therefore, is twofold: (1) establish the structure-function relationship of enamel as well as the dentine-enamel junction (DEJ) and (2) determine the effect of proteins on the enamel biomineralization process. A hierarchy in the enamel structure was established by means of various microscopy techniques (e.g. SEM, TEM, AFM). Mechanical properties (hardness and elastic modulus) associated with the microstructural features were also determined by nanoindentation. Furthermore, the DEJ was found to have a width in the range of micrometers to 10s of micrometers with continuous change in structure and mechanical properties. Indentation tests and contact fatigue tests using a spherical indenter have revealed that the structural features in the enamel and the DEJ played important roles in containing crack propagation emanating from the enamel tissue. To further understand the effect of this protein on the biominerailzation process, we have studied genetically engineered animals that express altered amelogenin which lack the known self-assembly properties. This in vivo study has revealed that, without the proper self-assembly of the amelogenin protein as demonstrated by the altered amelogenin, the crystal organization of the apatite phase was severely disrupted at the nucleation stage resulting in lower mineral density at the mature stage. Consequently measurably inferior mechanical properties were found in the mature enamel grown with altered amelogenin when compared to the age matched wild-type.

SDSL-ESR-based protein structure characterization.

PubMed

Strancar, Janez; Kavalenka, Aleh; Urbancic, Iztok; Ljubetic, Ajasja; Hemminga, Marcus A

2010-03-01

As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.

Application of stochastic automata networks for creation of continuous time Markov chain models of voltage gating of gap junction channels.

PubMed

Snipas, Mindaugas; Pranevicius, Henrikas; Pranevicius, Mindaugas; Pranevicius, Osvaldas; Paulauskas, Nerijus; Bukauskas, Feliksas F

2015-01-01

The primary goal of this work was to study advantages of numerical methods used for the creation of continuous time Markov chain models (CTMC) of voltage gating of gap junction (GJ) channels composed of connexin protein. This task was accomplished by describing gating of GJs using the formalism of the stochastic automata networks (SANs), which allowed for very efficient building and storing of infinitesimal generator of the CTMC that allowed to produce matrices of the models containing a distinct block structure. All of that allowed us to develop efficient numerical methods for a steady-state solution of CTMC models. This allowed us to accelerate CPU time, which is necessary to solve CTMC models, ~20 times.

Dissecting the relationship between protein structure and sequence variation

NASA Astrophysics Data System (ADS)

Shahmoradi, Amir; Wilke, Claus; Wilke Lab Team

2015-03-01

Over the past decade several independent works have shown that some structural properties of proteins are capable of predicting protein evolution. The strength and significance of these structure-sequence relations, however, appear to vary widely among different proteins, with absolute correlation strengths ranging from 0 . 1 to 0 . 8 . Here we present the results from a comprehensive search for the potential biophysical and structural determinants of protein evolution by studying more than 200 structural and evolutionary properties in a dataset of 209 monomeric enzymes. We discuss the main protein characteristics responsible for the general patterns of protein evolution, and identify sequence divergence as the main determinant of the strengths of virtually all structure-evolution relationships, explaining ~ 10 - 30 % of observed variation in sequence-structure relations. In addition to sequence divergence, we identify several protein structural properties that are moderately but significantly coupled with the strength of sequence-structure relations. In particular, proteins with more homogeneous back-bone hydrogen bond energies, large fractions of helical secondary structures and low fraction of beta sheets tend to have the strongest sequence-structure relation. BEACON-NSF center for the study of evolution in action.

Protein enriched pasta: structure and digestibility of its protein network.

PubMed

Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

2016-02-01

Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher β-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest.

CMsearch: simultaneous exploration of protein sequence space and structure space improves not only protein homology detection but also protein structure prediction.

PubMed

Cui, Xuefeng; Lu, Zhiwu; Wang, Sheng; Jing-Yan Wang, Jim; Gao, Xin

2016-06-15

Protein homology detection, a fundamental problem in computational biology, is an indispensable step toward predicting protein structures and understanding protein functions. Despite the advances in recent decades on sequence alignment, threading and alignment-free methods, protein homology detection remains a challenging open problem. Recently, network methods that try to find transitive paths in the protein structure space demonstrate the importance of incorporating network information of the structure space. Yet, current methods merge the sequence space and the structure space into a single space, and thus introduce inconsistency in combining different sources of information. We present a novel network-based protein homology detection method, CMsearch, based on cross-modal learning. Instead of exploring a single network built from the mixture of sequence and structure space information, CMsearch builds two separate networks to represent the sequence space and the structure space. It then learns sequence-structure correlation by simultaneously taking sequence information, structure information, sequence space information and structure space information into consideration. We tested CMsearch on two challenging tasks, protein homology detection and protein structure prediction, by querying all 8332 PDB40 proteins. Our results demonstrate that CMsearch is insensitive to the similarity metrics used to define the sequence and the structure spaces. By using HMM-HMM alignment as the sequence similarity metric, CMsearch clearly outperforms state-of-the-art homology detection methods and the CASP-winning template-based protein structure prediction methods. Our program is freely available for download from http://sfb.kaust.edu.sa/Pages/Software.aspx : xin.gao@kaust.edu.sa Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Identification of Conserved Water Sites in Protein Structures for Drug Design.

PubMed

Jukič, Marko; Konc, Janez; Gobec, Stanislav; Janežič, Dušanka

2017-12-26

Identification of conserved waters in protein structures is a challenging task with applications in molecular docking and protein stability prediction. As an alternative to computationally demanding simulations of proteins in water, experimental cocrystallized waters in the Protein Data Bank (PDB) in combination with a local structure alignment algorithm can be used for reliable prediction of conserved water sites. We developed the ProBiS H2O approach based on the previously developed ProBiS algorithm, which enables identification of conserved water sites in proteins using experimental protein structures from the PDB or a set of custom protein structures available to the user. With a protein structure, a binding site, or an individual water molecule as a query, ProBiS H2O collects similar proteins from the PDB and performs local or binding site-specific superimpositions of the query structure with similar proteins using the ProBiS algorithm. It collects the experimental water molecules from the similar proteins and transposes them to the query protein. Transposed waters are clustered by their mutual proximity, which enables identification of discrete sites in the query protein with high water conservation. ProBiS H2O is a robust and fast new approach that uses existing experimental structural data to identify conserved water sites on the interfaces of protein complexes, for example protein-small molecule interfaces, and elsewhere on the protein structures. It has been successfully validated in several reported proteins in which conserved water molecules were found to play an important role in ligand binding with applications in drug design.

Free-falling Crystals: Biological Macromolecular Crystal Growth Studies in Low Earth Orbit

NASA Technical Reports Server (NTRS)

Judge, Russell A.; Snell, E. H.; Pusey, M. L.; Whitaker, Ann F. (Technical Monitor)

2001-01-01

Spacecraft orbiting the earth experience a reduced acceleration environment due to being in a state of continuous free-fall. This state colloquially termed microgravity, has produced improved X-ray diffraction quality crystals of biological macromolecules. Improvements in X-ray diffraction resolution (detail) or signal to noise, provide greater detail in the three-dimensional molecular structure providing information about the molecule, how it works, how to improve its function or how to impede it. Greater molecular detail obtained by crystallization in microgravity, has important implications for structural biology. In this article we examine the theories behind macromolecule crystal quality improvement in microgravity using results obtained from studies with the model protein, chicken egg white lysozyme.

A new approach to the deposition of nanostructured biocatalytic films

NASA Astrophysics Data System (ADS)

Troitsky, V. I.; Berzina, T. S.; Pastorino, L.; Bernasconi, E.; Nicolini, C.

2003-06-01

In the present work, monolayer engineering was used to fabricate biocatalytic nanostructured thin films based on the enzyme penicillin G acylase. The biocatalytic films with enhanced characteristics were produced by the deposition of alternate-layer assemblies with a predetermined structure using a combination of Langmuir-Blodgett and adsorption techniques. The value of enzyme activity and the level of protein detachment were measured in dependence on the variation of film composition and on the sequence of layer alternation. As a result, highly active and stable structures were found, which could be promising candidates for practical applications. The method of modification of the deposition method to provide continuous film formation on large-area supports is discussed.

Functional assignment to JEV proteins using SVM.

PubMed

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

Functional assignment to JEV proteins using SVM

PubMed Central

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP). PMID:19052658

An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruan, Guihua, E-mail: guihuaruan@hotmail.com; Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004; Wu, Zhenwei

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N{sub α}-benzoyl-L-arginine ethyl ester to N{sub α}-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast andmore » easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.« less

Exploring Covalent Allosteric Inhibition of Antigen 85C from Mycobacterium tuberculosis by Ebselen Derivatives.

PubMed

Goins, Christopher M; Dajnowicz, Steven; Thanna, Sandeep; Sucheck, Steven J; Parks, Jerry M; Ronning, Donald R

2017-05-12

Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis (Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitor orientation that were unobserved in previous Ag85C ebselen structures. The k inact /K I values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein-inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein-inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally, this structural rearrangement due to covalent allosteric modification creates a sizable solvent network that encompasses the active site and extends to the modified Cys209 residue. In all, this study outlines factors that influence enzyme inhibition by ebselen and its derivatives while further highlighting the effects of the covalent modification of Cys209 by said inhibitors on the structure and stability of Ag85C. Furthermore, the results suggest a strategy for developing new classes of Ag85 inhibitors with increased specificity and potency.

Physics of protein folding

NASA Astrophysics Data System (ADS)

Finkelstein, A. V.; Galzitskaya, O. V.

2004-04-01

Protein physics is grounded on three fundamental experimental facts: protein, this long heteropolymer, has a well defined compact three-dimensional structure; this structure can spontaneously arise from the unfolded protein chain in appropriate environment; and this structure is separated from the unfolded state of the chain by the “all-or-none” phase transition, which ensures robustness of protein structure and therefore of its action. The aim of this review is to consider modern understanding of physical principles of self-organization of protein structures and to overview such important features of this process, as finding out the unique protein structure among zillions alternatives, nucleation of the folding process and metastable folding intermediates. Towards this end we will consider the main experimental facts and simple, mostly phenomenological theoretical models. We will concentrate on relatively small (single-domain) water-soluble globular proteins (whose structure and especially folding are much better studied and understood than those of large or membrane and fibrous proteins) and consider kinetic and structural aspects of transition of initially unfolded protein chains into their final solid (“native”) 3D structures.

Intuitive representation of surface properties of biomolecules using BioBlender.

PubMed

Andrei, Raluca Mihaela; Callieri, Marco; Zini, Maria Francesca; Loni, Tiziana; Maraziti, Giuseppe; Pan, Mike Chen; Zoppè, Monica

2012-03-28

In living cells, proteins are in continuous motion and interaction with the surrounding medium and/or other proteins and ligands. These interactions are mediated by protein features such as electrostatic and lipophilic potentials. The availability of protein structures enables the study of their surfaces and surface characteristics, based on atomic contribution. Traditionally, these properties are calculated by physico-chemical programs and visualized as range of colors that vary according to the tool used and imposes the necessity of a legend to decrypt it. The use of color to encode both characteristics makes the simultaneous visualization almost impossible, requiring these features to be visualized in different images. In this work, we describe a novel and intuitive code for the simultaneous visualization of these properties. Recent advances in 3D animation and rendering software have not yet been exploited for the representation of biomolecules in an intuitive, animated form. For our purpose we use Blender, an open-source, free, cross-platform application used professionally for 3D work. On the basis Blender, we developed BioBlender, dedicated to biological work: elaboration of protein motion with simultaneous visualization of their chemical and physical features. Electrostatic and lipophilic potentials are calculated using physico-chemical software and scripts, organized and accessed through BioBlender interface. A new visual code is introduced for molecular lipophilic potential: a range of optical features going from smooth-shiny for hydrophobic regions to rough-dull for hydrophilic ones. Electrostatic potential is represented as animated line particles that flow along field lines, proportional to the total charge of the protein. Our system permits visualization of molecular features and, in the case of moving proteins, their continuous perception, calculated for each conformation during motion. Using real world tactile/sight feelings, the nanoscale world of proteins becomes more understandable, familiar to our everyday life, making it easier to introduce "un-seen" phenomena (concepts) such as hydropathy or charges. Moreover, this representation contributes to gain insight into molecular functions by drawing viewer's attention to the most active regions of the protein. The program, available for Windows, Linux and MacOS, can be downloaded freely from the dedicated website http://www.bioblender.eu.

Intuitive representation of surface properties of biomolecules using BioBlender

PubMed Central

2012-01-01

Background In living cells, proteins are in continuous motion and interaction with the surrounding medium and/or other proteins and ligands. These interactions are mediated by protein features such as electrostatic and lipophilic potentials. The availability of protein structures enables the study of their surfaces and surface characteristics, based on atomic contribution. Traditionally, these properties are calculated by physico-chemical programs and visualized as range of colors that vary according to the tool used and imposes the necessity of a legend to decrypt it. The use of color to encode both characteristics makes the simultaneous visualization almost impossible, requiring these features to be visualized in different images. In this work, we describe a novel and intuitive code for the simultaneous visualization of these properties. Methods Recent advances in 3D animation and rendering software have not yet been exploited for the representation of biomolecules in an intuitive, animated form. For our purpose we use Blender, an open-source, free, cross-platform application used professionally for 3D work. On the basis Blender, we developed BioBlender, dedicated to biological work: elaboration of protein motion with simultaneous visualization of their chemical and physical features. Electrostatic and lipophilic potentials are calculated using physico-chemical software and scripts, organized and accessed through BioBlender interface. Results A new visual code is introduced for molecular lipophilic potential: a range of optical features going from smooth-shiny for hydrophobic regions to rough-dull for hydrophilic ones. Electrostatic potential is represented as animated line particles that flow along field lines, proportional to the total charge of the protein. Conclusions Our system permits visualization of molecular features and, in the case of moving proteins, their continuous perception, calculated for each conformation during motion. Using real world tactile/sight feelings, the nanoscale world of proteins becomes more understandable, familiar to our everyday life, making it easier to introduce "un-seen" phenomena (concepts) such as hydropathy or charges. Moreover, this representation contributes to gain insight into molecular functions by drawing viewer's attention to the most active regions of the protein. The program, available for Windows, Linux and MacOS, can be downloaded freely from the dedicated website http://www.bioblender.eu PMID:22536962

General overview on structure prediction of twilight-zone proteins.

PubMed

Khor, Bee Yin; Tye, Gee Jun; Lim, Theam Soon; Choong, Yee Siew

2015-09-04

Protein structure prediction from amino acid sequence has been one of the most challenging aspects in computational structural biology despite significant progress in recent years showed by critical assessment of protein structure prediction (CASP) experiments. When experimentally determined structures are unavailable, the predictive structures may serve as starting points to study a protein. If the target protein consists of homologous region, high-resolution (typically <1.5 Å) model can be built via comparative modelling. However, when confronted with low sequence similarity of the target protein (also known as twilight-zone protein, sequence identity with available templates is less than 30%), the protein structure prediction has to be initiated from scratch. Traditionally, twilight-zone proteins can be predicted via threading or ab initio method. Based on the current trend, combination of different methods brings an improved success in the prediction of twilight-zone proteins. In this mini review, the methods, progresses and challenges for the prediction of twilight-zone proteins were discussed.

Structure-Based Characterization of Multiprotein Complexes

PubMed Central

Wiederstein, Markus; Gruber, Markus; Frank, Karl; Melo, Francisco; Sippl, Manfred J.

2014-01-01

Summary Multiprotein complexes govern virtually all cellular processes. Their 3D structures provide important clues to their biological roles, especially through structural correlations among protein molecules and complexes. The detection of such correlations generally requires comprehensive searches in databases of known protein structures by means of appropriate structure-matching techniques. Here, we present a high-speed structure search engine capable of instantly matching large protein oligomers against the complete and up-to-date database of biologically functional assemblies of protein molecules. We use this tool to reveal unseen structural correlations on the level of protein quaternary structure and demonstrate its general usefulness for efficiently exploring complex structural relationships among known protein assemblies. PMID:24954616

Mathematical methods for protein science

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hart, W.; Istrail, S.; Atkins, J.

1997-12-31

Understanding the structure and function of proteins is a fundamental endeavor in molecular biology. Currently, over 100,000 protein sequences have been determined by experimental methods. The three dimensional structure of the protein determines its function, but there are currently less than 4,000 structures known to atomic resolution. Accordingly, techniques to predict protein structure from sequence have an important role in aiding the understanding of the Genome and the effects of mutations in genetic disease. The authors describe current efforts at Sandia to better understand the structure of proteins through rigorous mathematical analyses of simple lattice models. The efforts have focusedmore » on two aspects of protein science: mathematical structure prediction, and inverse protein folding.« less

Resource for structure related information on transmembrane proteins

NASA Astrophysics Data System (ADS)

Tusnády, Gábor E.; Simon, István

Transmembrane proteins are involved in a wide variety of vital biological processes including transport of water-soluble molecules, flow of information and energy production. Despite significant efforts to determine the structures of these proteins, only a few thousand solved structures are known so far. Here, we review the various resources for structure-related information on these types of proteins ranging from the 3D structure to the topology and from the up-to-date databases to the various Internet sites and servers dealing with structure prediction and structure analysis. Abbreviations: 3D, three dimensional; PDB, Protein Data Bank; TMP, transmembrane protein.

G-LoSA for Prediction of Protein-Ligand Binding Sites and Structures.

PubMed

Lee, Hui Sun; Im, Wonpil

2017-01-01

Recent advances in high-throughput structure determination and computational protein structure prediction have significantly enriched the universe of protein structure. However, there is still a large gap between the number of available protein structures and that of proteins with annotated function in high accuracy. Computational structure-based protein function prediction has emerged to reduce this knowledge gap. The identification of a ligand binding site and its structure is critical to the determination of a protein's molecular function. We present a computational methodology for predicting small molecule ligand binding site and ligand structure using G-LoSA, our protein local structure alignment and similarity measurement tool. All the computational procedures described here can be easily implemented using G-LoSA Toolkit, a package of standalone software programs and preprocessed PDB structure libraries. G-LoSA and G-LoSA Toolkit are freely available to academic users at http://compbio.lehigh.edu/GLoSA . We also illustrate a case study to show the potential of our template-based approach harnessing G-LoSA for protein function prediction.

A structural-alphabet-based strategy for finding structural motifs across protein families

PubMed Central

Wu, Chih Yuan; Chen, Yao Chi; Lim, Carmay

2010-01-01

Proteins with insignificant sequence and overall structure similarity may still share locally conserved contiguous structural segments; i.e. structural/3D motifs. Most methods for finding 3D motifs require a known motif to search for other similar structures or functionally/structurally crucial residues. Here, without requiring a query motif or essential residues, a fully automated method for discovering 3D motifs of various sizes across protein families with different folds based on a 16-letter structural alphabet is presented. It was applied to structurally non-redundant proteins bound to DNA, RNA, obligate/non-obligate proteins as well as free DNA-binding proteins (DBPs) and proteins with known structures but unknown function. Its usefulness was illustrated by analyzing the 3D motifs found in DBPs. A non-specific motif was found with a ‘corner’ architecture that confers a stable scaffold and enables diverse interactions, making it suitable for binding not only DNA but also RNA and proteins. Furthermore, DNA-specific motifs present ‘only’ in DBPs were discovered. The motifs found can provide useful guidelines in detecting binding sites and computational protein redesign. PMID:20525797

How cooperative are protein folding and unfolding transitions?

PubMed Central

Malhotra, Pooja

2016-01-01

Abstract A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two‐state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non‐cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier‐less “downhill” folding, as well as for continuous “uphill” unfolding transitions, indicate that gradual non‐cooperative processes may be ubiquitous features on the free energy landscape of protein folding. PMID:27522064

Expression, sorting, and segregation of Golgi proteins during germ cell differentiation in the testis

PubMed Central

Au, Catherine E.; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Simon, Paul H. G.; Kearney, Robert E.; Cameron, Pamela H.; Smith, Charles E.; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Ma, Kewei; Nilsson, Tommy; Bergeron, John J. M.

2015-01-01

The molecular basis of changes in structure, cellular location, and function of the Golgi apparatus during male germ cell differentiation is unknown. To deduce cognate Golgi proteins, we isolated germ cell Golgi fractions, and 1318 proteins were characterized, with 20 localized in situ. The most abundant protein, GL54D of unknown function, is characterized as a germ cell–specific Golgi-localized type II integral membrane glycoprotein. TM9SF3, also of unknown function, was revealed to be a universal Golgi marker for both somatic and germ cells. During acrosome formation, several Golgi proteins (GBF1, GPP34, GRASP55) localize to both the acrosome and Golgi, while GL54D, TM9SF3, and the Golgi trafficking protein TMED7/p27 are segregated from the acrosome. After acrosome formation, GL54D, TM9SF3, TMED4/p25, and TMED7/p27 continue to mark Golgi identity as it migrates away from the acrosome, while the others (GBF1, GPP34, GRASP55) remain in the acrosome and are progressively lost in later steps of differentiation. Cytoplasmic HSP70.2 and the endoplasmic reticulum luminal protein-folding enzyme PDILT are also Golgi recruited but only during acrosome formation. This resource identifies abundant Golgi proteins that are expressed differentially during mitosis, meiosis, and postacrosome Golgi migration, including the last step of differentiation. PMID:25808494

A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

PubMed

Katsura, Kazushige; Matsuda, Takayoshi; Tomabechi, Yuri; Yonemochi, Mayumi; Hanada, Kazuharu; Ohsawa, Noboru; Sakamoto, Kensaku; Takemoto, Chie; Shirouzu, Mikako

2017-11-01

Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

An overview of the structures of protein-DNA complexes

PubMed Central

Luscombe, Nicholas M; Austin, Susan E; Berman , Helen M; Thornton, Janet M

2000-01-01

On the basis of a structural analysis of 240 protein-DNA complexes contained in the Protein Data Bank (PDB), we have classified the DNA-binding proteins involved into eight different structural/functional groups, which are further classified into 54 structural families. Here we present this classification and review the functions, structures and binding interactions of these protein-DNA complexes. PMID:11104519

Functional structural motifs for protein-ligand, protein-protein, and protein-nucleic acid interactions and their connection to supersecondary structures.

PubMed

Kinjo, Akira R; Nakamura, Haruki

2013-01-01

Protein functions are mediated by interactions between proteins and other molecules. One useful approach to analyze protein functions is to compare and classify the structures of interaction interfaces of proteins. Here, we describe the procedures for compiling a database of interface structures and efficiently comparing the interface structures. To do so requires a good understanding of the data structures of the Protein Data Bank (PDB). Therefore, we also provide a detailed account of the PDB exchange dictionary necessary for extracting data that are relevant for analyzing interaction interfaces and secondary structures. We identify recurring structural motifs by classifying similar interface structures, and we define a coarse-grained representation of supersecondary structures (SSS) which represents a sequence of two or three secondary structure elements including their relative orientations as a string of four to seven letters. By examining the correspondence between structural motifs and SSS strings, we show that no SSS string has particularly high propensity to be found interaction interfaces in general, indicating any SSS can be used as a binding interface. When individual structural motifs are examined, there are some SSS strings that have high propensity for particular groups of structural motifs. In addition, it is shown that while the SSS strings found in particular structural motifs for nonpolymer and protein interfaces are as abundant as in other structural motifs that belong to the same subunit, structural motifs for nucleic acid interfaces exhibit somewhat stronger preference for SSS strings. In regard to protein folds, many motif-specific SSS strings were found across many folds, suggesting that SSS may be a useful description to investigate the universality of ligand binding modes.

Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

PubMed Central

2010-01-01

Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context. PMID:21129205

Folding domain B of protein A on a dynamically partitioned free energy landscape.

PubMed

Nelson, Erik D; Grishin, Nick V

2008-02-05

The B domain of staphylococcal protein A (BdpA) is a small helical protein that has been studied intensively in kinetics experiments and detailed computer simulations that include explicit water. The simulations indicate that BdpA needs to reorganize in crossing the transition barrier to facilitate folding its C-terminal helix (H3) onto the nucleus formed from helices H1 and H2. This process suggests frustration between two partially ordered forms of the protein, but recent varphi value measurements indicate that the transition structure is relatively constant over a broad range of temperatures. Here we develop a simplistic model to investigate the folding transition in which properties of the free energy landscape can be quantitatively compared with experimental data. The model is a continuation of the Muñoz-Eaton model to include the intermittency of contacts between structured parts of the protein, and the results compare variations in the landscape with denaturant and temperature to varphi value measurements and chevron plots of the kinetic rates. The topography of the model landscape (in particular, the feature of frustration) is consistent with detailed simulations even though variations in the varphi values are close to measured values. The transition barrier is smaller than indicated by the chevron data, but it agrees in order of magnitude with a similar alpha-carbon type of model. Discrepancies with the chevron plots are investigated from the point of view of solvent effects, and an approach is suggested to account for solvent participation in the model.

In nucleoli, the steady state of nucleolar proteins is leptomycin B-sensitive.

PubMed

Muro, Eleonora; Hoang, Thang Q; Jobart-Malfait, Aude; Hernandez-Verdun, Danièle

2008-05-01

The nucleolus is a dynamic structure. It has been demonstrated that nucleolar proteins rapidly associate with and dissociate from nucleolar components in continuous exchanges with the nucleoplasm using GFP (green fluorescent protein)-tagged proteins. However, how the exchanges within one nucleolus and between nucleoli within the nuclear volume occurred is still poorly understood. The movement of PAGFP (photoactivatable GFP)-tagged proteins that become visible after photoactivation can be followed. In the present study, we establish the protocol allowing quantification of the traffic of PAGFP-tagged nucleolar proteins in nuclei containing two nucleoli. The traffic in the activated area, at the periphery of the activated area and to the neighbouring nucleolus is measured. Protein B23 is rapidly replaced in the activated area, and at the periphery of the activated area the steady state suggests intranucleolar recycling of B23; this recycling is LMB (leptomycin B)-sensitive. The pool of activated B23 is equally distributed in the volume of the two nucleoli within 2 min. The three-dimensional distribution of the proteins Nop52 and fibrillarin is less rapid than that of B23 but is also LMB-sensitive. In contrast, traffic of fibrillarin from the nucleoli to the CB (Cajal body) was not modified by LMB. We propose that the steady state of nucleolar proteins in nucleoli depends on the affinity of the proteins for their partners and on intranucleolar recycling. This steady state can be impaired by LMB but not the uptake in the neighbouring nucleolus or the CB.

Untangling the web: Mechanisms underlying ER network formation

PubMed Central

Goyal, Uma; Blackstone, Craig

2013-01-01

The ER is a continuous membrane system consisting of the nuclear envelope, flat sheets often studded with ribosomes, and a polygonal network of highly-curved tubules extending throughout the cell. Although protein and lipid biosynthesis, protein modification, vesicular transport, Ca2+dynamics, and protein quality control have been investigated in great detail, mechanisms that generate the distinctive architecture of the ER have been uncovered only recently. Several protein families including the reticulons and REEPs/DP1/Yop1p harbor hydrophobic hairpin domains that shape high-curvature ER tubules and mediate intramembrane protein interactions. Members of the atlastin/RHD3/Sey1p family of dynamin-related GTPases interact with the ER-shaping proteins and mediate the formation of three-way junctions responsible for the polygonal structure of the tubular ER network, with Lunapark proteins acting antagonistically. Additional classes of tubular ER proteins including some REEPs and the M1 spastin ATPase interact with the microtubule cytoskeleton. Flat ER sheets possess a different complement of proteins such as p180, CLIMP-63 and kinectin implicated in shaping, cisternal stacking and cytoskeletal interactions. The ER is also in constant motion, and numerous signaling pathways as well as interactions among cytoskeletal elements, the plasma membrane, and organelles cooperate to position and shape the ER dynamically. Finally, many proteins involved in shaping the ER network are mutated in the most common forms of hereditary spastic paraplegia, indicating a particular importance for proper ER morphology and distribution in large, highly-polarized cells such as neurons. PMID:23602970

An Extended Structure of the APOBEC3G Catalytic Domain Suggests a Unique Holoenzyme Model

PubMed Central

Harjes, Elena; Gross, Phillip J.; Chen, Kuan-Ming; Lu, Yongjian; Shindo, Keisuke; Nowarski, Roni; Gross, John D.; Kotler, Moshe; Harris, Reuben S.; Matsuo, Hiroshi

2009-01-01

Summary Human APOBEC3G (A3G) belongs to a family of polynucleotide cytidine deaminases. This family includes APOBEC1 and AID, which edit APOB mRNA and antibody gene DNA, respectively. A3G deaminates cytidines to uridines in single-strand DNA and inhibits the replication of HIV-1, other retroviruses and retrotransposons. Although the mechanism of A3G-catalyzed DNA deamination has been investigated genetically and biochemically, atomic details are just starting to emerge. Here, we compare the DNA cytidine deaminase activities and NMR structures of two A3G catalytic domain constructs. The longer A3G191-384 protein is considerably more active than the shorter A3G198-384 variant. The longer structure has an α1 helix (residues 201–206) that was not apparent in the shorter protein and it contributes to catalytic activity through interactions with hydrophobic core structures (β1, β3, α5 and α6). Both A3G catalytic domain solution structures have a discontinuous β2 region that is clearly different than the continuous β2 strand of another family member APOBEC2. In addition, the longer A3G191-384 structure revealed part of the N-terminal pseudo-catalytic domain including the inter-domain linker and some of the last α-helix. These structured residues (191–196) enabled a novel full-length A3G model by providing physical overlap between the N-terminal pseudo-catalytic domain and the new C-terminal catalytic domain structure. Contrary to predictions, this structurally constrained model suggested that the two domains are tethered by structured residues and that the N- and C-terminal β2 regions are too distant from one another to participate in this interaction. PMID:19389408

Cellular Strategies of Protein Quality Control

PubMed Central

Chen, Bryan; Retzlaff, Marco; Roos, Thomas; Frydman, Judith

2011-01-01

Eukaryotic cells must contend with a continuous stream of misfolded proteins that compromise the cellular protein homeostasis balance and jeopardize cell viability. An elaborate network of molecular chaperones and protein degradation factors continually monitor and maintain the integrity of the proteome. Cellular protein quality control relies on three distinct yet interconnected strategies whereby misfolded proteins can either be refolded, degraded, or delivered to distinct quality control compartments that sequester potentially harmful misfolded species. Molecular chaperones play a critical role in determining the fate of misfolded proteins in the cell. Here, we discuss the spatial and temporal organization of cellular quality control strategies and their implications for human diseases linked to protein misfolding and aggregation. PMID:21746797

Evidence of structurally continuous collagen fibrils in tendons.

PubMed

Svensson, Rene B; Herchenhan, Andreas; Starborg, Tobias; Larsen, Michael; Kadler, Karl E; Qvortrup, Klaus; Magnusson, S Peter

2017-03-01

Tendons transmit muscle-generated force through an extracellular matrix of aligned collagen fibrils. The force applied by the muscle at one end of a microscopic fibril has to be transmitted through the macroscopic length of the tendon by mechanisms that are poorly understood. A key element in this structure-function relationship is the collagen fibril length. During embryogenesis short fibrils are produced but they grow rapidly with maturation. There is some controversy regarding fibril length in adult tendon, with mechanical data generally supporting discontinuity while structural investigations favor continuity. This study initially set out to trace the full length of individual fibrils in adult human tendons, using serial block face-scanning electron microscopy. But even with this advanced technique the required length could not be covered. Instead a statistical approach was used on a large volume of fibrils in shorter image stacks. Only a single end was observed after tracking 67.5mm of combined fibril lengths, in support of fibril continuity. To shed more light on this observation, the full length of a short tendon (mouse stapedius, 125μm) was investigated and continuity of individual fibrils was confirmed. In light of these results, possible mechanisms that could reconcile the opposing findings on fibril continuity are discussed. Connective tissues hold all parts of the body together and are mostly constructed from thin threads of the protein collagen (called fibrils). Connective tissues provide mechanical strength and one of the most demanding tissues in this regard are tendons, which transmit the forces generated by muscles. The length of the collagen fibrils is essential to the mechanical strength and to the type of damage the tissue may experience (slippage of short fibrils or breakage of longer ones). This in turn is important for understanding the repair processes after such damage occurs. Currently the issue of fibril length is contentious, but this study provides evidence that the fibrils are extremely long and likely continuous. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Glycomic characterization of basal tears and changes with diabetes and diabetic retinopathy.

PubMed

Nguyen-Khuong, Terry; Everest-Dass, Arun V; Kautto, Liisa; Zhao, Zhenjun; Willcox, Mark D P; Packer, Nicolle H

2015-03-01

As a secreted fluid, the state of tear glycosylation is particularly important in the role of immunity of the ocular surface. Tears are a valuable source of non-invasive biomarkers for disease and there are continued efforts to characterize their components thoroughly. In this study, a small volume of basal tears (5 μL) was collected from healthy controls, patients with diabetes without retinopathy and patients with diabetes and retinopathy. The detailed N- and O-linked tear protein glycome was characterized and the relative abundance of each structure determined. Of the 50 N-linked glycans found, 89% were complex with 50% containing a bisecting N-acetylglucosamine, 65% containing a core fucose whilst 33% were sialylated. Of the 8 O-linked glycans detected, 3 were of cores 1 and 5 of core 2 type, with a majority of them being sialylated (90%). Additionally, these glycan structures were profiled across the three diabetic disease groups. Whilst the higher abundant structures did not alter across the three groups, only five low abundance N-linked glycans and 1 O-linked glycan did alter with the onset of diabetes mellitus and diabetic retinopathy (DR). These results suggest the conservation of glycan types on basal tear proteins between individuals and point to only small changes in glycan expression on the proteins in tears with the development of diabetes and DR. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Course 12: Proteins: Structural, Thermodynamic and Kinetic Aspects

NASA Astrophysics Data System (ADS)

Finkelstein, A. V.

1 Introduction 2 Overview of protein architectures and discussion of physical background of their natural selection 2.1 Protein structures 2.2 Physical selection of protein structures 3 Thermodynamic aspects of protein folding 3.1 Reversible denaturation of protein structures 3.2 What do denatured proteins look like? 3.3 Why denaturation of a globular protein is the first-order phase transition 3.4 "Gap" in energy spectrum: The main characteristic that distinguishes protein chains from random polymers 4 Kinetic aspects of protein folding 4.1 Protein folding in vivo 4.2 Protein folding in vitro (in the test-tube) 4.3 Theory of protein folding rates and solution of the Levinthal paradox

An Evolution-Based Approach to De Novo Protein Design and Case Study on Mycobacterium tuberculosis

PubMed Central

Brender, Jeffrey R.; Czajka, Jeff; Marsh, David; Gray, Felicia; Cierpicki, Tomasz; Zhang, Yang

2013-01-01

Computational protein design is a reverse procedure of protein folding and structure prediction, where constructing structures from evolutionarily related proteins has been demonstrated to be the most reliable method for protein 3-dimensional structure prediction. Following this spirit, we developed a novel method to design new protein sequences based on evolutionarily related protein families. For a given target structure, a set of proteins having similar fold are identified from the PDB library by structural alignments. A structural profile is then constructed from the protein templates and used to guide the conformational search of amino acid sequence space, where physicochemical packing is accommodated by single-sequence based solvation, torsion angle, and secondary structure predictions. The method was tested on a computational folding experiment based on a large set of 87 protein structures covering different fold classes, which showed that the evolution-based design significantly enhances the foldability and biological functionality of the designed sequences compared to the traditional physics-based force field methods. Without using homologous proteins, the designed sequences can be folded with an average root-mean-square-deviation of 2.1 Å to the target. As a case study, the method is extended to redesign all 243 structurally resolved proteins in the pathogenic bacteria Mycobacterium tuberculosis, which is the second leading cause of death from infectious disease. On a smaller scale, five sequences were randomly selected from the design pool and subjected to experimental validation. The results showed that all the designed proteins are soluble with distinct secondary structure and three have well ordered tertiary structure, as demonstrated by circular dichroism and NMR spectroscopy. Together, these results demonstrate a new avenue in computational protein design that uses knowledge of evolutionary conservation from protein structural families to engineer new protein molecules of improved fold stability and biological functionality. PMID:24204234

ProBiS-database: precalculated binding site similarities and local pairwise alignments of PDB structures.

PubMed

Konc, Janez; Cesnik, Tomo; Konc, Joanna Trykowska; Penca, Matej; Janežič, Dušanka

2012-02-27

ProBiS-Database is a searchable repository of precalculated local structural alignments in proteins detected by the ProBiS algorithm in the Protein Data Bank. Identification of functionally important binding regions of the protein is facilitated by structural similarity scores mapped to the query protein structure. PDB structures that have been aligned with a query protein may be rapidly retrieved from the ProBiS-Database, which is thus able to generate hypotheses concerning the roles of uncharacterized proteins. Presented with uncharacterized protein structure, ProBiS-Database can discern relationships between such a query protein and other better known proteins in the PDB. Fast access and a user-friendly graphical interface promote easy exploration of this database of over 420 million local structural alignments. The ProBiS-Database is updated weekly and is freely available online at http://probis.cmm.ki.si/database.

A Method for WD40 Repeat Detection and Secondary Structure Prediction

PubMed Central

Wang, Yang; Jiang, Fan; Zhuo, Zhu; Wu, Xian-Hui; Wu, Yun-Dong

2013-01-01

WD40-repeat proteins (WD40s), as one of the largest protein families in eukaryotes, play vital roles in assembling protein-protein/DNA/RNA complexes. WD40s fold into similar β-propeller structures despite diversified sequences. A program WDSP (WD40 repeat protein Structure Predictor) has been developed to accurately identify WD40 repeats and predict their secondary structures. The method is designed specifically for WD40 proteins by incorporating both local residue information and non-local family-specific structural features. It overcomes the problem of highly diversified protein sequences and variable loops. In addition, WDSP achieves a better prediction in identifying multiple WD40-domain proteins by taking the global combination of repeats into consideration. In secondary structure prediction, the average Q3 accuracy of WDSP in jack-knife test reaches 93.7%. A disease related protein LRRK2 was used as a representive example to demonstrate the structure prediction. PMID:23776530

The MORPHEUS II protein crystallization screen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gorrec, Fabrice, E-mail: fgorrec@mrc-lmb.cam.ac.uk

2015-06-27

MORPHEUS II is a 96-condition initial crystallization screen formulated de novo. The screen incorporates reagents selected from the Protein Data Bank to yield crystals that are not observed in traditional conditions. In addition, the formulation facilitates the optimization and cryoprotection of crystals. High-quality macromolecular crystals are a prerequisite for the process of protein structure determination by X-ray diffraction. Unfortunately, the relative yield of diffraction-quality crystals from crystallization experiments is often very low. In this context, innovative crystallization screen formulations are continuously being developed. In the past, MORPHEUS, a screen in which each condition integrates a mix of additives selected frommore » the Protein Data Bank, a cryoprotectant and a buffer system, was developed. Here, MORPHEUS II, a follow-up to the original 96-condition initial screen, is described. Reagents were selected to yield crystals when none might be observed in traditional initial screens. Besides, the screen includes heavy atoms for experimental phasing and small polyols to ensure the cryoprotection of crystals. The suitability of the resulting novel conditions is shown by the crystallization of a broad variety of protein samples and their efficiency is compared with commercially available conditions.« less

Protein domain assignment from the recurrence of locally similar structures

PubMed Central

Tai, Chin-Hsien; Sam, Vichetra; Gibrat, Jean-Francois; Garnier, Jean; Munson, Peter J.

2010-01-01

Domains are basic units of protein structure and essential for exploring protein fold space and structure evolution. With the structural genomics initiative, the number of protein structures in the Protein Databank (PDB) is increasing dramatically and domain assignments need to be done automatically. Most existing structural domain assignment programs define domains using the compactness of the domains and/or the number and strength of intra-domain versus inter-domain contacts. Here we present a different approach based on the recurrence of locally similar structural pieces (LSSPs) found by one-against-all structure comparisons with a dataset of 6,373 protein chains from the PDB. Residues of the query protein are clustered using LSSPs via three different procedures to define domains. This approach gives results that are comparable to several existing programs that use geometrical and other structural information explicitly. Remarkably, most of the proteins that contribute the LSSPs defining a domain do not themselves contain the domain of interest. This study shows that domains can be defined by a collection of relatively small locally similar structural pieces containing, on average, four secondary structure elements. In addition, it indicates that domains are indeed made of recurrent small structural pieces that are used to build protein structures of many different folds as suggested by recent studies. PMID:21287617

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.

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

Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography.

PubMed

Coughlan, H D; Darmanin, C; Kirkwood, H J; Phillips, N W; Hoxley, D; Clark, J N; Vine, D J; Hofmann, F; Harder, R J; Maxey, E; Abbey, B

2017-01-01

The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals (Helliwell, 1984). Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to the diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. A possible mechanism to account for these observations is proposed.

Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography

DOE PAGES

Coughlan, H. D.; Darmanin, C.; Kirkwood, H. J.; ...

2017-01-01

The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals. Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to themore » diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. As a result, a possible mechanism to account for these observations is proposed.« less

Bragg coherent diffraction imaging and metrics for radiation damage in protein micro-crystallography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coughlan, H. D.; Darmanin, C.; Kirkwood, H. J.

The proliferation of extremely intense synchrotron sources has enabled ever higher-resolution structures to be obtained using data collected from smaller and often more imperfect biological crystals. Synchrotron beamlines now exist that are capable of measuring data from single crystals that are just a few micrometres in size. This provides renewed motivation to study and understand the radiation damage behaviour of small protein crystals. Reciprocal-space mapping and Bragg coherent diffractive imaging experiments have been performed on cryo-cooled microcrystals of hen egg-white lysozyme as they undergo radiation damage. Several well established metrics, such as intensity-loss and lattice expansion, are applied to themore » diffraction data and the results are compared with several new metrics that can be extracted from the coherent imaging experiments. Individually some of these metrics are inconclusive. However, combining metrics, the results suggest that radiation damage behaviour in protein micro-crystals differs from that of larger protein crystals and may allow them to continue to diffract for longer. As a result, a possible mechanism to account for these observations is proposed.« less

Protein Adsorption onto Nanomaterials for the Development of Biosensors and Analytical Devices: A Review

PubMed Central

Bhakta, Samir A.; Evans, Elizabeth; Benavidez, Tomás E.; Garcia, Carlos D.

2014-01-01

An important consideration for the development of biosensors is the adsorption of the bio recognition element to the surface of a substrate. As the first step in the immobilization process, adsorption affects most immobilization routes and much attention is given into the research of this process to maximize the overall activity of the bio sensor. The use of nanomaterials, specifically nanoparticles and nanostructured films, offers advantageous properties that can be fine-tuned for interaction with specific proteins to maximize activity, minimize structural changes, and enhance the catalytic step. In the biosensor field, protein-nanomaterial interactions are an emerging trend that span across many disciplines. This review addresses recent publications about the proteins most frequently used, their most relevant characteristics, and the conditions required to adsorb them to nanomaterials. When relevant and available, subsequent analytical figures of merits are discussed for selected biosensors. The general trend amongst the research papers allows concluding that the use of nanomaterials has already provided significant improvements in the analytical performance of many biosensors and that this research field will continue to grow. PMID:25892065

1H magic-angle spinning NMR evolves as a powerful new tool for membrane proteins

NASA Astrophysics Data System (ADS)

Schubeis, Tobias; Le Marchand, Tanguy; Andreas, Loren B.; Pintacuda, Guido

2018-02-01

Building on a decade of continuous advances of the community, the recent development of very fast (60 kHz and above) magic-angle spinning (MAS) probes has revolutionised the field of solid-state NMR. This new spinning regime reduces the 1H-1H dipolar couplings, so that direct detection of the larger magnetic moment available from 1H is now possible at high resolution, not only in deuterated molecules but also in fully-protonated substrates. Such capabilities allow rapid "fingerprinting" of samples with a ten-fold reduction of the required sample amounts with respect to conventional approaches, and permit extensive, robust and expeditious assignment of small-to-medium sized proteins (up to ca. 300 residues), and the determination of inter-nuclear proximities, relative orientations of secondary structural elements, protein-cofactor interactions, local and global dynamics. Fast MAS and 1H detection techniques have nowadays been shown to be applicable to membrane-bound systems. This paper reviews the strategies underlying this recent leap forward in sensitivity and resolution, describing its potential for the detailed characterization of membrane proteins.

Visualizing and Clustering Protein Similarity Networks: Sequences, Structures, and Functions.

PubMed

Mai, Te-Lun; Hu, Geng-Ming; Chen, Chi-Ming

2016-07-01

Research in the recent decade has demonstrated the usefulness of protein network knowledge in furthering the study of molecular evolution of proteins, understanding the robustness of cells to perturbation, and annotating new protein functions. In this study, we aimed to provide a general clustering approach to visualize the sequence-structure-function relationship of protein networks, and investigate possible causes for inconsistency in the protein classifications based on sequences, structures, and functions. Such visualization of protein networks could facilitate our understanding of the overall relationship among proteins and help researchers comprehend various protein databases. As a demonstration, we clustered 1437 enzymes by their sequences and structures using the minimum span clustering (MSC) method. The general structure of this protein network was delineated at two clustering resolutions, and the second level MSC clustering was found to be highly similar to existing enzyme classifications. The clustering of these enzymes based on sequence, structure, and function information is consistent with each other. For proteases, the Jaccard's similarity coefficient is 0.86 between sequence and function classifications, 0.82 between sequence and structure classifications, and 0.78 between structure and function classifications. From our clustering results, we discussed possible examples of divergent evolution and convergent evolution of enzymes. Our clustering approach provides a panoramic view of the sequence-structure-function network of proteins, helps visualize the relation between related proteins intuitively, and is useful in predicting the structure and function of newly determined protein sequences.

Structural determination of intact proteins using mass spectrometry

DOEpatents

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

2008-05-06

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

Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

PubMed

Hayashi, Takahiro; Hamachi, Itaru

2012-09-18

Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional affinity labeling method and allows for real-time monitoring of protein activity. With the high target specificity and biocompatibility of this technique, we have achieved individual labeling and imaging of endogenously expressed proteins in samples of high biological complexity. We also highlight applications in which our current approach enabled the monitoring of important biological events, such as ligand binding, in living cells. These novel chemical labeling techniques are expected to provide a molecular toolbox for studying a wide variety of proteins and beyond in living cells.

Metabolic Turnover of Synaptic Proteins: Kinetics, Interdependencies and Implications for Synaptic Maintenance

PubMed Central

Cohen, Laurie D.; Zuchman, Rina; Sorokina, Oksana; Müller, Anke; Dieterich, Daniela C.; Armstrong, J. Douglas; Ziv, Tamar; Ziv, Noam E.

2013-01-01

Chemical synapses contain multitudes of proteins, which in common with all proteins, have finite lifetimes and therefore need to be continuously replaced. Given the huge numbers of synaptic connections typical neurons form, the demand to maintain the protein contents of these connections might be expected to place considerable metabolic demands on each neuron. Moreover, synaptic proteostasis might differ according to distance from global protein synthesis sites, the availability of distributed protein synthesis facilities, trafficking rates and synaptic protein dynamics. To date, the turnover kinetics of synaptic proteins have not been studied or analyzed systematically, and thus metabolic demands or the aforementioned relationships remain largely unknown. In the current study we used dynamic Stable Isotope Labeling with Amino acids in Cell culture (SILAC), mass spectrometry (MS), Fluorescent Non–Canonical Amino acid Tagging (FUNCAT), quantitative immunohistochemistry and bioinformatics to systematically measure the metabolic half-lives of hundreds of synaptic proteins, examine how these depend on their pre/postsynaptic affiliation or their association with particular molecular complexes, and assess the metabolic load of synaptic proteostasis. We found that nearly all synaptic proteins identified here exhibited half-lifetimes in the range of 2–5 days. Unexpectedly, metabolic turnover rates were not significantly different for presynaptic and postsynaptic proteins, or for proteins for which mRNAs are consistently found in dendrites. Some functionally or structurally related proteins exhibited very similar turnover rates, indicating that their biogenesis and degradation might be coupled, a possibility further supported by bioinformatics-based analyses. The relatively low turnover rates measured here (∼0.7% of synaptic protein content per hour) are in good agreement with imaging-based studies of synaptic protein trafficking, yet indicate that the metabolic load synaptic protein turnover places on individual neurons is very substantial. PMID:23658807

Understand protein functions by comparing the similarity of local structural environments.

PubMed

Chen, Jiawen; Xie, Zhong-Ru; Wu, Yinghao

2017-02-01

The three-dimensional structures of proteins play an essential role in regulating binding between proteins and their partners, offering a direct relationship between structures and functions of proteins. It is widely accepted that the function of a protein can be determined if its structure is similar to other proteins whose functions are known. However, it is also observed that proteins with similar global structures do not necessarily correspond to the same function, while proteins with very different folds can share similar functions. This indicates that function similarity is originated from the local structural information of proteins instead of their global shapes. We assume that proteins with similar local environments prefer binding to similar types of molecular targets. In order to testify this assumption, we designed a new structural indicator to define the similarity of local environment between residues in different proteins. This indicator was further used to calculate the probability that a given residue binds to a specific type of structural neighbors, including DNA, RNA, small molecules and proteins. After applying the method to a large-scale non-redundant database of proteins, we show that the positive signal of binding probability calculated from the local structural indicator is statistically meaningful. In summary, our studies suggested that the local environment of residues in a protein is a good indicator to recognize specific binding partners of the protein. The new method could be a potential addition to a suite of existing template-based approaches for protein function prediction. Copyright © 2016 Elsevier B.V. All rights reserved.

Inferences from structural comparison: flexibility, secondary structure wobble and sequence alignment optimization.

PubMed

Zhang, Gaihua; Su, Zhen

2012-01-01

Work on protein structure prediction is very useful in biological research. To evaluate their accuracy, experimental protein structures or their derived data are used as the 'gold standard'. However, as proteins are dynamic molecular machines with structural flexibility such a standard may be unreliable. To investigate the influence of the structure flexibility, we analysed 3,652 protein structures of 137 unique sequences from 24 protein families. The results showed that (1) the three-dimensional (3D) protein structures were not rigid: the root-mean-square deviation (RMSD) of the backbone Cα of structures with identical sequences was relatively large, with the average of the maximum RMSD from each of the 137 sequences being 1.06 Å; (2) the derived data of the 3D structure was not constant, e.g. the highest ratio of the secondary structure wobble site was 60.69%, with the sequence alignments from structural comparisons of two proteins in the same family sometimes being completely different. Proteins may have several stable conformations and the data derived from resolved structures as a 'gold standard' should be optimized before being utilized as criteria to evaluate the prediction methods, e.g. sequence alignment from structural comparison. Helix/β-sheet transition exists in normal free proteins. The coil ratio of the 3D structure could affect its resolution as determined by X-ray crystallography.

A Molecular Genetics Laboratory Course Applying Bioinformatics and Cell Biology in the Context of Original Research

PubMed Central

Pruitt, Wendy M.; Robinson, Lucy C.

2008-01-01

Research based laboratory courses have been shown to stimulate student interest in science and to improve scientific skills. We describe here a project developed for a semester-long research-based laboratory course that accompanies a genetics lecture course. The project was designed to allow students to become familiar with the use of bioinformatics tools and molecular biology and genetic approaches while carrying out original research. Students were required to present their hypotheses, experiments, and results in a comprehensive lab report. The lab project concerned the yeast casein kinase 1 (CK1) protein kinase Yck2. CK1 protein kinases are present in all organisms and are well conserved in primary structure. These enzymes display sequence features that differ from other protein kinase subfamilies. Students identified such sequences within the CK1 subfamily, chose a sequence to analyze, used available structural data to determine possible functions for their sequences, and designed mutations within the sequences. After generating the mutant alleles, these were expressed in yeast and tested for function by using two growth assays. The student response to the project was positive, both in terms of knowledge and skills increases and interest in research, and several students are continuing the analysis of mutant alleles as summer projects. PMID:19047427

Discovery of new molecular entities able to strongly interfere with Hsp90 C-terminal domain.

PubMed

Terracciano, Stefania; Russo, Alessandra; Chini, Maria G; Vaccaro, Maria C; Potenza, Marianna; Vassallo, Antonio; Riccio, Raffaele; Bifulco, Giuseppe; Bruno, Ines

2018-01-26

Heat shock protein 90 (Hsp90) is an ATP dependent molecular chaperone deeply involved in the complex network of cellular signaling governing some key functions, such as cell proliferation and survival, invasion and angiogenesis. Over the past years the N-terminal protein domain has been fully investigated as attractive strategy against cancer, but despite the many efforts lavished in the field, none of the N-terminal binders (termed "classical inhibitors"), currently in clinical trials, have yet successfully reached the market, because of the detrimental heat shock response (HSR) that showed to induce; thus, recently, the selective inhibition of Hsp90 C-terminal domain has powerfully emerged as a more promising alternative strategy for anti-cancer therapy, not eliciting this cell rescue cascade. However, the structural complexity of the target protein and, mostly, the lack of a co-crystal structure of C-terminal domain-ligand, essential to drive the identification of new hits, represent the largest hurdles in the development of new selective C-terminal inhibitors. Continuing our investigations on the identification of new anticancer drug candidates, by using an orthogonal screening approach, here we describe two new potent C-terminal inhibitors able to induce cancer cell death and a considerable down-regulation of Hsp90 client oncoproteins, without triggering the undesired heat shock response.

Proteomic identification of rhythmic proteins in rice seedlings.

PubMed

Hwang, Heeyoun; Cho, Man-Ho; Hahn, Bum-Soo; Lim, Hyemin; Kwon, Yong-Kook; Hahn, Tae-Ryong; Bhoo, Seong Hee

2011-04-01

Many aspects of plant metabolism that are involved in plant growth and development are influenced by light-regulated diurnal rhythms as well as endogenous clock-regulated circadian rhythms. To identify the rhythmic proteins in rice, periodically grown (12h light/12h dark cycle) seedlings were harvested for three days at six-hour intervals. Continuous dark-adapted plants were also harvested for two days. Among approximately 3000 reproducible protein spots on each gel, proteomic analysis ascertained 354 spots (~12%) as light-regulated rhythmic proteins, in which 53 spots showed prolonged rhythm under continuous dark conditions. Of these 354 ascertained rhythmic protein spots, 74 diurnal spots and 10 prolonged rhythmic spots under continuous dark were identified by MALDI-TOF MS analysis. The rhythmic proteins were functionally classified into photosynthesis, central metabolism, protein synthesis, nitrogen metabolism, stress resistance, signal transduction and unknown. Comparative analysis of our proteomic data with the public microarray database (the Plant DIURNAL Project) and RT-PCR analysis of rhythmic proteins showed differences in rhythmic expression phases between mRNA and protein, suggesting that the clock-regulated proteins in rice are modulated by not only transcriptional but also post-transcriptional, translational, and/or post-translational processes. 2011 Elsevier B.V. All rights reserved.

Structure-based characterization of multiprotein complexes.

PubMed

Wiederstein, Markus; Gruber, Markus; Frank, Karl; Melo, Francisco; Sippl, Manfred J

2014-07-08

Multiprotein complexes govern virtually all cellular processes. Their 3D structures provide important clues to their biological roles, especially through structural correlations among protein molecules and complexes. The detection of such correlations generally requires comprehensive searches in databases of known protein structures by means of appropriate structure-matching techniques. Here, we present a high-speed structure search engine capable of instantly matching large protein oligomers against the complete and up-to-date database of biologically functional assemblies of protein molecules. We use this tool to reveal unseen structural correlations on the level of protein quaternary structure and demonstrate its general usefulness for efficiently exploring complex structural relationships among known protein assemblies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

An ambiguity principle for assigning protein structural domains.

PubMed

Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

2017-01-01

Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object-in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our "multipartitioning" approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules.

Ensemble-based evaluation for protein structure models.

PubMed

Jamroz, Michal; Kolinski, Andrzej; Kihara, Daisuke

2016-06-15

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. 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. https://bitbucket.org/mjamroz/flexscore dkihara@purdue.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

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

Cross-Linking/Mass Spectrometry for Studying Protein Structures and Protein-Protein Interactions: Where Are We Now and Where Should We Go from Here?

PubMed

Sinz, Andrea

2018-05-28

Structural mass spectrometry (MS) is gaining increasing importance for deriving valuable three-dimensional structural information on proteins and protein complexes, and it complements existing techniques, such as NMR spectroscopy and X-ray crystallography. Structural MS unites different MS-based techniques, such as hydrogen/deuterium exchange, native MS, ion-mobility MS, protein footprinting, and chemical cross-linking/MS, and it allows fundamental questions in structural biology to be addressed. In this Minireview, I will focus on the cross-linking/MS strategy. This method not only delivers tertiary structural information on proteins, but is also increasingly being used to decipher protein interaction networks, both in vitro and in vivo. Cross-linking/MS is currently one of the most promising MS-based approaches to derive structural information on very large and transient protein assemblies and intrinsically disordered proteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fourier-based classification of protein secondary structures.

PubMed

Shu, Jian-Jun; Yong, Kian Yan

2017-04-15

The correct prediction of protein secondary structures is one of the key issues in predicting the correct protein folded shape, which is used for determining gene function. Existing methods make use of amino acids properties as indices to classify protein secondary structures, but are faced with a significant number of misclassifications. The paper presents a technique for the classification of protein secondary structures based on protein "signal-plotting" and the use of the Fourier technique for digital signal processing. New indices are proposed to classify protein secondary structures by analyzing hydrophobicity profiles. The approach is simple and straightforward. Results show that the more types of protein secondary structures can be classified by means of these newly-proposed indices. Copyright © 2017 Elsevier Inc. All rights reserved.

ProTSAV: A protein tertiary structure analysis and validation server.

PubMed

Singh, Ankita; Kaushik, Rahul; Mishra, Avinash; Shanker, Asheesh; Jayaram, B

2016-01-01

Quality assessment of predicted model structures of proteins is as important as the protein tertiary structure prediction. A highly efficient quality assessment of predicted model structures directs further research on function. Here we present a new server ProTSAV, capable of evaluating predicted model structures based on some popular online servers and standalone tools. ProTSAV furnishes the user with a single quality score in case of individual protein structure along with a graphical representation and ranking in case of multiple protein structure assessment. The server is validated on ~64,446 protein structures including experimental structures from RCSB and predicted model structures for CASP targets and from public decoy sets. ProTSAV succeeds in predicting quality of protein structures with a specificity of 100% and a sensitivity of 98% on experimentally solved structures and achieves a specificity of 88%and a sensitivity of 91% on predicted protein structures of CASP11 targets under 2Å.The server overcomes the limitations of any single server/method and is seen to be robust in helping in quality assessment. ProTSAV is freely available at http://www.scfbio-iitd.res.in/software/proteomics/protsav.jsp. Copyright © 2015 Elsevier B.V. All rights reserved.

Recent developments in structural proteomics for protein structure determination.

PubMed

Liu, Hsuan-Liang; Hsu, Jyh-Ping

2005-05-01

The major challenges in structural proteomics include identifying all the proteins on the genome-wide scale, determining their structure-function relationships, and outlining the precise three-dimensional structures of the proteins. Protein structures are typically determined by experimental approaches such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. However, the knowledge of three-dimensional space by these techniques is still limited. Thus, computational methods such as comparative and de novo approaches and molecular dynamic simulations are intensively used as alternative tools to predict the three-dimensional structures and dynamic behavior of proteins. This review summarizes recent developments in structural proteomics for protein structure determination; including instrumental methods such as X-ray crystallography and NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulations.

Modularity in protein structures: study on all-alpha proteins.

PubMed

Khan, Taushif; Ghosh, Indira

2015-01-01

Modularity is known as one of the most important features of protein's robust and efficient design. The architecture and topology of proteins play a vital role by providing necessary robust scaffolds to support organism's growth and survival in constant evolutionary pressure. These complex biomolecules can be represented by several layers of modular architecture, but it is pivotal to understand and explore the smallest biologically relevant structural component. In the present study, we have developed a component-based method, using protein's secondary structures and their arrangements (i.e. patterns) in order to investigate its structural space. Our result on all-alpha protein shows that the known structural space is highly populated with limited set of structural patterns. We have also noticed that these frequently observed structural patterns are present as modules or "building blocks" in large proteins (i.e. higher secondary structure content). From structural descriptor analysis, observed patterns are found to be within similar deviation; however, frequent patterns are found to be distinctly occurring in diverse functions e.g. in enzymatic classes and reactions. In this study, we are introducing a simple approach to explore protein structural space using combinatorial- and graph-based geometry methods, which can be used to describe modularity in protein structures. Moreover, analysis indicates that protein function seems to be the driving force that shapes the known structure space.

Templating Biomineralization: Surface Directed Protein Self-assembly and External Magnetic Field Stimulation of Osteoblasts

NASA Astrophysics Data System (ADS)

Ba, Xiaolan

Biomineralization is a wide-spread phenomenon in the biological systems, which is the process of mineral formation by organisms through interaction between its organic contents and the inorganic minerals. The process is essential in a broad spectrum of biological phenomena ranging from bone and tooth formation to pathological mineralization under hypoxic conditions or cancerous formations. In this thesis I studied biomineralization at the earliest stages in order to obtain a better understanding of the fundamental principals involved. This knowledge is essential if we want to engineer devices which will increase bone regeneration or prevent unwanted mineral deposits. Extracellular matrix (ECM) proteins play an essential role during biomineralization in bone and engineered tissues. In this dissertation, I present an approach to mimic the ECM in vitro to probe the interactions of these proteins with calcium phosphate mineral and with each other. Early stage of mineralization is investigated by mechanical properties of the protein fibers using Scanning Probe Microscopy (SPM) and Shear Modulation Force Microscopy (SMFM). The development of mineral crystals on the protein matrices is also characterized by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Grazing Incidence X-ray Diffraction (GIXRD). The results demonstrate complementary actions of the two ECM proteins to collect cations and template calcium phosphate mineral, respectively. Magnets have been clinically used as an "induction source" in various bone or orthodontic treatments. However, the mechanism and effects of magnetic fields remain unclear. In this dissertation, I also undertake the present investigation to study the effects of 150 mT static magnetic fields (SMF) on ECM development and cell biomineralization using MC3T3-E1 osteobalst-like cells. Early stage of biomineralization is characterized by SPM, SMFM and confocal laser scanning microscopy (CSLM). Late stage of biomineralization is investigated by SEM, GIXRD and energy dispersive X-ray spectroscopy (EDXS). Gene expression during the exposure of SMF is also studies by RT-PCR. The results indicated that exposure to SMF induces osteoblasts to produce larger quantities of HA, with higher degree of crystalline order. The controlling and understanding of protein on the surface is of great interest in biomedical application such as implant medicine, biosensor design, food processing, and chromatographic separations. The adsorbed protein onto the surface significantly determines the performance of biomaterials in a biological environment. Recent studies have suggested that the preservation of the native secondary structure of protein adsorbed is essential for biological application. In order to manipulate protein adsorption and design biocompatible materials, the mechanisms underlying protein-surface interactions, especially how surface properties of materials induce conformational changes of adsorbed proteins, needs to be well understood. Here we demonstrated that even though SPS is a necessary condition, it is not sufficient. We show that low substrate conductivity as well as proper salt concentration are also critical in sustained protein adsorption continuously. These factors allow one to pattern regions of different conducting properties and for the first time patterns physiologically relevant protein structures. Here we show that we can achieve patterned biomineralized regimes, both with plasma proteins in a simple and robust manner without additional functionalization or application of electrochemical gradients. Since the data indicate that the patterns just need to differ in electrical conductivity, rather than surface chemistry, we propose that the creation of transient image charges, due to incomplete charge screening, may be responsible for sustain the driving force for continual protein absorption.

PS2007 Satellite Meeting on Photosynthetic Antennas, 19-22 July 2007, Drymen, Scotland

DOE Office of Scientific and Technical Information (OSTI.GOV)

Robert E. Blankenship

2009-06-04

A Satellite Workshop of the 14th International Congress on Photosynthesis on the topic of photosynthetic light-harvesting systems was held on 18-21 July 2007, at the Buchanan Arms Hotel in Drymen, Scotland, near Glasgow. This meeting continued the tradition of satellite light-harvesting conferences occurring prior to the last five international photosynthesis congresses in Japan, France, Hungary, Australia and Canada, dating from 1992. With an attendance of 124 participants, this Workshop represents an intimate gathering of scientists interested in a thorough coverage of the light-harvesting aspects of photosynthesis. A significant amount of time was set aside for discussion and poster sessions. Themore » organizers were: Richard J. Cogdell, UK (Chairperson), Alastair T. Gardiner, UK, Conrad W. Mullineaux, UK, Robert A. Niederman, USA, Robert E. Blankenship, USA, Harry Frank, USA, Bruno Robert, France. Sessions were focused on new concepts relating to the function, regulation, assembly, photoprotection and evolution of a wide variety of antenna systems. Cutting-edge scientific methods used to study these systems that were covered included time-resolved and single-molecule spectroscopy, structure determination by X-ray diffraction, NMR and electron and atomic force microscopy, molecular genetics, protein chemistry, model systems and theory. A central theme was how emerging high-resolution structural information on antenna proteins continues to provide an enhanced understanding of areas ranging from the kinetics of energy transfer processes to the assembly of the photosynthetic apparatus.« less

Differential permeation of artemia cysts and cucumber seeds by alcohols

NASA Technical Reports Server (NTRS)

Smith, C. W.; Siegel, S. M.

1975-01-01

The rate of penetration of the simpler alcohols into brine shrimp cysts and cucumber seeds was studied. In solutions below 70% the rate of penetration is related to lipid solvent capacity of the alcohol. In concentrations above 70%, particularly in absolute alcohols, methanol penetrates brine shrimp rapidly and ethanol penetrates slowly. All the other alcohols tested did not penetrate the dormant structures. Ethionine and deuteroxy-methanol did not affect the rate of penetration of methanol. It is suggested that in dehydrated membranes the lipid moiety is protected by a continuous sheet of protein. Methanol, which is fairly similar to water, is probably able to penetrate the membrane by initiating a conformation change in the protein, exposing the lipid which subsequently dissolves in the methanol thus destroying the membrane.

Coarse-grained molecular dynamics simulations for giant protein-DNA complexes

NASA Astrophysics Data System (ADS)

Takada, Shoji

Biomolecules are highly hierarchic and intrinsically flexible. Thus, computational modeling calls for multi-scale methodologies. We have been developing a coarse-grained biomolecular model where on-average 10-20 atoms are grouped into one coarse-grained (CG) particle. Interactions among CG particles are tuned based on atomistic interactions and the fluctuation matching algorithm. CG molecular dynamics methods enable us to simulate much longer time scale motions of much larger molecular systems than fully atomistic models. After broad sampling of structures with CG models, we can easily reconstruct atomistic models, from which one can continue conventional molecular dynamics simulations if desired. Here, we describe our CG modeling methodology for protein-DNA complexes, together with various biological applications, such as the DNA duplication initiation complex, model chromatins, and transcription factor dynamics on chromatin-like environment.

Analyzing Intracellular Binding and Diffusion with Continuous Fluorescence Photobleaching

PubMed Central

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W.; Knoch, Tobias A.; Waldeck, Waldemar; Langowski, Jörg

2003-01-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of ∼13 s. PMID:12719264

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

PubMed

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W; Knoch, Tobias A; Waldeck, Waldemar; Langowski, Jörg

2003-05-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

Domain fusion analysis by applying relational algebra to protein sequence and domain databases.

PubMed

Truong, Kevin; Ikura, Mitsuhiko

2003-05-06

Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at http://calcium.uhnres.utoronto.ca/pi. As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time.

The effect of enamel proteins on erosion

NASA Astrophysics Data System (ADS)

Baumann, T.; Carvalho, T. S.; Lussi, A.

2015-10-01

Enamel proteins form a scaffold for growing hydroxyapatite crystals during enamel formation. They are then almost completely degraded during enamel maturation, resulting in a protein content of only 1% (w/v) in mature enamel. Nevertheless, this small amount of remaining proteins has important effects on the mechanical and structural properties of enamel and on the electrostatic properties of its surface. To analyze how enamel proteins affect tooth erosion, human enamel specimens were deproteinated. Surface microhardness (SMH), surface reflection intensity (SRI) and calcium release of both deproteinated and control specimens were monitored while continuously eroding them. The deproteination itself already reduced the initial SMH and SRI of the enamel significantly (p < 0.001 and p < 0.01). During the course of erosion, the progression of all three evaluated parameters differed significantly between the two groups (p < 0.001 for each). The deproteinated enamel lost its SMH and SRI faster, and released more calcium than the control group, but these differences were only significant at later stages of erosion, where not only surface softening but surface loss can be observed. We conclude that enamel proteins have a significant effect on erosion, protecting the enamel and slowing down the progression of erosion when irreversible surface loss starts to occur.

The effect of enamel proteins on erosion

PubMed Central

Baumann, T.; Carvalho, T. S.; Lussi, A.

2015-01-01

Enamel proteins form a scaffold for growing hydroxyapatite crystals during enamel formation. They are then almost completely degraded during enamel maturation, resulting in a protein content of only 1% (w/v) in mature enamel. Nevertheless, this small amount of remaining proteins has important effects on the mechanical and structural properties of enamel and on the electrostatic properties of its surface. To analyze how enamel proteins affect tooth erosion, human enamel specimens were deproteinated. Surface microhardness (SMH), surface reflection intensity (SRI) and calcium release of both deproteinated and control specimens were monitored while continuously eroding them. The deproteination itself already reduced the initial SMH and SRI of the enamel significantly (p < 0.001 and p < 0.01). During the course of erosion, the progression of all three evaluated parameters differed significantly between the two groups (p < 0.001 for each). The deproteinated enamel lost its SMH and SRI faster, and released more calcium than the control group, but these differences were only significant at later stages of erosion, where not only surface softening but surface loss can be observed. We conclude that enamel proteins have a significant effect on erosion, protecting the enamel and slowing down the progression of erosion when irreversible surface loss starts to occur. PMID:26468660

Three-dimensional reconstruction of the size and shape of protein microcrystals using Bragg coherent diffractive imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coughlan, H. D.; Darmanin, C.; Kirkwood, H. J.

2016-03-14

Three-dimensional imaging of protein crystals during X-ray diffraction experiments opens up a range of possibilities for optimising crystal quality and gaining new insights into the fundamental processes that drive radiation damage. Obtaining this information at the appropriate lengthscales however is extremely challenging. One approach that has been recently demonstrated as a promising avenue for charactering the size and shape of protein crystals at nanometre lengthscales is Bragg Coherent Diffractive Imaging (BCDI). BCDI is a recently developed technique that is able to recover the phase of the continuous diffraction intensity signal around individual Bragg peaks. When data is collected at multiplemore » points on a rocking curve a Reciprocal Space Map (RSM) can be assembled and then inverted using BCDI to obtain a three-dimensional image of the crystal. The first demonstration of two-dimensional BCDI of protein crystals was reported by Boutet at al., recently this work was extended to the study of radiation damage of micron-sized crystals. Here we present the first three-dimensional reconstructions of a Lysozyme protein crystal using BDI. The results are validated against RSM and TEM data and have implications for both radiation damage studies and for developing new approaches to structure retrieval from micron-sized protein crystals.« less

An approach to large scale identification of non-obvious structural similarities between proteins

PubMed Central

Cherkasov, Artem; Jones, Steven JM

2004-01-01

Background A new sequence independent bioinformatics approach allowing genome-wide search for proteins with similar three dimensional structures has been developed. By utilizing the numerical output of the sequence threading it establishes putative non-obvious structural similarities between proteins. When applied to the testing set of proteins with known three dimensional structures the developed approach was able to recognize structurally similar proteins with high accuracy. Results The method has been developed to identify pathogenic proteins with low sequence identity and high structural similarity to host analogues. Such protein structure relationships would be hypothesized to arise through convergent evolution or through ancient horizontal gene transfer events, now undetectable using current sequence alignment techniques. The pathogen proteins, which could mimic or interfere with host activities, would represent candidate virulence factors. The developed approach utilizes the numerical outputs from the sequence-structure threading. It identifies the potential structural similarity between a pair of proteins by correlating the threading scores of the corresponding two primary sequences against the library of the standard folds. This approach allowed up to 64% sensitivity and 99.9% specificity in distinguishing protein pairs with high structural similarity. Conclusion Preliminary results obtained by comparison of the genomes of Homo sapiens and several strains of Chlamydia trachomatis have demonstrated the potential usefulness of the method in the identification of bacterial proteins with known or potential roles in virulence. PMID:15147578

Structure and function of seed storage proteins in faba bean (Vicia faba L.).

PubMed

Liu, Yujiao; Wu, Xuexia; Hou, Wanwei; Li, Ping; Sha, Weichao; Tian, Yingying

2017-05-01

The protein subunit is the most important basic unit of protein, and its study can unravel the structure and function of seed storage proteins in faba bean. In this study, we identified six specific protein subunits in Faba bean (cv. Qinghai 13) combining liquid chromatography (LC), liquid chromatography-electronic spray ionization mass (LC-ESI-MS/MS) and bio-information technology. The results suggested a diversity of seed storage proteins in faba bean, and a total of 16 proteins (four GroEL molecular chaperones and 12 plant-specific proteins) were identified from 97-, 96-, 64-, 47-, 42-, and 38-kD-specific protein subunits in faba bean based on the peptide sequence. We also analyzed the composition and abundance of the amino acids, the physicochemical characteristics, secondary structure, three-dimensional structure, transmembrane domain, and possible subcellular localization of these identified proteins in faba bean seed, and finally predicted function and structure. The three-dimensional structures were generated based on homologous modeling, and the protein function was analyzed based on the annotation from the non-redundant protein database (NR database, NCBI) and function analysis of optimal modeling. The objective of this study was to identify the seed storage proteins in faba bean and confirm the structure and function of these proteins. Our results can be useful for the study of protein nutrition and achieve breeding goals for optimal protein quality in faba bean.

Trends in structural coverage of the protein universe and the impact of the Protein Structure Initiative.

PubMed

Khafizov, Kamil; Madrid-Aliste, Carlos; Almo, Steven C; Fiser, Andras

2014-03-11

The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins--including proteins for which reliable homology models can be generated--on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long.

Rift Valley fever virus structural and non-structural proteins: Recombinant protein expression and immunoreactivity against antisera from sheep

USDA-ARS?s Scientific Manuscript database

The Rift Valley fever virus (RVFV) encodes structural proteins, nucleoprotein (N), N-terminus glycoprotein (Gn), C-terminus glycoprotein (Gc) and L protein, 78-kDa and non-structural proteins NSm and NSs. Using the baculovirus system we expressed the full-length coding sequence of N, NSs, NSm, Gc an...

Adjusting protein graphs based on graph entropy.

PubMed

Peng, Sheng-Lung; Tsay, Yu-Wei

2014-01-01

Measuring protein structural similarity attempts to establish a relationship of equivalence between polymer structures based on their conformations. In several recent studies, researchers have explored protein-graph remodeling, instead of looking a minimum superimposition for pairwise proteins. When graphs are used to represent structured objects, the problem of measuring object similarity become one of computing the similarity between graphs. Graph theory provides an alternative perspective as well as efficiency. Once a protein graph has been created, its structural stability must be verified. Therefore, a criterion is needed to determine if a protein graph can be used for structural comparison. In this paper, we propose a measurement for protein graph remodeling based on graph entropy. We extend the concept of graph entropy to determine whether a graph is suitable for representing a protein. The experimental results suggest that when applied, graph entropy helps a conformational on protein graph modeling. Furthermore, it indirectly contributes to protein structural comparison if a protein graph is solid.

Adjusting protein graphs based on graph entropy

PubMed Central

2014-01-01

Measuring protein structural similarity attempts to establish a relationship of equivalence between polymer structures based on their conformations. In several recent studies, researchers have explored protein-graph remodeling, instead of looking a minimum superimposition for pairwise proteins. When graphs are used to represent structured objects, the problem of measuring object similarity become one of computing the similarity between graphs. Graph theory provides an alternative perspective as well as efficiency. Once a protein graph has been created, its structural stability must be verified. Therefore, a criterion is needed to determine if a protein graph can be used for structural comparison. In this paper, we propose a measurement for protein graph remodeling based on graph entropy. We extend the concept of graph entropy to determine whether a graph is suitable for representing a protein. The experimental results suggest that when applied, graph entropy helps a conformational on protein graph modeling. Furthermore, it indirectly contributes to protein structural comparison if a protein graph is solid. PMID:25474347

Hepatic immunohistochemical localization of the tight junction protein ZO-1 in rat models of cholestasis.

PubMed Central

Anderson, J. M.; Glade, J. L.; Stevenson, B. R.; Boyer, J. L.; Mooseker, M. S.

1989-01-01

Structural alterations in hepatocyte tight junctions accompanying cholestasis were investigated using immunolocalization of ZO-1, the first known protein component of the tight junction. Disruption in the paracellular barrier function of the tight junction has been proposed to allow reflux of bile into the blood. Cholestasis was induced in 210 to 235 g male Sprague-Dawley rats either by five consecutive daily subcutaneous injections of 17-alpha-ethinyl estradiol (0.5 mg/kg/d in propylene glycol) or ligation of the common bile duct for 72 hours. The structural organization of the tight junction was assessed in each model by indirect immunofluorescent and immunoperoxidase staining for ZO-1 on frozen sections of liver and compared with controls. In control, sham-operated, and estradiol-injected animals, ZO-1 localizes in a uniform continuous manner along the margins of the canaliculi. In contrast, bile duct ligation results in the appearance of numerous discontinuities in ZO-1 staining accompanied by dilation or collapse of the lumenal space. Tissue content of the ZO-1 protein, as determined by quantitative immunoblotting, was unaffected in either cholestatic model compared with controls. These findings indicate that the molecular organization of the tight junction can be assessed from immunostaining patterns of ZO-1 in frozen sections of cholestatic livers. Under these experimental conditions, the organization of the tight junction at the level of the ZO-1 protein is altered by bile duct obstruction but not by ethinyl estradiol. Images Figure 1 Figure 2 PMID:2719075

Structural deformation upon protein-protein interaction: A structural alphabet approach

PubMed Central

Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

2008-01-01

Background In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. Results In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Conclusion Our study provides qualitative information about induced fit. These results could be of help for flexible docking. PMID:18307769

Structural deformation upon protein-protein interaction: a structural alphabet approach.

PubMed

Martin, Juliette; Regad, Leslie; Lecornet, Hélène; Camproux, Anne-Claude

2008-02-28

In a number of protein-protein complexes, the 3D structures of bound and unbound partners significantly differ, supporting the induced fit hypothesis for protein-protein binding. In this study, we explore the induced fit modifications on a set of 124 proteins available in both bound and unbound forms, in terms of local structure. The local structure is described thanks to a structural alphabet of 27 structural letters that allows a detailed description of the backbone. Using a control set to distinguish induced fit from experimental error and natural protein flexibility, we show that the fraction of structural letters modified upon binding is significantly greater than in the control set (36% versus 28%). This proportion is even greater in the interface regions (41%). Interface regions preferentially involve coils. Our analysis further reveals that some structural letters in coil are not favored in the interface. We show that certain structural letters in coil are particularly subject to modifications at the interface, and that the severity of structural change also varies. These information are used to derive a structural letter substitution matrix that summarizes the local structural changes observed in our data set. We also illustrate the usefulness of our approach to identify common binding motifs in unrelated proteins. Our study provides qualitative information about induced fit. These results could be of help for flexible docking.

Stepwise Evolution of Coral Biomineralization Revealed with Genome-Wide Proteomics and Transcriptomics

PubMed Central

Sawada, Hitoshi; Satoh, Noriyuki

2016-01-01

Despite the importance of stony corals in many research fields related to global issues, such as marine ecology, climate change, paleoclimatogy, and metazoan evolution, very little is known about the evolutionary origin of coral skeleton formation. In order to investigate the evolution of coral biomineralization, we have identified skeletal organic matrix proteins (SOMPs) in the skeletal proteome of the scleractinian coral, Acropora digitifera, for which large genomic and transcriptomic datasets are available. Scrupulous gene annotation was conducted based on comparisons of functional domain structures among metazoans. We found that SOMPs include not only coral-specific proteins, but also protein families that are widely conserved among cnidarians and other metazoans. We also identified several conserved transmembrane proteins in the skeletal proteome. Gene expression analysis revealed that expression of these conserved genes continues throughout development. Therefore, these genes are involved not only skeleton formation, but also in basic cellular functions, such as cell-cell interaction and signaling. On the other hand, genes encoding coral-specific proteins, including extracellular matrix domain-containing proteins, galaxins, and acidic proteins, were prominently expressed in post-settlement stages, indicating their role in skeleton formation. Taken together, the process of coral skeleton formation is hypothesized as: 1) formation of initial extracellular matrix between epithelial cells and substrate, employing pre-existing transmembrane proteins; 2) additional extracellular matrix formation using novel proteins that have emerged by domain shuffling and rapid molecular evolution and; 3) calcification controlled by coral-specific SOMPs. PMID:27253604

Continuous desalting of refolded protein solution improves capturing in ion exchange chromatography: A seamless process.

PubMed

Walch, Nicole; Jungbauer, Alois

2017-06-01

Truly continuous biomanufacturing processes enable an uninterrupted feed stream throughout the whole production without the need for holding tanks. We have utilized microporous anion and cation exchangers into which only salts, but not proteins, can penetrate into the pores for desalting of protein solutions, while diafiltration or dilution is usually employed for feed adjustments. Anion exchange and cation exchange chromatography columns were connected in series to remove both anions and cations. To increase operation performance, a continuous process was developed comprised of four columns. Continuous mode was achieved by staggered cycle operation, where one set of columns, consisting of one anion exchange and one cation exchange column, was loaded during the regeneration of the second set. Refolding, desalting and subsequent ion exchange capturing with a scFv as the model protein was demonstrated. The refolding solution was successfully desalted resulting in a consistent conductivity below 0.5 mS/cm from initial values of 10 to 11 mS/cm. With continuous operation process time could be reduced by 39% while productivity was increased to 163% compared to batch operation. Desalting of the protein solution resulted in up to 7-fold higher binding capacities in the subsequent ion exchange capture step with conventional protein binding resins. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Life in the fast lane for protein crystallization and X-ray crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2005-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high-rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today's high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from the efforts of the Southeast Collaboratory for Structural Genomics (SECSG).

Life in the Fast Lane for Protein Crystallization and X-Ray Crystallography

NASA Technical Reports Server (NTRS)

Pusey, Marc L.; Liu, Zhi-Jie; Tempel, Wolfram; Praissman, Jeremy; Lin, Dawei; Wang, Bi-Cheng; Gavira, Jose A.; Ng, Joseph D.

2004-01-01

The common goal for structural genomic centers and consortiums is to decipher as quickly as possible the three-dimensional structures for a multitude of recombinant proteins derived from known genomic sequences. Since X-ray crystallography is the foremost method to acquire atomic resolution for macromolecules, the limiting step is obtaining protein crystals that can be useful of structure determination. High-throughput methods have been developed in recent years to clone, express, purify, crystallize and determine the three-dimensional structure of a protein gene product rapidly using automated devices, commercialized kits and consolidated protocols. However, the average number of protein structures obtained for most structural genomic groups has been very low compared to the total number of proteins purified. As more entire genomic sequences are obtained for different organisms from the three kingdoms of life, only the proteins that can be crystallized and whose structures can be obtained easily are studied. Consequently, an astonishing number of genomic proteins remain unexamined. In the era of high-throughput processes, traditional methods in molecular biology, protein chemistry and crystallization are eclipsed by automation and pipeline practices. The necessity for high rate production of protein crystals and structures has prevented the usage of more intellectual strategies and creative approaches in experimental executions. Fundamental principles and personal experiences in protein chemistry and crystallization are minimally exploited only to obtain "low-hanging fruit" protein structures. We review the practical aspects of today s high-throughput manipulations and discuss the challenges in fast pace protein crystallization and tools for crystallography. Structural genomic pipelines can be improved with information gained from low-throughput tactics that may help us reach the higher-bearing fruits. Examples of recent developments in this area are reported from the efforts of the Southeast Collaboratory for Structural Genomics (SECSG).

Use of a robot for high-throughput crystallization of membrane proteins in lipidic mesophases.

PubMed

Li, Dianfan; Boland, Coilín; Walsh, Kilian; Caffrey, Martin

2012-09-01

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural & Functional Biology (MS&FB) Group(1-3). JoVE video articles describing the method are available(1,4). The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&FB Group(5,6). The other two have recently become available and are included here for completeness. An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 °C) under ambient conditions.

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

An ambiguity principle for assigning protein structural domains

PubMed Central

Postic, Guillaume; Ghouzam, Yassine; Chebrek, Romain; Gelly, Jean-Christophe

2017-01-01

Ambiguity is the quality of being open to several interpretations. For an image, it arises when the contained elements can be delimited in two or more distinct ways, which may cause confusion. We postulate that it also applies to the analysis of protein three-dimensional structure, which consists in dividing the molecule into subunits called domains. Because different definitions of what constitutes a domain can be used to partition a given structure, the same protein may have different but equally valid domain annotations. However, knowledge and experience generally displace our ability to accept more than one way to decompose the structure of an object—in this case, a protein. This human bias in structure analysis is particularly harmful because it leads to ignoring potential avenues of research. We present an automated method capable of producing multiple alternative decompositions of protein structure (web server and source code available at www.dsimb.inserm.fr/sword/). Our innovative algorithm assigns structural domains through the hierarchical merging of protein units, which are evolutionarily preserved substructures that describe protein architecture at an intermediate level, between domain and secondary structure. To validate the use of these protein units for decomposing protein structures into domains, we set up an extensive benchmark made of expert annotations of structural domains and including state-of-the-art domain parsing algorithms. The relevance of our “multipartitioning” approach is shown through numerous examples of applications covering protein function, evolution, folding, and structure prediction. Finally, we introduce a measure for the structural ambiguity of protein molecules. PMID:28097215

Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy.

PubMed

Huang, Xiaoshuai; Fan, Junchao; Li, Liuju; Liu, Haosen; Wu, Runlong; Wu, Yi; Wei, Lisi; Mao, Heng; Lal, Amit; Xi, Peng; Tang, Liqiang; Zhang, Yunfeng; Liu, Yanmei; Tan, Shan; Chen, Liangyi

2018-06-01

To increase the temporal resolution and maximal imaging time of super-resolution (SR) microscopy, we have developed a deconvolution algorithm for structured illumination microscopy based on Hessian matrixes (Hessian-SIM). It uses the continuity of biological structures in multiple dimensions as a priori knowledge to guide image reconstruction and attains artifact-minimized SR images with less than 10% of the photon dose used by conventional SIM while substantially outperforming current algorithms at low signal intensities. Hessian-SIM enables rapid imaging of moving vesicles or loops in the endoplasmic reticulum without motion artifacts and with a spatiotemporal resolution of 88 nm and 188 Hz. Its high sensitivity allows the use of sub-millisecond excitation pulses followed by dark recovery times to reduce photobleaching of fluorescent proteins, enabling hour-long time-lapse SR imaging of actin filaments in live cells. Finally, we observed the structural dynamics of mitochondrial cristae and structures that, to our knowledge, have not been observed previously, such as enlarged fusion pores during vesicle exocytosis.

Unraveling the meaning of chemical shifts in protein NMR.

PubMed

Berjanskii, Mark V; Wishart, David S

2017-11-01

Chemical shifts are among the most informative parameters in protein NMR. They provide wealth of information about protein secondary and tertiary structure, protein flexibility, and protein-ligand binding. In this report, we review the progress in interpreting and utilizing protein chemical shifts that has occurred over the past 25years, with a particular focus on the large body of work arising from our group and other Canadian NMR laboratories. More specifically, this review focuses on describing, assessing, and providing some historical context for various chemical shift-based methods to: (1) determine protein secondary and super-secondary structure; (2) derive protein torsion angles; (3) assess protein flexibility; (4) predict residue accessible surface area; (5) refine 3D protein structures; (6) determine 3D protein structures and (7) characterize intrinsically disordered proteins. This review also briefly covers some of the methods that we previously developed to predict chemical shifts from 3D protein structures and/or protein sequence data. It is hoped that this review will help to increase awareness of the considerable utility of NMR chemical shifts in structural biology and facilitate more widespread adoption of chemical-shift based methods by the NMR spectroscopists, structural biologists, protein biophysicists, and biochemists worldwide. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman. Copyright © 2017 Elsevier B.V. All rights reserved.

Computational Prediction of Protein-Protein Interactions

PubMed Central

Ehrenberger, Tobias; Cantley, Lewis C.; Yaffe, Michael B.

2015-01-01

The prediction of protein-protein interactions and kinase-specific phosphorylation sites on individual proteins is critical for correctly placing proteins within signaling pathways and networks. The importance of this type of annotation continues to increase with the continued explosion of genomic and proteomic data, particularly with emerging data categorizing posttranslational modifications on a large scale. A variety of computational tools are available for this purpose. In this chapter, we review the general methodologies for these types of computational predictions and present a detailed user-focused tutorial of one such method and computational tool, Scansite, which is freely available to the entire scientific community over the Internet. PMID:25859943

Efficient protein structure search using indexing methods

PubMed Central

2013-01-01

Understanding functions of proteins is one of the most important challenges in many studies of biological processes. The function of a protein can be predicted by analyzing the functions of structurally similar proteins, thus finding structurally similar proteins accurately and efficiently from a large set of proteins is crucial. A protein structure can be represented as a vector by 3D-Zernike Descriptor (3DZD) which compactly represents the surface shape of the protein tertiary structure. This simplified representation accelerates the searching process. However, computing the similarity of two protein structures is still computationally expensive, thus it is hard to efficiently process many simultaneous requests of structurally similar protein search. This paper proposes indexing techniques which substantially reduce the search time to find structurally similar proteins. In particular, we first exploit two indexing techniques, i.e., iDistance and iKernel, on the 3DZDs. After that, we extend the techniques to further improve the search speed for protein structures. The extended indexing techniques build and utilize an reduced index constructed from the first few attributes of 3DZDs of protein structures. To retrieve top-k similar structures, top-10 × k similar structures are first found using the reduced index, and top-k structures are selected among them. We also modify the indexing techniques to support θ-based nearest neighbor search, which returns data points less than θ to the query point. The results show that both iDistance and iKernel significantly enhance the searching speed. In top-k nearest neighbor search, the searching time is reduced 69.6%, 77%, 77.4% and 87.9%, respectively using iDistance, iKernel, the extended iDistance, and the extended iKernel. In θ-based nearest neighbor serach, the searching time is reduced 80%, 81%, 95.6% and 95.6% using iDistance, iKernel, the extended iDistance, and the extended iKernel, respectively. PMID:23691543

Efficient protein structure search using indexing methods.

PubMed

Kim, Sungchul; Sael, Lee; Yu, Hwanjo

2013-01-01

Understanding functions of proteins is one of the most important challenges in many studies of biological processes. The function of a protein can be predicted by analyzing the functions of structurally similar proteins, thus finding structurally similar proteins accurately and efficiently from a large set of proteins is crucial. A protein structure can be represented as a vector by 3D-Zernike Descriptor (3DZD) which compactly represents the surface shape of the protein tertiary structure. This simplified representation accelerates the searching process. However, computing the similarity of two protein structures is still computationally expensive, thus it is hard to efficiently process many simultaneous requests of structurally similar protein search. This paper proposes indexing techniques which substantially reduce the search time to find structurally similar proteins. In particular, we first exploit two indexing techniques, i.e., iDistance and iKernel, on the 3DZDs. After that, we extend the techniques to further improve the search speed for protein structures. The extended indexing techniques build and utilize an reduced index constructed from the first few attributes of 3DZDs of protein structures. To retrieve top-k similar structures, top-10 × k similar structures are first found using the reduced index, and top-k structures are selected among them. We also modify the indexing techniques to support θ-based nearest neighbor search, which returns data points less than θ to the query point. The results show that both iDistance and iKernel significantly enhance the searching speed. In top-k nearest neighbor search, the searching time is reduced 69.6%, 77%, 77.4% and 87.9%, respectively using iDistance, iKernel, the extended iDistance, and the extended iKernel. In θ-based nearest neighbor serach, the searching time is reduced 80%, 81%, 95.6% and 95.6% using iDistance, iKernel, the extended iDistance, and the extended iKernel, respectively.

Structure-based barcoding of proteins.

PubMed

Metri, Rahul; Jerath, Gaurav; Kailas, Govind; Gacche, Nitin; Pal, Adityabarna; Ramakrishnan, Vibin

2014-01-01

A reduced representation in the format of a barcode has been developed to provide an overview of the topological nature of a given protein structure from 3D coordinate file. The molecular structure of a protein coordinate file from Protein Data Bank is first expressed in terms of an alpha-numero code and further converted to a barcode image. The barcode representation can be used to compare and contrast different proteins based on their structure. The utility of this method has been exemplified by comparing structural barcodes of proteins that belong to same fold family, and across different folds. In addition to this, we have attempted to provide an illustration to (i) the structural changes often seen in a given protein molecule upon interaction with ligands and (ii) Modifications in overall topology of a given protein during evolution. The program is fully downloadable from the website http://www.iitg.ac.in/probar/. © 2013 The Protein Society.

Shaping up the protein folding funnel by local interaction: lesson from a structure prediction study.

PubMed

Chikenji, George; Fujitsuka, Yoshimi; Takada, Shoji

2006-02-28

Predicting protein tertiary structure by folding-like simulations is one of the most stringent tests of how much we understand the principle of protein folding. Currently, the most successful method for folding-based structure prediction is the fragment assembly (FA) method. Here, we address why the FA method is so successful and its lesson for the folding problem. To do so, using the FA method, we designed a structure prediction test of "chimera proteins." In the chimera proteins, local structural preference is specific to the target sequences, whereas nonlocal interactions are only sequence-independent compaction forces. We find that these chimera proteins can find the native folds of the intact sequences with high probability indicating dominant roles of the local interactions. We further explore roles of local structural preference by exact calculation of the HP lattice model of proteins. From these results, we suggest principles of protein folding: For small proteins, compact structures that are fully compatible with local structural preference are few, one of which is the native fold. These local biases shape up the funnel-like energy landscape.

Shaping up the protein folding funnel by local interaction: Lesson from a structure prediction study

PubMed Central

Chikenji, George; Fujitsuka, Yoshimi; Takada, Shoji

2006-01-01

Predicting protein tertiary structure by folding-like simulations is one of the most stringent tests of how much we understand the principle of protein folding. Currently, the most successful method for folding-based structure prediction is the fragment assembly (FA) method. Here, we address why the FA method is so successful and its lesson for the folding problem. To do so, using the FA method, we designed a structure prediction test of “chimera proteins.” In the chimera proteins, local structural preference is specific to the target sequences, whereas nonlocal interactions are only sequence-independent compaction forces. We find that these chimera proteins can find the native folds of the intact sequences with high probability indicating dominant roles of the local interactions. We further explore roles of local structural preference by exact calculation of the HP lattice model of proteins. From these results, we suggest principles of protein folding: For small proteins, compact structures that are fully compatible with local structural preference are few, one of which is the native fold. These local biases shape up the funnel-like energy landscape. PMID:16488978

Penostatin derivatives, a novel kind of protein phosphatase 1b inhibitors isolated from solid cultures of the entomogenous fungus Isaria tenuipes.

PubMed

Chen, Yu-Peng; Yang, Chun-Gui; Wei, Pei-Yao; Li, Lin; Luo, Du-Qiang; Zheng, Zhi-Hui; Lu, Xin-Hua

2014-01-29

Protein tyrosine phosphatase 1B (PTP1B) is implicated as a negative regulator of insulin receptor (IR) signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. Therefore, small molecular inhibitors of PTP1B can be considered as an attractive approach for the design of new therapeutic agents of type II diabetes diseases. In a continuing search for new protein phosphatase inhibitors from fungi, we have isolated a new compound, named penostatin J (1), together with three known ones, penostatin C (2), penostatin A (3), and penostatin B (4), from cultures of the entomogenous fungus Isaria tenuipes. The structure of penostatin J (1) was elucidated by extensive spectroscopic analysis. We also demonstrate for the first time that penostatin derivatives exhibit the best PTP1B inhibitory action. These findings suggest that penostatin derivatives are a potential novel kind of PTP1B inhibitors.

Analysis and Chemistry of Novel Protein Oxidation Markers in Vivo.

PubMed

Henning, Christian; Liehr, Kristin; Girndt, Matthias; Ulrich, Christof; Glomb, Marcus A

2018-05-09

Proteins continually undergo spontaneous oxidation reactions, which lead to changes in structure and function. The quantitative assessment of protein oxidation adducts provides information on the level of exposure to reactive precursor compounds with a high oxidizing potential and reactive oxygen species (ROS). In the present work, we introduce N 6 -(2-hydroxyethyl)lysine as a novel marker based on the ratio of glycolaldehyde and its oxidized form glyoxal. The high analytical potential was proven with a first set of patients undergoing hemodialysis versus healthy controls, in comparison with well-established parameters for oxidative stress. In vitro experiments with N 1 - t-BOC-lysine and N 1 - t-BOC-arginine enlightened the mechanistic relationship of glycolaldehyde and glyoxal. Oxidation was strongly dependent on the catalytic action of the ε-amino moiety of lysine. Investigations on the formation of N 6 -carboxymethyl lysine revealed glycolaldehyde-imine as the more reactive precursor, even though an additional oxidative step is required. As a result, a novel and very effective alternative mechanism was unraveled.

Modeling complexes of modeled proteins.

PubMed

Anishchenko, Ivan; Kundrotas, Petras J; Vakser, Ilya A

2017-03-01

Structural characterization of proteins is essential for understanding life processes at the molecular level. However, only a fraction of known proteins have experimentally determined structures. This fraction is even smaller for protein-protein complexes. Thus, structural modeling of protein-protein interactions (docking) primarily has to rely on modeled structures of the individual proteins, which typically are less accurate than the experimentally determined ones. Such "double" modeling is the Grand Challenge of structural reconstruction of the interactome. Yet it remains so far largely untested in a systematic way. We present a comprehensive validation of template-based and free docking on a set of 165 complexes, where each protein model has six levels of structural accuracy, from 1 to 6 Å C α RMSD. Many template-based docking predictions fall into acceptable quality category, according to the CAPRI criteria, even for highly inaccurate proteins (5-6 Å RMSD), although the number of such models (and, consequently, the docking success rate) drops significantly for models with RMSD > 4 Å. The results show that the existing docking methodologies can be successfully applied to protein models with a broad range of structural accuracy, and the template-based docking is much less sensitive to inaccuracies of protein models than the free docking. Proteins 2017; 85:470-478. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Salvage of failed protein targets by reductive alkylation.

PubMed

Tan, Kemin; Kim, Youngchang; Hatzos-Skintges, Catherine; Chang, Changsoo; Cuff, Marianne; Chhor, Gekleng; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw; An, Hao; Babnigg, Gyorgy; Bigelow, Lance; Joachimiak, Grazyna; Li, Hui; Mack, Jamey; Makowska-Grzyska, Magdalena; Maltseva, Natalia; Mulligan, Rory; Tesar, Christine; Zhou, Min; Joachimiak, Andrzej

2014-01-01

The growth of diffraction-quality single crystals is of primary importance in protein X-ray crystallography. Chemical modification of proteins can alter their surface properties and crystallization behavior. The Midwest Center for Structural Genomics (MCSG) has previously reported how reductive methylation of lysine residues in proteins can improve crystallization of unique proteins that initially failed to produce diffraction-quality crystals. Recently, this approach has been expanded to include ethylation and isopropylation in the MCSG protein crystallization pipeline. Applying standard methods, 180 unique proteins were alkylated and screened using standard crystallization procedures. Crystal structures of 12 new proteins were determined, including the first ethylated and the first isopropylated protein structures. In a few cases, the structures of native and methylated or ethylated states were obtained and the impact of reductive alkylation of lysine residues was assessed. Reductive methylation tends to be more efficient and produces the most alkylated protein structures. Structures of methylated proteins typically have higher resolution limits. A number of well-ordered alkylated lysine residues have been identified, which make both intermolecular and intramolecular contacts. The previous report is updated and complemented with the following new data; a description of a detailed alkylation protocol with results, structural features, and roles of alkylated lysine residues in protein crystals. These contribute to improved crystallization properties of some proteins.