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

The Role of Proanthocyanidins Complex in Structure and Nutrition Interaction in Alfalfa Forage

PubMed Central

Jonker, Arjan; Yu, Peiqiang

2016-01-01

Alfalfa (Medicago sativa L.) is one of the main forages grown in the world. Alfalfa is a winter hardy, drought tolerant, N-fixing legume with a good longevity, high yield, high nutrient levels, high digestibility, unique structural to non-structural components ratio, high dry matter intake, and high animal productivity per hectare. However, its main limitation is its excessively rapid initial rate of protein degradation in the rumen, which results in pasture bloat and inefficient use of protein with consequent excessive excretions of nitrogen into the environment. Proanthocyanidins are secondary plant metabolites that can bind with protein and thereby reduce the rate and extent of ruminal protein degradation. However, these secondary metabolites do not accumulate in alfalfa. This review aims to firstly describe the events involved in the rapid release of protein from alfalfa and its effect on ruminant nutrition, environmental pollution, and pasture bloat; secondly, to describe occurrence, structure, functions and benefits of moderate amounts of proanthocyanidin; and finally, to describe the development of alfalfa which accumulates moderate amounts of proanthocyanidins. The emphasis of this review focuses on the role of proanthocyanidins compounds in structure and nutrition interaction in ruminant livestock systems. PMID:27223279

The Role of Proanthocyanidins Complex in Structure and Nutrition Interaction in Alfalfa Forage.

PubMed

Jonker, Arjan; Yu, Peiqiang

2016-05-23

Alfalfa (Medicago sativa L.) is one of the main forages grown in the world. Alfalfa is a winter hardy, drought tolerant, N-fixing legume with a good longevity, high yield, high nutrient levels, high digestibility, unique structural to non-structural components ratio, high dry matter intake, and high animal productivity per hectare. However, its main limitation is its excessively rapid initial rate of protein degradation in the rumen, which results in pasture bloat and inefficient use of protein with consequent excessive excretions of nitrogen into the environment. Proanthocyanidins are secondary plant metabolites that can bind with protein and thereby reduce the rate and extent of ruminal protein degradation. However, these secondary metabolites do not accumulate in alfalfa. This review aims to firstly describe the events involved in the rapid release of protein from alfalfa and its effect on ruminant nutrition, environmental pollution, and pasture bloat; secondly, to describe occurrence, structure, functions and benefits of moderate amounts of proanthocyanidin; and finally, to describe the development of alfalfa which accumulates moderate amounts of proanthocyanidins. The emphasis of this review focuses on the role of proanthocyanidins compounds in structure and nutrition interaction in ruminant livestock systems.

Herbaspirillum seropedicae signal transduction protein PII is structurally similar to the enteric GlnK.

PubMed

Machado Benelli, Elaine; Buck, Martin; Polikarpov, Igor; Maltempi de Souza, Emanuel; Cruz, Leonardo M; Pedrosa, Fábio O

2002-07-01

PII-like proteins are signal transduction proteins found in bacteria, archaea and eukaryotes. They mediate a variety of cellular responses. A second PII-like protein, called GlnK, has been found in several organisms. In the diazotroph Herbaspirillum seropedicae, PII protein is involved in sensing nitrogen levels and controlling nitrogen fixation genes. In this work, the crystal structure of the unliganded H. seropedicae PII was solved by X-ray diffraction. H. seropedicae PII has a Gly residue, Gly108 preceding Pro109 and the main-chain forms a beta turn. The glycine at position 108 allows a bend in the C-terminal main-chain, thereby modifying the surface of the cleft between monomers and potentially changing function. The structure suggests that the C-terminal region of PII proteins may be involved in specificity of function, and nonenteric diazotrophs are found to have the C-terminal consensus XGXDAX(107-112). We are also proposing binding sites for ATP and 2-oxoglutarate based on the structural alignment of PII with PII-ATP/GlnK-ATP, 5-carboxymethyl-2-hydroxymuconate isomerase and 4-oxalocrotonate tautomerase bound to the inhibitor 2-oxo-3-pentynoate.

Parmodel: a web server for automated comparative modeling of proteins.

PubMed

Uchôa, Hugo Brandão; Jorge, Guilherme Eberhart; Freitas Da Silveira, Nelson José; Camera, João Carlos; Canduri, Fernanda; De Azevedo, Walter Filgueira

2004-12-24

Parmodel is a web server for automated comparative modeling and evaluation of protein structures. The aim of this tool is to help inexperienced users to perform modeling, assessment, visualization, and optimization of protein models as well as crystallographers to evaluate structures solved experimentally. It is subdivided in four modules: Parmodel Modeling, Parmodel Assessment, Parmodel Visualization, and Parmodel Optimization. The main module is the Parmodel Modeling that allows the building of several models for a same protein in a reduced time, through the distribution of modeling processes on a Beowulf cluster. Parmodel automates and integrates the main softwares used in comparative modeling as MODELLER, Whatcheck, Procheck, Raster3D, Molscript, and Gromacs. This web server is freely accessible at .

From gene to structure: The protein factory of the NBICS Centre of Kurchatov Institute

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

Boyko, K. M.; Lipkin, A. V.; Popov, V. O., E-mail: vpopov@inbi.ras.ru

2013-05-15

The Protein Factory was established at the Centre for Nano, Bio, Info, Cognitive, and Social Sciences and Technologies (NBICS Centre) of the National Research Centre 'Kurchatov Institute' in 2010. The Protein Factory, together with the Centre for Synchrotron Radiation and Nanotechnology, promote research on structural biology. This paper presents the technology platforms developed at the Protein Factory and the facilities available for researchers. The main projects currently being performed at the Protein Factory are briefly described.

Improvement on a simplified model for protein folding simulation.

PubMed

Zhang, Ming; Chen, Changjun; He, Yi; Xiao, Yi

2005-11-01

Improvements were made on a simplified protein model--the Ramachandran model-to achieve better computer simulation of protein folding. To check the validity of such improvements, we chose the ultrafast folding protein Engrailed Homeodomain as an example and explored several aspects of its folding. The engrailed homeodomain is a mainly alpha-helical protein of 61 residues from Drosophila melanogaster. We found that the simplified model of Engrailed Homeodomain can fold into a global minimum state with a tertiary structure in good agreement with its native structure.

Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients

PubMed Central

Aw-Yong, Kam Leng; Sam, I-Ching; Koh, Mia Tuang

2016-01-01

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142–156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41–55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates. PMID:27806091

Beyond Membrane Protein Structure: Drug Discovery, Dynamics and Difficulties.

PubMed

Biggin, Philip C; Aldeghi, Matteo; Bodkin, Michael J; Heifetz, Alexander

2016-01-01

Most of the previous content of this book has focused on obtaining the structures of membrane proteins. In this chapter we explore how those structures can be further used in two key ways. The first is their use in structure based drug design (SBDD) and the second is how they can be used to extend our understanding of their functional activity via the use of molecular dynamics. Both aspects now heavily rely on computations. This area is vast, and alas, too large to consider in depth in a single book chapter. Thus where appropriate we have referred the reader to recent reviews for deeper assessment of the field. We discuss progress via the use of examples from two main drug target areas; G-protein coupled receptors (GPCRs) and ion channels. We end with a discussion of some of the main challenges in the area.

Proteins as sponges: a statistical journey along protein structure organization principles.

PubMed

Paola, Luisa Di; Paci, Paola; Santoni, Daniele; Ruvo, Micol De; Giuliani, Alessandro

2012-02-27

The analysis of a large database of protein structures by means of topological and shape indexes inspired by complex network and fractal analysis shed light on some organizational principles of proteins. Proteins appear much more similar to "fractal" sponges than to closely packed spheres, casting doubts on the tenability of the hydrophobic core concept. Principal component analysis highlighted three main order parameters shaping the protein universe: (1) "size", with the consequent generation of progressively less dense and more empty structures at an increasing number of residues, (2) "microscopic structuring", linked to the existence of a spectrum going from the prevalence of heterologous (different hydrophobicity) to the prevalence of homologous (similar hydrophobicity) contacts, and (3) "fractal shape", an organizing protein data set along a continuum going from approximately linear to very intermingled structures. Perhaps the time has come for seriously taking into consideration the real relevance of time-honored principles like the hydrophobic core and hydrophobic effect.

Structural genomics: keeping up with expanding knowledge of the protein universe.

PubMed

Grabowski, Marek; Joachimiak, Andrzej; Otwinowski, Zbyszek; Minor, Wladek

2007-06-01

Structural characterization of the protein universe is the main mission of Structural Genomics (SG) programs. However, progress in gene sequencing technology, set in motion in the 1990s, has resulted in rapid expansion of protein sequence space--a twelvefold increase in the past seven years. For the SG field, this creates new challenges and necessitates a re-assessment of its strategies. Nevertheless, despite the growth of sequence space, at present nearly half of the content of the Swiss-Prot database and over 40% of Pfam protein families can be structurally modeled based on structures determined so far, with SG projects making an increasingly significant contribution. The SG contribution of new Pfam structures nearly doubled from 27.2% in 2003 to 51.6% in 2006.

Tools to evaluate the conformation of protein products.

PubMed

Manta, Bruno; Obal, Gonzalo; Ricciardi, Alejandro; Pritsch, Otto; Denicola, Ana

2011-06-01

Production of recombinant proteins is a process intensively used in the research laboratory. In addition, the main biotechnology market products are recombinant proteins and monoclonal antibodies. The biological (and clinical) properties of the protein product strongly depend on the conformation of the polypeptide. Therefore, assessment of the correct conformation of the produced protein is crucial. There is no single method to assess every aspect of protein structure or function. Depending on the protein, the methods of choice vary. There are general methods to evaluate not only mass and primary sequence of the protein, but also higher-order structure. This review outlines the principal techniques for determining the conformation of a protein from structural (biophysical methods) to functional (in vitro binding assays) analyses. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Guidelines to reach high-quality purified recombinant proteins.

PubMed

Oliveira, Carla; Domingues, Lucília

2018-01-01

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization-denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS) and circular dichroism (CD)-are revisited with focus on the protein and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.

Protein structure estimation from NMR data by matrix completion.

PubMed

Li, Zhicheng; Li, Yang; Lei, Qiang; Zhao, Qing

2017-09-01

Knowledge of protein structures is very important to understand their corresponding physical and chemical properties. Nuclear Magnetic Resonance (NMR) spectroscopy is one of the main methods to measure protein structure. In this paper, we propose a two-stage approach to calculate the structure of a protein from a highly incomplete distance matrix, where most data are obtained from NMR. We first randomly "guess" a small part of unobservable distances by utilizing the triangle inequality, which is crucial for the second stage. Then we use matrix completion to calculate the protein structure from the obtained incomplete distance matrix. We apply the accelerated proximal gradient algorithm to solve the corresponding optimization problem. Furthermore, the recovery error of our method is analyzed, and its efficiency is demonstrated by several practical examples.

Challenges in NMR-based structural genomics

NASA Astrophysics Data System (ADS)

Sue, Shih-Che; Chang, Chi-Fon; Huang, Yao-Te; Chou, Ching-Yu; Huang, Tai-huang

2005-05-01

Understanding the functions of the vast number of proteins encoded in many genomes that have been completely sequenced recently is the main challenge for biologists in the post-genomics era. Since the function of a protein is determined by its exact three-dimensional structure it is paramount to determine the 3D structures of all proteins. This need has driven structural biologists to undertake the structural genomics project aimed at determining the structures of all known proteins. Several centers for structural genomics studies have been established throughout the world. Nuclear magnetic resonance (NMR) spectroscopy has played a major role in determining protein structures in atomic details and in a physiologically relevant solution state. Since the number of new genes being discovered daily far exceeds the number of structures determined by both NMR and X-ray crystallography, a high-throughput method for speeding up the process of protein structure determination is essential for the success of the structural genomics effort. In this article we will describe NMR methods currently being employed for protein structure determination. We will also describe methods under development which may drastically increase the throughput, as well as point out areas where opportunities exist for biophysicists to make significant contribution in this important field.

Discrete-continuous duality of protein structure space.

PubMed

Sadreyev, Ruslan I; Kim, Bong-Hyun; Grishin, Nick V

2009-06-01

Recently, the nature of protein structure space has been widely discussed in the literature. The traditional discrete view of protein universe as a set of separate folds has been criticized in the light of growing evidence that almost any arrangement of secondary structures is possible and the whole protein space can be traversed through a path of similar structures. Here we argue that the discrete and continuous descriptions are not mutually exclusive, but complementary: the space is largely discrete in evolutionary sense, but continuous geometrically when purely structural similarities are quantified. Evolutionary connections are mainly confined to separate structural prototypes corresponding to folds as islands of structural stability, with few remaining traceable links between the islands. However, for a geometric similarity measure, it is usually possible to find a reasonable cutoff that yields paths connecting any two structures through intermediates.

PROGEN: An automated modelling algorithm for the generation of complete protein structures from the α-carbon atomic coordinates

NASA Astrophysics Data System (ADS)

Mandal, Chhabinath; Linthicum, D. Scott

1993-04-01

A modelling algorithm (PROGEN) for the generation of complete protein atomic coordinates from only the α-carbon coordinates is described. PROGEN utilizes an optimal geometry parameter (OGP) database for the positioning of atoms for each amino acid of the polypeptide model. The OGP database was established by examining the statistical correlations between 23 different intra-peptide and inter-peptide geometric parameters relative to the α-carbon distances for each amino acid in a library of 19 known proteins from the Brookhaven Protein Database (BPDB). The OGP files for specific amino acids and peptides were used to generate the atomic positions, with respect to α-carbons, for main-chain and side-chain atoms in the modelled structure. Refinement of the initial model was accomplished using energy minimization (EM) and molecular dynamics techniques. PROGEN was tested using 60 known proteins in the BPDB, representing a wide spectrum of primary and secondary structures. Comparison between PROGEN models and BPDB crystal reference structures gave r.m.s.d. values for peptide main-chain atoms between 0.29 and 0.76 Å, with a grand average of 0.53 Å for all 60 models. The r.m.s.d. for all non-hydrogen atoms ranged between 1.44 and 1.93 Å for the 60 polypeptide models. PROGEN was also able to make the correct assignment of cis- or trans-proline configurations in the protein structures examined. PROGEN offers a fully automatic building and refinement procedure and requires no special or specific structural considerations for the protein to be modelled.

High-Resolution Mapping of a Repeat Protein Folding Free Energy Landscape.

PubMed

Fossat, Martin J; Dao, Thuy P; Jenkins, Kelly; Dellarole, Mariano; Yang, Yinshan; McCallum, Scott A; Garcia, Angel E; Barrick, Doug; Roumestand, Christian; Royer, Catherine A

2016-12-06

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the conformational ensemble along the entire folding reaction coordinate. Simulations can provide this level of insight for small proteins. In contrast, with the exception of hydrogen exchange, which does not monitor folding directly, experimental studies of protein folding have not yielded such structural and energetic detail. NMR can provide residue specific atomic level structural information, but its implementation in protein folding studies using chemical or temperature perturbation is problematic. Here we present a highly detailed structural and energetic map of the entire folding landscape of the leucine-rich repeat protein, pp32 (Anp32), obtained by combining pressure-dependent site-specific 1 H- 15 N HSQC data with coarse-grained molecular dynamics simulations. The results obtained using this equilibrium approach demonstrate that the main barrier to folding of pp32 is quite broad and lies near the unfolded state, with structure apparent only in the C-terminal region. Significant deviation from two-state unfolding under pressure reveals an intermediate on the folded side of the main barrier in which the N-terminal region is disordered. A nonlinear temperature dependence of the population of this intermediate suggests a large heat capacity change associated with its formation. The combination of pressure, which favors the population of folding intermediates relative to chemical denaturants; NMR, which allows their observation; and constrained structure-based simulations yield unparalleled insight into protein folding mechanisms. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Comparison of the structural basis for thermal stability between archaeal and bacterial proteins.

PubMed

Ding, Yanrui; Cai, Yujie; Han, Yonggang; Zhao, Bingqiang

2012-01-01

In this study, the structural basis for thermal stability in archaeal and bacterial proteins was investigated. There were many common factors that confer resistance to high temperature in both archaeal and bacterial proteins. These factors include increases in the Lys content, the bends and blanks of secondary structure, the Glu content of salt bridge; decreases in the number of main-side chain hydrogen bond and exposed surface area, and changes in the bends and blanks of amino acids. Certainly, the utilization of charged amino acids to form salt bridges is a primary factor. In both heat-resistant archaeal and bacterial proteins, most Glu and Asp participate in the formation of salt bridges. Other factors may influence either archaeal or bacterial protein thermostability, which includes the more frequent occurrence of shorter 3(10)-helices and increased hydrophobicity in heat-resistant archaeal proteins. However, there were increases in average helix length, the Glu content in salt bridges, temperature factors and decreases in the number of main-side chain hydrogen bonds, uncharged-uncharged hydrogen bonds, hydrophobicity, and buried and exposed polar surface area in heat-resistant bacterial proteins. Evidently, there are few similarities and many disparities between the heat-resistant mechanisms of archaeal and bacterial proteins.

Structural genomics: keeping up with expanding knowledge of the protein universe

PubMed Central

Grabowski, Marek; Joachimiak, Andrzej; Otwinowski, Zbyszek; Minor, Wladek

2010-01-01

Structural characterization of the protein universe is the main mission of Structural Genomics (SG) programs. However, progress in gene sequencing technology, set in motion in the 1990s, has resulted in rapid expansion of protein sequence space — a twelvefold increase in the past seven years. For the SG field, this creates new challenges and necessitates a reassessment of its strategies. Nevertheless, despite the growth of sequence space, at present nearly half of the content of the Swiss-Prot database and over 40% of Pfam protein families can be structurally modeled based on structures determined so far, with SG projects making an increasingly significant contribution. The SG contribution of new Pfam structures nearly doubled from 27.2% in 2003 to 51.6% in 2006. PMID:17587562

The use of supramolecular structures as protein ligands.

PubMed

Stopa, Barbara; Jagusiak, Anna; Konieczny, Leszek; Piekarska, Barbara; Rybarska, Janina; Zemanek, Grzegorz; Król, Marcin; Piwowar, Piotr; Roterman, Irena

2013-11-01

Congo red dye as well as other eagerly self-assembling organic molecules which form rod-like or ribbon-like supramolecular structures in water solutions, appears to represent a new class of protein ligands with possible wide-ranging medical applications. Such molecules associate with proteins as integral clusters and preferentially penetrate into areas of low molecular stability. Abnormal, partly unfolded proteins are the main binding target for such ligands, while well packed molecules are generally inaccessible. Of particular interest is the observation that local susceptibility for binding supramolecular ligands may be promoted in some proteins as a consequence of function-derived structural changes, and that such complexation may alter the activity profile of target proteins. Examples are presented in this paper.

Structure elucidation of dimeric transmembrane domains of bitopic proteins.

PubMed

Bocharov, Eduard V; Volynsky, Pavel E; Pavlov, Konstantin V; Efremov, Roman G; Arseniev, Alexander S

2010-01-01

The interaction between transmembrane helices is of great interest because it directly determines biological activity of a membrane protein. Either destroying or enhancing such interactions can result in many diseases related to dysfunction of different tissues in human body. One much studied form of membrane proteins known as bitopic protein is a dimer containing two membrane-spanning helices associating laterally. Establishing structure-function relationship as well as rational design of new types of drugs targeting membrane proteins requires precise structural information about this class of objects. At present time, to investigate spatial structure and internal dynamics of such transmembrane helical dimers, several strategies were developed based mainly on a combination of NMR spectroscopy, optical spectroscopy, protein engineering and molecular modeling. These approaches were successfully applied to homo- and heterodimeric transmembrane fragments of several bitopic proteins, which play important roles in normal and in pathological conditions of human organism.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae.

PubMed

Albornos, Lucía; Martín, Ignacio; Iglesias, Rebeca; Jiménez, Teresa; Labrador, Emilia; Dopico, Berta

2012-11-07

Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found.

ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae

PubMed Central

2012-01-01

Background Many proteins with tandem repeats in their sequence have been described and classified according to the length of the repeats: I) Repeats of short oligopeptides (from 2 to 20 amino acids), including structural cell wall proteins and arabinogalactan proteins. II) Repeats that range in length from 20 to 40 residues, including proteins with a well-established three-dimensional structure often involved in mediating protein-protein interactions. (III) Longer repeats in the order of 100 amino acids that constitute structurally and functionally independent units. Here we analyse ShooT specific (ST) proteins, a family of proteins with tandem repeats of unknown function that were first found in Leguminosae, and their possible similarities to other proteins with tandem repeats. Results ST protein sequences were only found in dicotyledonous plants, limited to several plant families, mainly the Fabaceae and the Asteraceae. ST mRNAs accumulate mainly in the roots and under biotic interactions. Most ST proteins have one or several Domain(s) of Unknown Function 2775 (DUF2775). All deduced ST proteins have a signal peptide, indicating that these proteins enter the secretory pathway, and the mature proteins have tandem repeat oligopeptides that share a hexapeptide (E/D)FEPRP followed by 4 partially conserved amino acids, which could determine a putative N-glycosylation signal, and a fully conserved tyrosine. In a phylogenetic tree, the sequences clade according to taxonomic group. A possible involvement in symbiosis and abiotic stress as well as in plant cell elongation is suggested, although different STs could play different roles in plant development. Conclusions We describe a new family of proteins called ST whose presence is limited to the plant kingdom, specifically to a few families of dicotyledonous plants. They present 20 to 40 amino acid tandem repeat sequences with different characteristics (signal peptide, DUF2775 domain, conservative repeat regions) from the described group of 20 to 40 amino acid tandem repeat proteins and also from known cell wall proteins with repeat sequences. Several putative roles in plant physiology can be inferred from the characteristics found. PMID:23134664

A novel Multi-Agent Ada-Boost algorithm for predicting protein structural class with the information of protein secondary structure.

PubMed

Fan, Ming; Zheng, Bin; Li, Lihua

2015-10-01

Knowledge of the structural class of a given protein is important for understanding its folding patterns. Although a lot of efforts have been made, it still remains a challenging problem for prediction of protein structural class solely from protein sequences. The feature extraction and classification of proteins are the main problems in prediction. In this research, we extended our earlier work regarding these two aspects. In protein feature extraction, we proposed a scheme by calculating the word frequency and word position from sequences of amino acid, reduced amino acid, and secondary structure. For an accurate classification of the structural class of protein, we developed a novel Multi-Agent Ada-Boost (MA-Ada) method by integrating the features of Multi-Agent system into Ada-Boost algorithm. Extensive experiments were taken to test and compare the proposed method using four benchmark datasets in low homology. The results showed classification accuracies of 88.5%, 96.0%, 88.4%, and 85.5%, respectively, which are much better compared with the existing methods. The source code and dataset are available on request.

Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines.

PubMed

Su, Qiudong; Guo, Minzhuo; Jia, Zhiyuan; Qiu, Feng; Lu, Xuexin; Gao, Yan; Meng, Qingling; Tian, Ruiguang; Bi, Shengli; Yi, Yao

2016-07-01

Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular comparison of the structural proteins encoding gene clusters of two related Lactobacillus delbrueckii bacteriophages.

PubMed Central

Vasala, A; Dupont, L; Baumann, M; Ritzenthaler, P; Alatossava, T

1993-01-01

Virulent phage LL-H and temperate phage mv4 are two related bacteriophages of Lactobacillus delbrueckii. The gene clusters encoding structural proteins of these two phages have been sequenced and further analyzed. Six open reading frames (ORF-1 to ORF-6) were detected. Protein sequencing and Western immunoblotting experiments confirmed that ORF-3 (g34) encoded the main capsid protein Gp34. The presence of a putative late promoter in front of the phage LL-H g34 gene was suggested by primer extension experiments. Comparative sequence analysis between phage LL-H and phage mv4 revealed striking similarities in the structure and organization of this gene cluster, suggesting that the genes encoding phage structural proteins belong to a highly conservative module. Images PMID:8497043

Structural characterization of metal binding to a cold-adapted frataxin.

PubMed

Noguera, Martín E; Roman, Ernesto A; Rigal, Juan B; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

2015-06-01

Frataxin is an evolutionary conserved protein that participates in iron metabolism. Deficiency of this small protein in humans causes a severe neurodegenerative disease known as Friedreich's ataxia. A number of studies indicate that frataxin binds iron and regulates Fe-S cluster biosynthesis. Previous structural studies showed that metal binding occurs mainly in a region of high density of negative charge. However, a comprehensive characterization of the binding sites is required to gain further insights into the mechanistic details of frataxin function. In this work, we have solved the X-ray crystal structures of a cold-adapted frataxin from a psychrophilic bacterium in the presence of cobalt or europium ions. We have identified a number of metal-binding sites, mainly solvent exposed, several of which had not been observed in previous studies on mesophilic homologues. No major structural changes were detected upon metal binding, although the structures exhibit significant changes in crystallographic B-factors. The analysis of these B-factors, in combination with crystal packing and RMSD among structures, suggests the existence of localized changes in the internal motions. Based on these results, we propose that bacterial frataxins possess binding sites of moderate affinity for a quick capture and transfer of iron to other proteins and for the regulation of Fe-S cluster biosynthesis, modulating interactions with partner proteins.

Universal partitioning of the hierarchical fold network of 50-residue segments in proteins

PubMed Central

Ito, Jun-ichi; Sonobe, Yuki; Ikeda, Kazuyoshi; Tomii, Kentaro; Higo, Junichi

2009-01-01

Background Several studies have demonstrated that protein fold space is structured hierarchically and that power-law statistics are satisfied in relation between the numbers of protein families and protein folds (or superfamilies). We examined the internal structure and statistics in the fold space of 50 amino-acid residue segments taken from various protein folds. We used inter-residue contact patterns to measure the tertiary structural similarity among segments. Using this similarity measure, the segments were classified into a number (Kc) of clusters. We examined various Kc values for the clustering. The special resolution to differentiate the segment tertiary structures increases with increasing Kc. Furthermore, we constructed networks by linking structurally similar clusters. Results The network was partitioned persistently into four regions for Kc ≥ 1000. This main partitioning is consistent with results of earlier studies, where similar partitioning was reported in classifying protein domain structures. Furthermore, the network was partitioned naturally into several dozens of sub-networks (i.e., communities). Therefore, intra-sub-network clusters were mutually connected with numerous links, although inter-sub-network ones were rarely done with few links. For Kc ≥ 1000, the major sub-networks were about 40; the contents of the major sub-networks were conserved. This sub-partitioning is a novel finding, suggesting that the network is structured hierarchically: Segments construct a cluster, clusters form a sub-network, and sub-networks constitute a region. Additionally, the network was characterized by non-power-law statistics, which is also a novel finding. Conclusion Main findings are: (1) The universe of 50 residue segments found here was characterized by non-power-law statistics. Therefore, the universe differs from those ever reported for the protein domains. (2) The 50-residue segments were partitioned persistently and universally into some dozens (ca. 40) of major sub-networks, irrespective of the number of clusters. (3) These major sub-networks encompassed 90% of all segments. Consequently, the protein tertiary structure is constructed using the dozens of elements (sub-networks). PMID:19454039

Metamorphic Proteins: Emergence of Dual Protein Folds from One Primary Sequence.

PubMed

Lella, Muralikrishna; Mahalakshmi, Radhakrishnan

2017-06-20

Every amino acid exhibits a different propensity for distinct structural conformations. Hence, decoding how the primary amino acid sequence undergoes the transition to a defined secondary structure and its final three-dimensional fold is presently considered predictable with reasonable certainty. However, protein sequences that defy the first principles of secondary structure prediction (they attain two different folds) have recently been discovered. Such proteins, aptly named metamorphic proteins, decrease the conformational constraint by increasing flexibility in the secondary structure and thereby result in efficient functionality. In this review, we discuss the major factors driving the conformational switch related both to protein sequence and to structure using illustrative examples. We discuss the concept of an evolutionary transition in sequence and structure, the functional impact of the tertiary fold, and the pressure of intrinsic and external factors that give rise to metamorphic proteins. We mainly focus on the major components of protein architecture, namely, the α-helix and β-sheet segments, which are involved in conformational switching within the same or highly similar sequences. These chameleonic sequences are widespread in both cytosolic and membrane proteins, and these folds are equally important for protein structure and function. We discuss the implications of metamorphic proteins and chameleonic peptide sequences in de novo peptide design.

Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: Application to Anabaena Sensory Rhodopsin

NASA Astrophysics Data System (ADS)

Ward, Meaghan E.; Brown, Leonid S.; Ladizhansky, Vladimir

2015-04-01

Studies of the structure, dynamics, and function of membrane proteins (MPs) have long been considered one of the main applications of solid-state NMR (SSNMR). Advances in instrumentation, and the plethora of new SSNMR methodologies developed over the past decade have resulted in a number of high-resolution structures and structural models of both bitopic and polytopic α-helical MPs. The necessity to retain lipids in the sample, the high proportion of one type of secondary structure, differential dynamics, and the possibility of local disorder in the loop regions all create challenges for structure determination. In this Perspective article we describe our recent efforts directed at determining the structure and functional dynamics of Anabaena Sensory Rhodopsin, a heptahelical transmembrane (7TM) protein. We review some of the established and emerging methods which can be utilized for SSNMR-based structure determination, with a particular focus on those used for ASR, a bacterial protein which shares its 7TM architecture with G-protein coupled receptors.

Development of the field of structural physiology

PubMed Central

FUJIYOSHI, Yoshinori

2015-01-01

Electron crystallography is especially useful for studying the structure and function of membrane proteins — key molecules with important functions in neural and other cells. Electron crystallography is now an established technique for analyzing the structures of membrane proteins in lipid bilayers that closely simulate their natural biological environment. Utilizing cryo-electron microscopes with helium-cooled specimen stages that were developed through a personal motivation to understand the functions of neural systems from a structural point of view, the structures of membrane proteins can be analyzed at a higher than 3 Å resolution. This review covers four objectives. First, I introduce the new research field of structural physiology. Second, I recount some of the struggles involved in developing cryo-electron microscopes. Third, I review the structural and functional analyses of membrane proteins mainly by electron crystallography using cryo-electron microscopes. Finally, I discuss multifunctional channels named “adhennels” based on structures analyzed using electron and X-ray crystallography. PMID:26560835

How Egg Case Proteins Can Protect Cuttlefish Offspring?

PubMed Central

Cornet, Valérie; Henry, Joël; Goux, Didier; Duval, Emilie; Bernay, Benoit; Le Corguillé, Gildas; Corre, Erwan; Zatylny-Gaudin, Céline

2015-01-01

Sepia officinalis egg protection is ensured by a complex capsule produced by the female accessory genital glands and the ink bag. Our study is focused on the proteins constituting the main egg case. De novo transcriptomes from female genital glands provided essential databases for protein identification. A proteomic approach in SDS-PAGE coupled with MS unveiled a new egg case protein family: SepECPs, for Sepia officinalis Egg Case Proteins. N-glycosylation was demonstrated by PAS staining SDS-PAGE gels. These glycoproteins are mainly produced in the main nidamental glands. SepECPs share high sequence homology, especially in the signal peptide and the three cysteine-rich domains. SepECPs have a high number of cysteines, with conserved motifs involved in 3D-structure. SDS-PAGE showed that SepECPs could form dimers; this result was confirmed by TEM observations, which also revealed a protein network. This network is similar to the capsule network, and it associates these structural proteins with polysaccharides, melanin and bacteria to form a tight mesh. Its hardness and elasticity provide physical protection to the embryo. In addition, SepECPs also have bacteriostatic antimicrobial activity on GRAM- bacteria. By observing the SepECP / Vibrio aestuarianus complex in SEM, we demonstrated the ability of these proteins to agglomerate bacteria and thus inhibit their growth. These original proteins identified from the outer egg case ensure the survival of the species by providing physical and chemical protection to the embryos released in the environment without any maternal protection. PMID:26168161

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.

Cloning, production, and purification of proteins for a medium-scale structural genomics project.

PubMed

Quevillon-Cheruel, Sophie; Collinet, Bruno; Trésaugues, Lionel; Minard, Philippe; Henckes, Gilles; Aufrère, Robert; Blondeau, Karine; Zhou, Cong-Zhao; Liger, Dominique; Bettache, Nabila; Poupon, Anne; Aboulfath, Ilham; Leulliot, Nicolas; Janin, Joël; van Tilbeurgh, Herman

2007-01-01

The South-Paris Yeast Structural Genomics Pilot Project (http://www.genomics.eu.org) aims at systematically expressing, purifying, and determining the three-dimensional structures of Saccharomyces cerevisiae proteins. We have already cloned 240 yeast open reading frames in the Escherichia coli pET system. Eighty-two percent of the targets can be expressed in E. coli, and 61% yield soluble protein. We have currently purified 58 proteins. Twelve X-ray structures have been solved, six are in progress, and six other proteins gave crystals. In this chapter, we present the general experimental flowchart applied for this project. One of the main difficulties encountered in this pilot project was the low solubility of a great number of target proteins. We have developed parallel strategies to recover these proteins from inclusion bodies, including refolding, coexpression with chaperones, and an in vitro expression system. A limited proteolysis protocol, developed to localize flexible regions in proteins that could hinder crystallization, is also described.

Predicting nucleic acid binding interfaces from structural models of proteins

PubMed Central

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2011-01-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared to patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. PMID:22086767

Hill-Climbing search and diversification within an evolutionary approach to protein structure prediction.

PubMed

Chira, Camelia; Horvath, Dragos; Dumitrescu, D

2011-07-30

Proteins are complex structures made of amino acids having a fundamental role in the correct functioning of living cells. The structure of a protein is the result of the protein folding process. However, the general principles that govern the folding of natural proteins into a native structure are unknown. The problem of predicting a protein structure with minimum-energy starting from the unfolded amino acid sequence is a highly complex and important task in molecular and computational biology. Protein structure prediction has important applications in fields such as drug design and disease prediction. The protein structure prediction problem is NP-hard even in simplified lattice protein models. An evolutionary model based on hill-climbing genetic operators is proposed for protein structure prediction in the hydrophobic - polar (HP) model. Problem-specific search operators are implemented and applied using a steepest-ascent hill-climbing approach. Furthermore, the proposed model enforces an explicit diversification stage during the evolution in order to avoid local optimum. The main features of the resulting evolutionary algorithm - hill-climbing mechanism and diversification strategy - are evaluated in a set of numerical experiments for the protein structure prediction problem to assess their impact to the efficiency of the search process. Furthermore, the emerging consolidated model is compared to relevant algorithms from the literature for a set of difficult bidimensional instances from lattice protein models. The results obtained by the proposed algorithm are promising and competitive with those of related methods.

Sequence and structure insights of kazal type thrombin inhibitor protein: Studied with phylogeny, homology modeling and dynamic MM/GBSA studies.

PubMed

Jadhav, Aparna; Dash, RadhaCharan; Hirwani, Raj; Abdin, Malik

2018-03-01

Despite the wide medical importance of serine protease inhibitors, many of kazal type proteins are still to be explored. These thrombin inhibiting proteins are found in the digestive system of hematophagous organisms mainly Arthropods. We studied one of such protein i.e. Kazal type-1 protein from sand-fly Phlebotomus papatasi as its structure and interaction with thrombin is unclear. Initially, Dipetalin a kazal-follistasin domain protein was run through PSI-BLAST to retrieve related sequences. Using this set of sequence a phylogenetic tree was constructed, which identified a distantly related kazal type-1 protein. A three-dimensional structure was predicted for this protein and was aligned with Rhodniin for further evaluation. To have a comparative understanding of it's binding at the thrombin active site, the aligned kazal model-thrombin and rhodniin-thrombin complexes were subjected to molecular dynamics simulations. Dynamics analysis with reference to main chain RMSD, H-chain residue RMSF and total energy showed rhodniin-thrombin complex as a more stable system. Further, the MM/GBSA method was applied that calculated the binding free energy (ΔG binding ) for rhodniin and kazal model as -220.32kcal/Mol and -90.70kcal/Mol, respectively. Thus, it shows that kazal model has weaker bonding with thrombin, unlike rhodniin. Copyright © 2017 Elsevier B.V. All rights reserved.

Chemistry of gluten proteins.

PubMed

Wieser, Herbert

2007-04-01

Gluten proteins play a key role in determining the unique baking quality of wheat by conferring water absorption capacity, cohesivity, viscosity and elasticity on dough. Gluten proteins can be divided into two main fractions according to their solubility in aqueous alcohols: the soluble gliadins and the insoluble glutenins. Both fractions consist of numerous, partially closely related protein components characterized by high glutamine and proline contents. Gliadins are mainly monomeric proteins with molecular weights (MWs) around 28,000-55,000 and can be classified according to their different primary structures into the alpha/beta-, gamma- and omega-type. Disulphide bonds are either absent or present as intrachain crosslinks. The glutenin fraction comprises aggregated proteins linked by interchain disulphide bonds; they have a varying size ranging from about 500,000 to more than 10 million. After reduction of disulphide bonds, the resulting glutenin subunits show a solubility in aqueous alcohols similar to gliadins. Based on primary structure, glutenin subunits have been divided into the high-molecular-weight (HMW) subunits (MW=67,000-88,000) and low-molecular-weight (LMW) subunits (MW=32,000-35,000). Each gluten protein type consists or two or three different structural domains; one of them contains unique repetitive sequences rich in glutamine and proline. Native glutenins are composed of a backbone formed by HMW subunit polymers and of LMW subunit polymers branched off from HMW subunits. Non-covalent bonds such as hydrogen bonds, ionic bonds and hydrophobic bonds are important for the aggregation of gliadins and glutenins and implicate structure and physical properties of dough.

Folding a Protein with Equal Probability of Being Helix or Hairpin

PubMed Central

Lin, Chun-Yu; Chen, Nan-Yow; Mou, Chung Yu

2012-01-01

We explore the possibility for the native structure of a protein being inherently multiconformational in an ab initio coarse-grained model. Based on the Wang-Landau algorithm, the complete free energy landscape for the designed sequence 2DX4: INYWLAHAKAGYIVHWTA is constructed. It is shown that 2DX4 possesses two nearly degenerate native structures: one is a helix structure with the other a hairpinlike structure, and their free energy difference is <2% of that of local minima. Two degenerate native structures are stabilized by an energy barrier of ∼10 kcal/mol. Furthermore, the hydrogen-bond and dipole-dipole interactions are found to be two major competing interactions in transforming one conformation into the other. Our results indicate that two degenerate native structures are stabilized by subtle balance between different interactions in proteins. In particular, for small proteins, balance between the hydrogen-bond and dipole-dipole interactions happens for proteins of sizes being ∼18 amino acids and is shown to the main driving mechanism for the occurrence of degeneracy. These results provide important clues to the study of native structures of proteins. PMID:22828336

Applications of graph theory in protein structure identification

PubMed Central

2011-01-01

There is a growing interest in the identification of proteins on the proteome wide scale. Among different kinds of protein structure identification methods, graph-theoretic methods are very sharp ones. Due to their lower costs, higher effectiveness and many other advantages, they have drawn more and more researchers’ attention nowadays. Specifically, graph-theoretic methods have been widely used in homology identification, side-chain cluster identification, peptide sequencing and so on. This paper reviews several methods in solving protein structure identification problems using graph theory. We mainly introduce classical methods and mathematical models including homology modeling based on clique finding, identification of side-chain clusters in protein structures upon graph spectrum, and de novo peptide sequencing via tandem mass spectrometry using the spectrum graph model. In addition, concluding remarks and future priorities of each method are given. PMID:22165974

Crystallization of PTP Domains.

PubMed

Levy, Colin; Adams, James; Tabernero, Lydia

2016-01-01

Protein crystallography is the most powerful method to obtain atomic resolution information on the three-dimensional structure of proteins. An essential step towards determining the crystallographic structure of a protein is to produce good quality crystals from a concentrated sample of purified protein. These crystals are then used to obtain X-ray diffraction data necessary to determine the 3D structure by direct phasing or molecular replacement if the model of a homologous protein is available. Here, we describe the main approaches and techniques to obtain suitable crystals for X-ray diffraction. We include tools and guidance on how to evaluate and design the protein construct, how to prepare Se-methionine derivatized protein, how to assess the stability and quality of the sample, and how to crystallize and prepare crystals for diffraction experiments. While general strategies for protein crystallization are summarized, specific examples of the application of these strategies to the crystallization of PTP domains are discussed.

Network biology discovers pathogen contact points in host protein-protein interactomes.

PubMed

Ahmed, Hadia; Howton, T C; Sun, Yali; Weinberger, Natascha; Belkhadir, Youssef; Mukhtar, M Shahid

2018-06-13

In all organisms, major biological processes are controlled by complex protein-protein interactions networks (interactomes), yet their structural complexity presents major analytical challenges. Here, we integrate a compendium of over 4300 phenotypes with Arabidopsis interactome (AI-1 MAIN ). We show that nodes with high connectivity and betweenness are enriched and depleted in conditional and essential phenotypes, respectively. Such nodes are located in the innermost layers of AI-1 MAIN and are preferential targets of pathogen effectors. We extend these network-centric analyses to Cell Surface Interactome (CSI LRR ) and predict its 35 most influential nodes. To determine their biological relevance, we show that these proteins physically interact with pathogen effectors and modulate plant immunity. Overall, our findings contrast with centrality-lethality rule, discover fast information spreading nodes, and highlight the structural properties of pathogen targets in two different interactomes. Finally, this theoretical framework could possibly be applicable to other inter-species interactomes to reveal pathogen contact points.

Therapeutic approaches against common structural features of toxic oligomers shared by multiple amyloidogenic proteins.

PubMed

Guerrero-Muñoz, Marcos J; Castillo-Carranza, Diana L; Kayed, Rakez

2014-04-15

Impaired proteostasis is one of the main features of all amyloid diseases, which are associated with the formation of insoluble aggregates from amyloidogenic proteins. The aggregation process can be caused by overproduction or poor clearance of these proteins. However, numerous reports suggest that amyloid oligomers are the most toxic species, rather than insoluble fibrillar material, in Alzheimer's, Parkinson's, and Prion diseases, among others. Although the exact protein that aggregates varies between amyloid disorders, they all share common structural features that can be used as therapeutic targets. In this review, we focus on therapeutic approaches against shared features of toxic oligomeric structures and future directions. Copyright © 2014 Elsevier Inc. All rights reserved.

Structural characterizations of human periostin dimerization and cysteinylation.

PubMed

Liu, Jianmei; Zhang, Junying; Xu, Fei; Lin, Zhaohan; Li, Zhiqiang; Liu, Heli

2018-05-12

Human periostin plays a multifaceted role in remodeling the extracellular matrix milieu by interacting with other proteins and itself in both a heterophilic and homophilic manner. However, the structural mechanism for its extensive interactions has remained elusive. Here, we report the crystal structures of human periostin (EMI-Fas1 I- IV ) and its Cys60Ala mutant. In combination with multi-angle light scattering analysis and biochemical assays, the crystal structures reveal that periostin mainly exists as a dimer in solution and its homophilic interaction is mainly mediated by the EMI domain. Furthermore, Cys60 undergoes cysteinylation as confirmed by mass spectroscopy, and this site hardly affects the homophilic interaction. Also, the structures yield insights into how periostin forms heterophilic interactions with other proteins under physiological or pathological conditions. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Finding the target sites of RNA-binding proteins

PubMed Central

Li, Xiao; Kazan, Hilal; Lipshitz, Howard D; Morris, Quaid D

2014-01-01

RNA–protein interactions differ from DNA–protein interactions because of the central role of RNA secondary structure. Some RNA-binding domains (RBDs) recognize their target sites mainly by their shape and geometry and others are sequence-specific but are sensitive to secondary structure context. A number of small- and large-scale experimental approaches have been developed to measure RNAs associated in vitro and in vivo with RNA-binding proteins (RBPs). Generalizing outside of the experimental conditions tested by these assays requires computational motif finding. Often RBP motif finding is done by adapting DNA motif finding methods; but modeling secondary structure context leads to better recovery of RBP-binding preferences. Genome-wide assessment of mRNA secondary structure has recently become possible, but these data must be combined with computational predictions of secondary structure before they add value in predicting in vivo binding. There are two main approaches to incorporating structural information into motif models: supplementing primary sequence motif models with preferred secondary structure contexts (e.g., MEMERIS and RNAcontext) and directly modeling secondary structure recognized by the RBP using stochastic context-free grammars (e.g., CMfinder and RNApromo). The former better reconstruct known binding preferences for sequence-specific RBPs but are not suitable for modeling RBPs that recognize shape and geometry of RNAs. Future work in RBP motif finding should incorporate interactions between multiple RBDs and multiple RBPs in binding to RNA. WIREs RNA 2014, 5:111–130. doi: 10.1002/wrna.1201 PMID:24217996

High-resolution structure of infectious prion protein: the final frontier

PubMed Central

Diaz-Espinoza, Rodrigo; Soto, Claudio

2014-01-01

Prions are the proteinaceous infectious agents responsible for the transmission of prion diseases. The main or sole component of prions is the misfolded prion protein (PrPSc), which is able to template the conversion of the host’s natively folded form of the protein (PrPC). The detailed mechanism of prion replication and the high-resolution structure of PrPSc are unknown. The currently available information on PrPSc structure comes mostly from low-resolution biophysical techniques, which have resulted in quite divergent models. Recent advances in the production of infectious prions, using very pure recombinant protein, offer new hope for PrPSc structural studies. This review highlights the importance of, challenges for and recent progress toward elucidating the elusive structure of PrPSc, arguably the major pending milestone to reach in understanding prions. PMID:22472622

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

NMR studies of structure, hydrogen exchange, and main-chain dynamics in a disrupted-core mutant of thioredoxin.

PubMed Central

De Lorimier, R.; Hellinga, H. W.; Spicer, L. D.

1996-01-01

Core-packing mutants of proteins often approach molten globule states, and hence may have attributes of folding intermediates. We have studied a core-packing mutant of thioredoxin, L78K, in which a leucine residue is substituted by lysine, using 15N heteronuclear two- and three-dimensional NMR. Chemical shift differences between the mutant and wild-type main-chain resonances reveal that structural changes caused by the mutation are localized within 12 A of the altered side chain. The majority of resonances are unchanged, as are many 1H-1H NOEs indicative of the main-chain fold, suggesting that the structure of L78K is largely similar to wild type. Hydrogen exchange studies reveal that residues comprising the central beta-sheet of both mutant and wild-type proteins constitute a local unfolding unit, but with the unfolding/folding equilibrium approximately 12 times larger in L78K. The dynamics of main-chain NH bonds in L78K were studied by 15N spin relaxation and compared with a previous study of wild type. Order parameters for angular motion of NH bonds in the mutant are on average lower than in wild type, suggesting greater spatial freedom on a rapid time scale, but may also be related to different rotational correlation times in the two proteins. There is also evidence of greater conformational exchange in the mutant. Differences between mutant and wild type in hydrogen exchange and main-chain dynamics are not confined to the vicinity of the mutation. We infer that mispacking of the protein core in one location affects local dynamics and stability throughout. PMID:8976564

Protein local structure alignment under the discrete Fréchet distance.

PubMed

Zhu, Binhai

2007-12-01

Protein structure alignment is a fundamental problem in computational and structural biology. While there has been lots of experimental/heuristic methods and empirical results, very few results are known regarding the algorithmic/complexity aspects of the problem, especially on protein local structure alignment. A well-known measure to characterize the similarity of two polygonal chains is the famous Fréchet distance, and with the application of protein-related research, a related discrete Fréchet distance has been used recently. In this paper, following the recent work of Jiang et al. we investigate the protein local structural alignment problem using bounded discrete Fréchet distance. Given m proteins (or protein backbones, which are 3D polygonal chains), each of length O(n), our main results are summarized as follows: * If the number of proteins, m, is not part of the input, then the problem is NP-complete; moreover, under bounded discrete Fréchet distance it is NP-hard to approximate the maximum size common local structure within a factor of n(1-epsilon). These results hold both when all the proteins are static and when translation/rotation are allowed. * If the number of proteins, m, is a constant, then there is a polynomial time solution for the problem.

High-throughput Crystallography for Structural Genomics

PubMed Central

Joachimiak, Andrzej

2009-01-01

Protein X-ray crystallography recently celebrated its 50th anniversary. The structures of myoglobin and hemoglobin determined by Kendrew and Perutz provided the first glimpses into the complex protein architecture and chemistry. Since then, the field of structural molecular biology has experienced extraordinary progress and now over 53,000 proteins structures have been deposited into the Protein Data Bank. In the past decade many advances in macromolecular crystallography have been driven by world-wide structural genomics efforts. This was made possible because of third-generation synchrotron sources, structure phasing approaches using anomalous signal and cryo-crystallography. Complementary progress in molecular biology, proteomics, hardware and software for crystallographic data collection, structure determination and refinement, computer science, databases, robotics and automation improved and accelerated many processes. These advancements provide the robust foundation for structural molecular biology and assure strong contribution to science in the future. In this report we focus mainly on reviewing structural genomics high-throughput X-ray crystallography technologies and their impact. PMID:19765976

Protein complex prediction in large ontology attributed protein-protein interaction networks.

PubMed

Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian; Li, Yanpeng; Xu, Bo

2013-01-01

Protein complexes are important for unraveling the secrets of cellular organization and function. Many computational approaches have been developed to predict protein complexes in protein-protein interaction (PPI) networks. However, most existing approaches focus mainly on the topological structure of PPI networks, and largely ignore the gene ontology (GO) annotation information. In this paper, we constructed ontology attributed PPI networks with PPI data and GO resource. After constructing ontology attributed networks, we proposed a novel approach called CSO (clustering based on network structure and ontology attribute similarity). Structural information and GO attribute information are complementary in ontology attributed networks. CSO can effectively take advantage of the correlation between frequent GO annotation sets and the dense subgraph for protein complex prediction. Our proposed CSO approach was applied to four different yeast PPI data sets and predicted many well-known protein complexes. The experimental results showed that CSO was valuable in predicting protein complexes and achieved state-of-the-art performance.

Extensive structural change of the envelope protein of dengue virus induced by a tuned ionic strength: conformational and energetic analyses

NASA Astrophysics Data System (ADS)

Degrève, Léo; Fuzo, Carlos A.; Caliri, Antonio

2012-12-01

The Dengue has become a global public health threat, with over 100 million infections annually; to date there is no specific vaccine or any antiviral drug. The structures of the envelope (E) proteins of the four known serotype of the dengue virus (DENV) are already known, but there are insufficient molecular details of their structural behavior in solution in the distinct environmental conditions in which the DENVs are submitted, from the digestive tract of the mosquito up to its replication inside the host cell. Such detailed knowledge becomes important because of the multifunctional character of the E protein: it mediates the early events in cell entry, via receptor endocytosis and, as a class II protein, participates determinately in the process of membrane fusion. The proposed infection mechanism asserts that once in the endosome, at low pH, the E homodimers dissociate and insert into the endosomal lipid membrane, after an extensive conformational change, mainly on the relative arrangement of its three domains. In this work we employ all-atom explicit solvent Molecular Dynamics simulations to specify the thermodynamic conditions in that the E proteins are induced to experience extensive structural changes, such as during the process of reducing pH. We study the structural behavior of the E protein monomer at acid pH solution of distinct ionic strength. Extensive simulations are carried out with all the histidine residues in its full protonated form at four distinct ionic strengths. The results are analyzed in detail from structural and energetic perspectives, and the virtual protein movements are described by means of the principal component analyses. As the main result, we found that at acid pH and physiological ionic strength, the E protein suffers a major structural change; for lower or higher ionic strengths, the crystal structure is essentially maintained along of all extensive simulations. On the other hand, at basic pH, when all histidine residues are in the unprotonated form, the protein structure is very stable for ionic strengths ranging from 0 to 225 mM. Therefore, our findings support the hypothesis that the histidines constitute the hot points that induce configurational changes of E protein in acid pH, and give extra motivation to the development of new ideas for antivirus compound design.

Structure and formation of highly luminescent protein-stabilized gold clusters† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc05086k

PubMed Central

Chevrier, D. M.; Thanthirige, V. D.; Luo, Z.; Driscoll, S.; Cho, P.; MacDonald, M. A.; Yao, Q.; Guda, R.; Xie, J.; Johnson, E. R.; Chatt, A.; Zheng, N.

2018-01-01

Highly luminescent gold clusters simultaneously synthesized and stabilized by protein molecules represent a remarkable category of nanoscale materials with promising applications in bionanotechnology as sensors. Nevertheless, the atomic structure and luminescence mechanism of these gold clusters are still unknown after several years of developments. Herein, we report findings on the structure, luminescence and biomolecular self-assembly of gold clusters stabilized by the large globular protein, bovine serum albumin. We highlight the surprising identification of interlocked gold-thiolate rings as the main gold structural unit. Importantly, such gold clusters are in a rigidified state within the protein scaffold, offering an explanation for their highly luminescent character. Combined free-standing cluster synthesis (without protecting protein scaffold) with rigidifying and un-rigidifying experiments, were designed to further verify the luminescence mechanism and gold atomic structure within the protein. Finally, the biomolecular self-assembly process of the protein-stabilized gold clusters was elucidated by time-dependent X-ray absorption spectroscopy measurements and density functional theory calculations. PMID:29732064

Construction of ontology augmented networks for protein complex prediction.

PubMed

Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian

2013-01-01

Protein complexes are of great importance in understanding the principles of cellular organization and function. The increase in available protein-protein interaction data, gene ontology and other resources make it possible to develop computational methods for protein complex prediction. Most existing methods focus mainly on the topological structure of protein-protein interaction networks, and largely ignore the gene ontology annotation information. In this article, we constructed ontology augmented networks with protein-protein interaction data and gene ontology, which effectively unified the topological structure of protein-protein interaction networks and the similarity of gene ontology annotations into unified distance measures. After constructing ontology augmented networks, a novel method (clustering based on ontology augmented networks) was proposed to predict protein complexes, which was capable of taking into account the topological structure of the protein-protein interaction network, as well as the similarity of gene ontology annotations. Our method was applied to two different yeast protein-protein interaction datasets and predicted many well-known complexes. The experimental results showed that (i) ontology augmented networks and the unified distance measure can effectively combine the structure closeness and gene ontology annotation similarity; (ii) our method is valuable in predicting protein complexes and has higher F1 and accuracy compared to other competing methods.

3D structure of eukaryotic flagella/cilia by cryo-electron tomography.

PubMed

Ishikawa, Takashi

2013-01-01

Flagella/cilia are motile organelles with more than 400 proteins. To understand the mechanism of such complex systems, we need methods to describe molecular arrange-ments and conformations three-dimensionally in vivo. Cryo-electron tomography enabled us such a 3D structural analysis. Our group has been working on 3D structure of flagella/cilia using this method and revealed highly ordered and beautifully organized molecular arrangement. 3D structure gave us insights into the mechanism to gener-ate bending motion with well defined waveforms. In this review, I summarize our recent structural studies on fla-gella/cilia by cryo-electron tomography, mainly focusing on dynein microtubule-based ATPase motor proteins and the radial spoke, a regulatory protein complex.

3D structure of eukaryotic flagella/cilia by cryo-electron tomography

PubMed Central

Ishikawa, Takashi

2013-01-01

Flagella/cilia are motile organelles with more than 400 proteins. To understand the mechanism of such complex systems, we need methods to describe molecular arrange-ments and conformations three-dimensionally in vivo. Cryo-electron tomography enabled us such a 3D structural analysis. Our group has been working on 3D structure of flagella/cilia using this method and revealed highly ordered and beautifully organized molecular arrangement. 3D structure gave us insights into the mechanism to gener-ate bending motion with well defined waveforms. In this review, I summarize our recent structural studies on fla-gella/cilia by cryo-electron tomography, mainly focusing on dynein microtubule-based ATPase motor proteins and the radial spoke, a regulatory protein complex. PMID:27493552

Did Convergent Protein Evolution Enable Phytoplasmas to Generate 'Zombie Plants'?

PubMed

Rümpler, Florian; Gramzow, Lydia; Theißen, Günter; Melzer, Rainer

2015-12-01

Phytoplasmas are pathogenic bacteria that reprogram plant development such that leaf-like structures instead of floral organs develop. Infected plants are sterile and mainly serve to propagate phytoplasmas and thus have been termed 'zombie plants'. The developmental reprogramming relies on specific interactions of the phytoplasma protein SAP54 with a small subset of MADS-domain transcription factors. Here, we propose that SAP54 folds into a structure that is similar to that of the K-domain, a protein-protein interaction domain of MADS-domain proteins. We suggest that undergoing convergent structural and sequence evolution, SAP54 evolved to mimic the K-domain. Given the high specificity of resulting developmental alterations, phytoplasmas might be used to study flower development in genetically intractable plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Camps 2.0: exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins.

PubMed

Neumann, Sindy; Hartmann, Holger; Martin-Galiano, Antonio J; Fuchs, Angelika; Frishman, Dmitrij

2012-03-01

Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ∼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/. Copyright © 2011 Wiley Periodicals, Inc.

Predicting nucleic acid binding interfaces from structural models of proteins.

PubMed

Dror, Iris; Shazman, Shula; Mukherjee, Srayanta; Zhang, Yang; Glaser, Fabian; Mandel-Gutfreund, Yael

2012-02-01

The function of DNA- and RNA-binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure-based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high-resolution three-dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I-TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high-resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I-TASSER produces high-quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low-resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Copyright © 2011 Wiley Periodicals, Inc.

Protein Multifunctionality: Principles and Mechanisms

PubMed Central

Zaretsky, Joseph Z.; Wreschner, Daniel H.

2008-01-01

In the review, the nature of protein multifunctionality is analyzed. In the first part of the review the principles of structural/functional organization of protein are discussed. In the second part, the main mechanisms involved in development of multiple functions on a single gene product(s) are analyzed. The last part represents a number of examples showing that multifunctionality is a basic feature of biologically active proteins. PMID:21566747

Coordination of genomic structure and transcription by the main bacterial nucleoid-associated protein HU

PubMed Central

Berger, Michael; Farcas, Anca; Geertz, Marcel; Zhelyazkova, Petya; Brix, Klaudia; Travers, Andrew; Muskhelishvili, Georgi

2010-01-01

The histone-like protein HU is a highly abundant DNA architectural protein that is involved in compacting the DNA of the bacterial nucleoid and in regulating the main DNA transactions, including gene transcription. However, the coordination of the genomic structure and function by HU is poorly understood. Here, we address this question by comparing transcript patterns and spatial distributions of RNA polymerase in Escherichia coli wild-type and hupA/B mutant cells. We demonstrate that, in mutant cells, upregulated genes are preferentially clustered in a large chromosomal domain comprising the ribosomal RNA operons organized on both sides of OriC. Furthermore, we show that, in parallel to this transcription asymmetry, mutant cells are also impaired in forming the transcription foci—spatially confined aggregations of RNA polymerase molecules transcribing strong ribosomal RNA operons. Our data thus implicate HU in coordinating the global genomic structure and function by regulating the spatial distribution of RNA polymerase in the nucleoid. PMID:20010798

Mapping of ligand-binding cavities in proteins.

PubMed

Andersson, C David; Chen, Brian Y; Linusson, Anna

2010-05-01

The complex interactions between proteins and small organic molecules (ligands) are intensively studied because they play key roles in biological processes and drug activities. Here, we present a novel approach to characterize and map the ligand-binding cavities of proteins without direct geometric comparison of structures, based on Principal Component Analysis of cavity properties (related mainly to size, polarity, and charge). This approach can provide valuable information on the similarities and dissimilarities, of binding cavities due to mutations, between-species differences and flexibility upon ligand-binding. The presented results show that information on ligand-binding cavity variations can complement information on protein similarity obtained from sequence comparisons. The predictive aspect of the method is exemplified by successful predictions of serine proteases that were not included in the model construction. The presented strategy to compare ligand-binding cavities of related and unrelated proteins has many potential applications within protein and medicinal chemistry, for example in the characterization and mapping of "orphan structures", selection of protein structures for docking studies in structure-based design, and identification of proteins for selectivity screens in drug design programs. 2009 Wiley-Liss, Inc.

Adsorption of GA module onto graphene and graphene oxide: A molecular dynamics simulation study

NASA Astrophysics Data System (ADS)

Chen, Junlang; Wang, Xiaogang; Dai, Chaoqing; Chen, Shude; Tu, Yusong

2014-08-01

Using all-atom molecular dynamics (MD) simulation, we have investigated the adsorption of protein GA module (GA53) onto graphene oxide (GO), compared with similar adsorption onto pristine graphene (PG). We find that: (1) the protein GA53 can be easily and firmly adsorbed onto the surface of GO and PG, but the binding sites are not specific; the main difference is that the secondary structure of GA53 can be well preserved in protein-GO system, while GA53 will partially lose its secondary structure after adsorbed on PG. (2) in protein-GO system, hydroxyl and epoxy groups increase the distance between protein and GO, which weaken their vdW interactions, meanwhile, hydrogen bonds and electrostatic interactions enhance their binding affinity. In protein-PG system, strong vdW interactions between residues of GA53 and PG have destroyed its secondary structure. (3) π-π stacking interactions still exist between aromatic residues and both the basal plane of GO and PG. In comparison with PG, our results suggest that GO presents better biocompatibility to preserve protein secondary structure when simultaneously absorbing protein.

Layers: A molecular surface peeling algorithm and its applications to analyze protein structures

PubMed Central

Karampudi, Naga Bhushana Rao; Bahadur, Ranjit Prasad

2015-01-01

We present an algorithm ‘Layers’ to peel the atoms of proteins as layers. Using Layers we show an efficient way to transform protein structures into 2D pattern, named residue transition pattern (RTP), which is independent of molecular orientations. RTP explains the folding patterns of proteins and hence identification of similarity between proteins is simple and reliable using RTP than with the standard sequence or structure based methods. Moreover, Layers generates a fine-tunable coarse model for the molecular surface by using non-random sampling. The coarse model can be used for shape comparison, protein recognition and ligand design. Additionally, Layers can be used to develop biased initial configuration of molecules for protein folding simulations. We have developed a random forest classifier to predict the RTP of a given polypeptide sequence. Layers is a standalone application; however, it can be merged with other applications to reduce the computational load when working with large datasets of protein structures. Layers is available freely at http://www.csb.iitkgp.ernet.in/applications/mol_layers/main. PMID:26553411

Caseoperoxidase, Mixed β-Casein-SDS-Hemin-Imidazole Complex: A Nano Artificial Enzyme

PubMed Central

Moosavi-Movahedi, Zainab; Gharibi, Hussein; Hadi-Alijanvand, Hamid; Akbarzadeh, Mohammad; Esmaili, Mansoore; Atri, Maliheh S.; Sefidbakht, Yahya; Bohlooli, Mousa; Nazari, Khodadad; Javadian, Soheila; Hong, Jun; Saboury, Ali A.; Sheibani, Nader; Moosavi-Movahedi, Ali A.

2016-01-01

A novel peroxidase-like artificial enzyme, named “caseoperoxidase”, was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme-imidazole-β-casein-SDS exhibited high activity growth and kcat performance towards the native horseradish peroxidase (HRP) demonstrated by the steady state kinetics using UV-Vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel β-casein (Cβ-casein), with a flexible structure and exalted hydrophobicity, was selected as an appropriate apo-protein for the heme active site using a homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation, and indicated that the obtained structure has a good protective role for the heme active-site. Additional further experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme. PMID:25562503

Secure web book to store structural genomics research data.

PubMed

Manjasetty, Babu A; Höppner, Klaus; Mueller, Uwe; Heinemann, Udo

2003-01-01

Recently established collaborative structural genomics programs aim at significantly accelerating the crystal structure analysis of proteins. These large-scale projects require efficient data management systems to ensure seamless collaboration between different groups of scientists working towards the same goal. Within the Berlin-based Protein Structure Factory, the synchrotron X-ray data collection and the subsequent crystal structure analysis tasks are located at BESSY, a third-generation synchrotron source. To organize file-based communication and data transfer at the BESSY site of the Protein Structure Factory, we have developed the web-based BCLIMS, the BESSY Crystallography Laboratory Information Management System. BCLIMS is a relational data management system which is powered by MySQL as the database engine and Apache HTTP as the web server. The database interface routines are written in Python programing language. The software is freely available to academic users. Here we describe the storage, retrieval and manipulation of laboratory information, mainly pertaining to the synchrotron X-ray diffraction experiments and the subsequent protein structure analysis, using BCLIMS.

Structure of the N-terminal domain of the protein Expansion: an ‘Expansion’ to the Smad MH2 fold

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

Beich-Frandsen, Mads; Aragón, Eric; Llimargas, Marta

2015-04-01

Expansion is a modular protein that is conserved in protostomes. The first structure of the N-terminal domain of Expansion has been determined at 1.6 Å resolution and the new Nα-MH2 domain was found to belong to the Smad/FHA superfamily of structures. Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of proteinmore » belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.« less

Protein Tertiary Structure Prediction Based on Main Chain Angle Using a Hybrid Bees Colony Optimization Algorithm

NASA Astrophysics Data System (ADS)

Mahmood, Zakaria N.; Mahmuddin, Massudi; Mahmood, Mohammed Nooraldeen

Encoding proteins of amino acid sequence to predict classified into their respective families and subfamilies is important research area. However for a given protein, knowing the exact action whether hormonal, enzymatic, transmembranal or nuclear receptors does not depend solely on amino acid sequence but on the way the amino acid thread folds as well. This study provides a prototype system that able to predict a protein tertiary structure. Several methods are used to develop and evaluate the system to produce better accuracy in protein 3D structure prediction. The Bees Optimization algorithm which inspired from the honey bees food foraging method, is used in the searching phase. In this study, the experiment is conducted on short sequence proteins that have been used by the previous researches using well-known tools. The proposed approach shows a promising result.

NMR studies of protein-nucleic acid interactions.

PubMed

Varani, Gabriele; Chen, Yu; Leeper, Thomas C

2004-01-01

Protein-DNA and protein-RNA complexes play key functional roles in every living organism. Therefore, the elucidation of their structure and dynamics is an important goal of structural and molecular biology. Nuclear magnetic resonance (NMR) studies of protein and nucleic acid complexes have common features with studies of protein-protein complexes: the interaction surfaces between the molecules must be carefully delineated, the relative orientation of the two species needs to be accurately and precisely determined, and close intermolecular contacts defined by nuclear Overhauser effects (NOEs) must be obtained. However, differences in NMR properties (e.g., chemical shifts) and biosynthetic pathways for sample productions generate important differences. Chemical shift differences between the protein and nucleic acid resonances can aid the NMR structure determination process; however, the relatively limited dispersion of the RNA ribose resonances makes the process of assigning intermolecular NOEs more difficult. The analysis of the resulting structures requires computational tools unique to nucleic acid interactions. This chapter summarizes the most important elements of the structure determination by NMR of protein-nucleic acid complexes and their analysis. The main emphasis is on recent developments (e.g., residual dipolar couplings and new Web-based analysis tools) that have facilitated NMR studies of these complexes and expanded the type of biological problems to which NMR techniques of structural elucidation can now be applied.

Bio-mimicking galactose oxidase and hemocyanin, two dioxygen-processing copper proteins.

PubMed

Gamez, Patrick; Koval, Iryna A; Reedijk, Jan

2004-12-21

The modelling of the active sites of metalloproteins is one of the most challenging tasks in bio-inorganic chemistry. Copper proteins form part of this stimulating field of research as copper enzymes are mainly involved in oxidation bio-reactions. Thus, the understanding of the structure-function relationship of their active sites will allow the design of effective and environmental friendly oxidation catalysts. This perspective illustrates some outstanding structural and functional synthetic models of the active site of copper proteins, with special attention given to models of galactose oxidase and hemocyanin.

Intracellular and extracellular microtubule associated protein tau as a therapeutic target in Alzheimer disease and other tauopathies.

PubMed

Avila, Jesús; Pallas, Noemí; Bolós, Marta; Sayas, C Laura; Hernandez, Felix

2016-06-01

Microtubule associated protein tau, a protein mainly expressed in neurons, plays an important role in several diseases related to dementia, named tauopathies. Alzheimer disease is the most relevant tauopathy. The role of tau protein in dementia is now a topic under discussion, and is the focus of this review. We have covered two major areas: tau pathology and tau as a therapeutic target. Tau pathology is mainly related to a gain of toxic function due to an abnormal accumulation, aberrant modifications (such as hyperphosphorylation and truncation, among others) and self-aggregation of tau into oligomers or larger structures. Also, tau can be found extracellularly in a toxic form. Tau-based therapy is mainly centered on avoiding the gain of these toxic functions of tau. Tau therapies are focused on lowering tau levels, mainly of modified tau species that could be toxic for neurons (phosphorylated, truncated or aggregated tau), in intracellular or extracellular form. Decreasing the levels of those toxic species is a possible therapeutic strategy.

High pressure effects on protein structure and function.

PubMed

Mozhaev, V V; Heremans, K; Frank, J; Masson, P; Balny, C

1996-01-01

Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and of rather limited value in experimental biochemistry. The goal of this overview is to show that pressure is a powerful tool for the study of proteins and modulation of enzymatic activity.

α-Crystallins Are Small Heat Shock Proteins: Functional and Structural Properties.

PubMed

Tikhomirova, T S; Selivanova, O M; Galzitskaya, O V

2017-02-01

During its life cycle, a cell can be subjected to various external negative effects. Many proteins provide cell protection, including small heat shock proteins (sHsp) that have chaperone-like activity. These proteins have several important functions involving prevention of apoptosis and retention of cytoskeletal integrity; also, sHsp take part in the recovery of enzyme activity. The action mechanism of sHsp is based on the binding of hydrophobic regions exposed to the surface of a molten globule. α-Crystallins presented in chordate cells as two αA- and αB-isoforms are the most studied small heat shock proteins. In this review, we describe the main functions of α-crystallins, features of their secondary and tertiary structures, and examples of their partners in protein-protein interactions.

NMR in structural genomics to increase structural coverage of the protein universe: Delivered by Prof. Kurt Wüthrich on 7 July 2013 at the 38th FEBS Congress in St. Petersburg, Russia.

PubMed

Serrano, Pedro; Dutta, Samit K; Proudfoot, Andrew; Mohanty, Biswaranjan; Susac, Lukas; Martin, Bryan; Geralt, Michael; Jaroszewski, Lukasz; Godzik, Adam; Elsliger, Marc; Wilson, Ian A; Wüthrich, Kurt

2016-11-01

For more than a decade, the Joint Center for Structural Genomics (JCSG; www.jcsg.org) worked toward increased three-dimensional structure coverage of the protein universe. This coordinated quest was one of the main goals of the four high-throughput (HT) structure determination centers of the Protein Structure Initiative (PSI; www.nigms.nih.gov/Research/specificareas/PSI). To achieve the goals of the PSI, the JCSG made use of the complementarity of structure determination by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy to increase and diversify the range of targets entering the HT structure determination pipeline. The overall strategy, for both techniques, was to determine atomic resolution structures for representatives of large protein families, as defined by the Pfam database, which had no structural coverage and could make significant contributions to biological and biomedical research. Furthermore, the experimental structures could be leveraged by homology modeling to further expand the structural coverage of the protein universe and increase biological insights. Here, we describe what could be achieved by this structural genomics approach, using as an illustration the contributions from 20 NMR structure determinations out of a total of 98 JCSG NMR structures, which were selected because they are the first three-dimensional structure representations of the respective Pfam protein families. The information from this small sample is representative for the overall results from crystal and NMR structure determination in the JCSG. There are five new folds, which were classified as domains of unknown functions (DUF), three of the proteins could be functionally annotated based on three-dimensional structure similarity with previously characterized proteins, and 12 proteins showed only limited similarity with previous deposits in the Protein Data Bank (PDB) and were classified as DUFs. © 2016 Federation of European Biochemical Societies.

Molecular Simulation-Based Structural Prediction of Protein Complexes in Mass Spectrometry: The Human Insulin Dimer

PubMed Central

Li, Jinyu; Rossetti, Giulia; Dreyer, Jens; Raugei, Simone; Ippoliti, Emiliano; Lüscher, Bernhard; Carloni, Paolo

2014-01-01

Protein electrospray ionization (ESI) mass spectrometry (MS)-based techniques are widely used to provide insight into structural proteomics under the assumption that non-covalent protein complexes being transferred into the gas phase preserve basically the same intermolecular interactions as in solution. Here we investigate the applicability of this assumption by extending our previous structural prediction protocol for single proteins in ESI-MS to protein complexes. We apply our protocol to the human insulin dimer (hIns2) as a test case. Our calculations reproduce the main charge and the collision cross section (CCS) measured in ESI-MS experiments. Molecular dynamics simulations for 0.075 ms show that the complex maximizes intermolecular non-bonded interactions relative to the structure in water, without affecting the cross section. The overall gas-phase structure of hIns2 does exhibit differences with the one in aqueous solution, not inferable from a comparison with calculated CCS. Hence, care should be exerted when interpreting ESI-MS proteomics data based solely on NMR and/or X-ray structural information. PMID:25210764

Understanding the structural and energetic basis of PD-1 and monoclonal antibodies bound to PD-L1: A molecular modeling perspective.

PubMed

Shi, Danfeng; Zhou, Shuangyan; Liu, Xuewei; Zhao, Chenxi; Liu, Huanxiang; Yao, Xiaojun

2018-03-01

The inhibitors blocking the interaction between programmed cell death protein 1(PD-1) and programmed death-ligand 1(PD-L1) can activate the immune response of T cell and eliminate cancer cells. The crystallographic studies have provided structural insights of the interactive interfaces between PD-L1 and its protein ligands. However, the hotspot residues on PD-L1 as well as structural and energetic basis for different protein ligands still need to be further investigated. Molecular modeling methods including molecular dynamics simulation, per-residue free energy decomposition, virtual alanine scanning mutagenesis and residue-residue contact analysis were used to qualitatively and quantitatively analyze the interactions between PD-L1 and different protein ligands. The results of virtual alanine scanning mutagenesis suggest that Y56, Q66, M115, D122, Y123, R125 are the hotspot residues on PD-L1. The residue-residue contact analysis further shows that PD-1 interacts with PD-L1 mainly by F and G strands while monoclonal antibodies like avelumab and BMS-936559 mainly interact with PD-L1 by CDR2 and CDR3 loops of the heavy chain. A structurally similar β-hairpin peptide with 13 or 14 residues was extracted from each protein ligand and these β-hairpin peptides were found tightly binding to the putative hotspot residues on PD-L1. This study recognizes the hotspot residues on PD-L1 and uncovers the common structural and energetic basis of different protein ligands binding to PD-L1. These results will be valuable for the design of small molecule or peptide inhibitors targeting on PD-L1. Copyright © 2017 Elsevier B.V. All rights reserved.

Amino Acid Distribution Rules Predict Protein Fold: Protein Grammar for Beta-Strand Sandwich-Like Structures

PubMed Central

Kister, Alexander

2015-01-01

We present an alternative approach to protein 3D folding prediction based on determination of rules that specify distribution of “favorable” residues, that are mainly responsible for a given fold formation, and “unfavorable” residues, that are incompatible with that fold, in polypeptide sequences. The process of determining favorable and unfavorable residues is iterative. The starting assumptions are based on the general principles of protein structure formation as well as structural features peculiar to a protein fold under investigation. The initial assumptions are tested one-by-one for a set of all known proteins with a given structure. The assumption is accepted as a “rule of amino acid distribution” for the protein fold if it holds true for all, or near all, structures. If the assumption is not accepted as a rule, it can be modified to better fit the data and then tested again in the next step of the iterative search algorithm, or rejected. We determined the set of amino acid distribution rules for a large group of beta sandwich-like proteins characterized by a specific arrangement of strands in two beta sheets. It was shown that this set of rules is highly sensitive (~90%) and very specific (~99%) for identifying sequences of proteins with specified beta sandwich fold structure. The advantage of the proposed approach is that it does not require that query proteins have a high degree of homology to proteins with known structure. So long as the query protein satisfies residue distribution rules, it can be confidently assigned to its respective protein fold. Another advantage of our approach is that it allows for a better understanding of which residues play an essential role in protein fold formation. It may, therefore, facilitate rational protein engineering design. PMID:25625198

G23D: Online tool for mapping and visualization of genomic variants on 3D protein structures.

PubMed

Solomon, Oz; Kunik, Vered; Simon, Amos; Kol, Nitzan; Barel, Ortal; Lev, Atar; Amariglio, Ninette; Somech, Raz; Rechavi, Gidi; Eyal, Eran

2016-08-26

Evaluation of the possible implications of genomic variants is an increasingly important task in the current high throughput sequencing era. Structural information however is still not routinely exploited during this evaluation process. The main reasons can be attributed to the partial structural coverage of the human proteome and the lack of tools which conveniently convert genomic positions, which are the frequent output of genomic pipelines, to proteins and structure coordinates. We present G23D, a tool for conversion of human genomic coordinates to protein coordinates and protein structures. G23D allows mapping of genomic positions/variants on evolutionary related (and not only identical) protein three dimensional (3D) structures as well as on theoretical models. By doing so it significantly extends the space of variants for which structural insight is feasible. To facilitate interpretation of the variant consequence, pathogenic variants, functional sites and polymorphism sites are displayed on protein sequence and structure diagrams alongside the input variants. G23D also provides modeling of the mutant structure, analysis of intra-protein contacts and instant access to functional predictions and predictions of thermo-stability changes. G23D is available at http://www.sheba-cancer.org.il/G23D . G23D extends the fraction of variants for which structural analysis is applicable and provides better and faster accessibility for structural data to biologists and geneticists who routinely work with genomic information.

The impact of thermal treatment on the stability of freeze-dried amorphous pharmaceuticals: II. Aggregation in an IgG1 fusion protein.

PubMed

Wang, Bingquan; Cicerone, Marcus T; Aso, Yukio; Pikal, Michael J

2010-02-01

The objective of this research was to investigate the impact of thermal treatment on storage stability of an IgG1 fusion protein. IgG1 protein formulations were prepared by freeze-drying the protein with sucrose. Some samples were used as controls, and others were subjected to a further heat treatment (annealing). The protein structure was investigated with Fourier transform infrared spectroscopy (FTIR), and protein aggregation was monitored with size exclusion HPLC. Enthalpy recovery was studied using DSC, and global mobility represented by the structural relaxation time constant (tau(beta)) was characterized by a thermal activity monitor (TAM). The local mobility of the protein system was monitored by both (13)C solid-state NMR and neutron backscattering. Annealing increased the storage stability of the protein, as shown by the smaller aggregation rate and less total aggregation at the end of a storage period. The structural relaxation time constant of an annealed sample was significantly higher than the unannealed control sample, suggesting a decrease in global mobility of the protein system upon annealing. However, annealing does not significantly impact the protein secondary structure or the local mobility. Given the similar protein native structure and specific surface area, the improved stability upon annealing is mainly a result of reduced global molecular mobility. (c) 2009 Wiley-Liss, Inc. and the American Pharmacists Association.

CADB: Conformation Angles DataBase of proteins

PubMed Central

Sheik, S. S.; Ananthalakshmi, P.; Bhargavi, G. Ramya; Sekar, K.

2003-01-01

Conformation Angles DataBase (CADB) provides an online resource to access data on conformation angles (both main-chain and side-chain) of protein structures in two data sets corresponding to 25% and 90% sequence identity between any two proteins, available in the Protein Data Bank. In addition, the database contains the necessary crystallographic parameters. The package has several flexible options and display facilities to visualize the main-chain and side-chain conformation angles for a particular amino acid residue. The package can also be used to study the interrelationship between the main-chain and side-chain conformation angles. A web based JAVA graphics interface has been deployed to display the user interested information on the client machine. The database is being updated at regular intervals and can be accessed over the World Wide Web interface at the following URL: http://144.16.71.148/cadb/. PMID:12520049

Ultrastructural and biochemical investigations of protein mobilization of Mucuna pruriens (L.) DC. cotyledons and embryo axis.

PubMed

Muccifora, Simonetta; Guerranti, Roberto; Muzzi, Chiara; Hope-Onyekwere, Nnadozies S; Pagani, Roberto; Leoncini, Roberto; Bellani, Lorenza M

2010-03-01

The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.

Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

PubMed

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

2017-10-01

While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Legume Lectins: Proteins with Diverse Applications

PubMed Central

Lagarda-Diaz, Irlanda; Guzman-Partida, Ana Maria; Vazquez-Moreno, Luz

2017-01-01

Lectins are a diverse class of proteins distributed extensively in nature. Among these proteins; legume lectins display a variety of interesting features including antimicrobial; insecticidal and antitumor activities. Because lectins recognize and bind to specific glycoconjugates present on the surface of cells and intracellular structures; they can serve as potential target molecules for developing practical applications in the fields of food; agriculture; health and pharmaceutical research. This review presents the current knowledge of the main structural characteristics of legume lectins and the relationship of structure to the exhibited specificities; provides an overview of their particular antimicrobial; insecticidal and antitumor biological activities and describes possible applications based on the pattern of recognized glyco-targets. PMID:28604616

Dynameomics: data-driven methods and models for utilizing large-scale protein structure repositories for improving fragment-based loop prediction.

PubMed

Rysavy, Steven J; Beck, David A C; Daggett, Valerie

2014-11-01

Protein function is intimately linked to protein structure and dynamics yet experimentally determined structures frequently omit regions within a protein due to indeterminate data, which is often due protein dynamics. We propose that atomistic molecular dynamics simulations provide a diverse sampling of biologically relevant structures for these missing segments (and beyond) to improve structural modeling and structure prediction. Here we make use of the Dynameomics data warehouse, which contains simulations of representatives of essentially all known protein folds. We developed novel computational methods to efficiently identify, rank and retrieve small peptide structures, or fragments, from this database. We also created a novel data model to analyze and compare large repositories of structural data, such as contained within the Protein Data Bank and the Dynameomics data warehouse. Our evaluation compares these structural repositories for improving loop predictions and analyzes the utility of our methods and models. Using a standard set of loop structures, containing 510 loops, 30 for each loop length from 4 to 20 residues, we find that the inclusion of Dynameomics structures in fragment-based methods improves the quality of the loop predictions without being dependent on sequence homology. Depending on loop length, ∼ 25-75% of the best predictions came from the Dynameomics set, resulting in lower main chain root-mean-square deviations for all fragment lengths using the combined fragment library. We also provide specific cases where Dynameomics fragments provide better predictions for NMR loop structures than fragments from crystal structures. Online access to these fragment libraries is available at http://www.dynameomics.org/fragments. © 2014 The Protein Society.

[NMR structure and dynamics of the chimeric protein SH3-F2].

PubMed

Kutyshenko, V P; Gushchina, L V; Khristoforov, V S; Prokhorov, D A; Timchenko, M A; Kudrevatykh, Iu A; Fediukina, D V; Filimonov, V V

2010-01-01

For the further elucidation of structural and dynamic principles of protein self-organization and protein-ligand interactions the design of new chimeric protein SH3-F2 was made and genetically engineered construct was created. The SH3-F2 amino acid sequence consists of polyproline ligand mgAPPLPPYSA, GG linker and the sequence of spectrin SH3 domain circular permutant S19-P20s. Structural and dynamics properties of the protein were studied by high-resolution NMR. According to NMR data the tertiary structure of the chimeric protein SH3-F2 has the topology which is typical of SH3 domains in the complex with the ligand, forming polyproline type II helix, located in the conservative region of binding in the orientation II. The polyproline ligand closely adjoins with the protein globule and is stabilized by hydrophobic interactions. However the interaction of ligand and the part of globule relative to SH3 domain is not too large because the analysis of protein dynamic characteristics points to the low amplitude, high-frequency ligand tumbling in relation to the slow intramolecular motions of the main globule. The constructed chimera permits to carry out further structural and thermodynamic investigations of polyproline helix properties and its interaction with regulatory domains.

Processing of Cholinesterase-like α/β-Hydrolase Fold Proteins: Alterations Associated with Congenital Disorders

PubMed Central

De Jaco, Antonella; Comoletti, Davide; Dubi, Noga; Camp, Shelley; Taylor, Palmer

2016-01-01

The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β-hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold. PMID:21933121

Quality assessment of protein model-structures based on structural and functional similarities.

PubMed

Konopka, Bogumil M; Nebel, Jean-Christophe; Kotulska, Malgorzata

2012-09-21

Experimental determination of protein 3D structures is expensive, time consuming and sometimes impossible. A gap between number of protein structures deposited in the World Wide Protein Data Bank and the number of sequenced proteins constantly broadens. Computational modeling is deemed to be one of the ways to deal with the problem. Although protein 3D structure prediction is a difficult task, many tools are available. These tools can model it from a sequence or partial structural information, e.g. contact maps. Consequently, biologists have the ability to generate automatically a putative 3D structure model of any protein. However, the main issue becomes evaluation of the model quality, which is one of the most important challenges of structural biology. GOBA--Gene Ontology-Based Assessment is a novel Protein Model Quality Assessment Program. It estimates the compatibility between a model-structure and its expected function. GOBA is based on the assumption that a high quality model is expected to be structurally similar to proteins functionally similar to the prediction target. Whereas DALI is used to measure structure similarity, protein functional similarity is quantified using standardized and hierarchical description of proteins provided by Gene Ontology combined with Wang's algorithm for calculating semantic similarity. Two approaches are proposed to express the quality of protein model-structures. One is a single model quality assessment method, the other is its modification, which provides a relative measure of model quality. Exhaustive evaluation is performed on data sets of model-structures submitted to the CASP8 and CASP9 contests. The validation shows that the method is able to discriminate between good and bad model-structures. The best of tested GOBA scores achieved 0.74 and 0.8 as a mean Pearson correlation to the observed quality of models in our CASP8 and CASP9-based validation sets. GOBA also obtained the best result for two targets of CASP8, and one of CASP9, compared to the contest participants. Consequently, GOBA offers a novel single model quality assessment program that addresses the practical needs of biologists. In conjunction with other Model Quality Assessment Programs (MQAPs), it would prove useful for the evaluation of single protein models.

Unifying the microscopic picture of His-containing turns: from gas phase model peptides to crystallized proteins.

PubMed

Sohn, Woon Yong; Habka, Sana; Gloaguen, Eric; Mons, Michel

2017-07-14

The presence in crystallized proteins of a local anchoring between the side chain of a His residue, located in the central position of a γ- or β-turn, and its local main chain environment, was assessed by the comparison of protein structures with relevant isolated model peptides. Gas phase laser spectroscopy, combined with relevant quantum chemistry methods, was used to characterize the γ- and β-turn structures in these model peptides. A conformer-selective NH stretch infrared study provided evidence for the formation in vacuo of two types of short-range H-bonded motifs, labelled ε-6 δ and δ- δ 7/π H , bridging the His side chain (in its gauche+ rotamer) to the neighbouring NH(i) and CO(i) sites of the backbone; each side chain-backbone motif was found to be specific of the tautomer (ε or δ) adopted by the His side chain in its neutral form. A close comparison between β- and γ-turns, selected from the Protein Data Bank, and the gas phase models demonstrated that a significant proportion of the gauche+ His rotamer distribution of proteins was well described by the corresponding gas phase H-bonded structures. This is consistent with the persistence of local 6 δ and δ 7/π H intramolecular interactions in proteins, emphasizing the relevance of gas phase data to secondary structures that are poorly accessible to solvents, e.g., in the case of a specific compact topology (Xxx-His β-turns). Deviations from the gas phase structures were also observed, mainly in His-Xxx β-turns, and assigned to solvent accessible turn structures. They were well accounted for by theoretical models of microhydrated turns, in which a few solvent molecules take over the gas phase motifs, constituting a water-mediated local anchoring of the His side chain to the backbone. Finally, the present gas phase benchmark models also pinpointed weaknesses in the protein structure determination by X-ray diffraction analysis; in particular, besides the lack of tautomer information, inaccuracies in the description of imidazole ring flip rotamerism were identified.

The Structure and Function of Non-Collagenous Bone Proteins

NASA Technical Reports Server (NTRS)

Hook, Magnus

1997-01-01

The long-term goal for this program is to determine the structural and functional relationships of bone proteins and proteins that interact with bone. This information will used to design useful pharmacological compounds that will have a beneficial effect in osteoporotic patients and in the osteoporotic-like effects experienced on long duration space missions. The first phase of this program, funded under a cooperative research agreement with NASA through the Texas Medical Center, aimed to develop powerful recombinant expression systems and purification methods for production of large amounts of target proteins. Proteins expressed in sufficient'amount and purity would be characterized by a variety of structural methods, and made available for crystallization studies. In order to increase the likelihood of crystallization and subsequent high resolution solution of structures, we undertook to develop expression of normal and mutant forms of proteins by bacterial and mammalian cells. In addition to the main goals of this program, we would also be able to provide reagents for other related studies, including development of anti-fibrotic and anti-metastatic therapeutics.

Hydrogen atoms in protein structures: high-resolution X-ray diffraction structure of the DFPase

PubMed Central

2013-01-01

Background Hydrogen atoms represent about half of the total number of atoms in proteins and are often involved in substrate recognition and catalysis. Unfortunately, X-ray protein crystallography at usual resolution fails to access directly their positioning, mainly because light atoms display weak contributions to diffraction. However, sub-Ångstrom diffraction data, careful modeling and a proper refinement strategy can allow the positioning of a significant part of hydrogen atoms. Results A comprehensive study on the X-ray structure of the diisopropyl-fluorophosphatase (DFPase) was performed, and the hydrogen atoms were modeled, including those of solvent molecules. This model was compared to the available neutron structure of DFPase, and differences in the protein and the active site solvation were noticed. Conclusions A further examination of the DFPase X-ray structure provides substantial evidence about the presence of an activated water molecule that may constitute an interesting piece of information as regard to the enzymatic hydrolysis mechanism. PMID:23915572

The Role of Protein Loops and Linkers in Conformational Dynamics and Allostery.

PubMed

Papaleo, Elena; Saladino, Giorgio; Lambrughi, Matteo; Lindorff-Larsen, Kresten; Gervasio, Francesco Luigi; Nussinov, Ruth

2016-06-08

Proteins are dynamic entities that undergo a plethora of conformational changes that may take place on a wide range of time scales. These changes can be as small as the rotation of one or a few side-chain dihedral angles or involve concerted motions in larger portions of the three-dimensional structure; both kinds of motions can be important for biological function and allostery. It is becoming increasingly evident that "connector regions" are important components of the dynamic personality of protein structures. These regions may be either disordered loops, i.e., poorly structured regions connecting secondary structural elements, or linkers that connect entire protein domains. Experimental and computational studies have, however, revealed that these regions are not mere connectors, and their role in allostery and conformational changes has been emerging in the last few decades. Here we provide a detailed overview of the structural properties and classification of loops and linkers, as well as a discussion of the main computational methods employed to investigate their function and dynamical properties. We also describe their importance for protein dynamics and allostery using as examples key proteins in cellular biology and human diseases such as kinases, ubiquitinating enzymes, and transcription factors.

Mapping of Ligand-Binding Cavities in Proteins

PubMed Central

Andersson, C. David; Chen, Brian Y.; Linusson, Anna

2010-01-01

The complex interactions between proteins and small organic molecules (ligands) are intensively studied because they play key roles in biological processes and drug activities. Here, we present a novel approach to characterise and map the ligand-binding cavities of proteins without direct geometric comparison of structures, based on Principal Component Analysis of cavity properties (related mainly to size, polarity and charge). This approach can provide valuable information on the similarities, and dissimilarities, of binding cavities due to mutations, between-species differences and flexibility upon ligand-binding. The presented results show that information on ligand-binding cavity variations can complement information on protein similarity obtained from sequence comparisons. The predictive aspect of the method is exemplified by successful predictions of serine proteases that were not included in the model construction. The presented strategy to compare ligand-binding cavities of related and unrelated proteins has many potential applications within protein and medicinal chemistry, for example in the characterisation and mapping of “orphan structures”, selection of protein structures for docking studies in structure-based design and identification of proteins for selectivity screens in drug design programs. PMID:20034113

Heat of supersaturation-limited amyloid burst directly monitored by isothermal titration calorimetry.

PubMed

Ikenoue, Tatsuya; Lee, Young-Ho; Kardos, József; Yagi, Hisashi; Ikegami, Takahisa; Naiki, Hironobu; Goto, Yuji

2014-05-06

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with β2-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.

The structure of the casein micelle of milk and its changes during processing.

PubMed

Dalgleish, Douglas G; Corredig, Milena

2012-01-01

The majority of the protein in cow's milk is contained in the particles known as casein micelles. This review describes the main structural features of these particles and the different models that have been used to define the interior structures. The reactions of the micelles during processing operations are described in terms of the structural models.

Structural Prediction and In Silico Physicochemical Characterization for Mouse Caltrin I and Bovine Caltrin Proteins

PubMed Central

Grasso, Ernesto J.; Sottile, Adolfo E.; Coronel, Carlos E.

2016-01-01

It is known that caltrin (calcium transport inhibitor) protein binds to sperm cells during ejaculation and inhibits extracellular Ca2+ uptake. Although the sequence and some biological features of mouse caltrin I and bovine caltrin are known, their physicochemical properties and tertiary structure are mainly unknown. We predicted the 3D structures of mouse caltrin I and bovine caltrin by molecular homology modeling and threading. Surface electrostatic potentials and electric fields were calculated using the Poisson–Boltzmann equation. Several different bioinformatics tools and available web servers were used to thoroughly analyze the physicochemical characteristics of both proteins, such as their Kyte and Doolittle hydropathy scores and helical wheel projections. The results presented in this work significantly aid further understanding of the molecular mechanisms of caltrin proteins modulating physiological processes associated with fertilization. PMID:27812283

Physics-based protein-structure prediction using a hierarchical protocol based on the UNRES force field: assessment in two blind tests.

PubMed

Ołdziej, S; Czaplewski, C; Liwo, A; Chinchio, M; Nanias, M; Vila, J A; Khalili, M; Arnautova, Y A; Jagielska, A; Makowski, M; Schafroth, H D; Kaźmierkiewicz, R; Ripoll, D R; Pillardy, J; Saunders, J A; Kang, Y K; Gibson, K D; Scheraga, H A

2005-05-24

Recent improvements in the protein-structure prediction method developed in our laboratory, based on the thermodynamic hypothesis, are described. The conformational space is searched extensively at the united-residue level by using our physics-based UNRES energy function and the conformational space annealing method of global optimization. The lowest-energy coarse-grained structures are then converted to an all-atom representation and energy-minimized with the ECEPP/3 force field. The procedure was assessed in two recent blind tests of protein-structure prediction. During the first blind test, we predicted large fragments of alpha and alpha+beta proteins [60-70 residues with C(alpha) rms deviation (rmsd) <6 A]. However, for alpha+beta proteins, significant topological errors occurred despite low rmsd values. In the second exercise, we predicted whole structures of five proteins (two alpha and three alpha+beta, with sizes of 53-235 residues) with remarkably good accuracy. In particular, for the genomic target TM0487 (a 102-residue alpha+beta protein from Thermotoga maritima), we predicted the complete, topologically correct structure with 7.3-A C(alpha) rmsd. So far this protein is the largest alpha+beta protein predicted based solely on the amino acid sequence and a physics-based potential-energy function and search procedure. For target T0198, a phosphate transport system regulator PhoU from T. maritima (a 235-residue mainly alpha-helical protein), we predicted the topology of the whole six-helix bundle correctly within 8 A rmsd, except the 32 C-terminal residues, most of which form a beta-hairpin. These and other examples described in this work demonstrate significant progress in physics-based protein-structure prediction.

Structure-related statistical singularities along protein sequences: a correlation study.

PubMed

Colafranceschi, Mauro; Colosimo, Alfredo; Zbilut, Joseph P; Uversky, Vladimir N; Giuliani, Alessandro

2005-01-01

A data set composed of 1141 proteins representative of all eukaryotic protein sequences in the Swiss-Prot Protein Knowledge base was coded by seven physicochemical properties of amino acid residues. The resulting numerical profiles were submitted to correlation analysis after the application of a linear (simple mean) and a nonlinear (Recurrence Quantification Analysis, RQA) filter. The main RQA variables, Recurrence and Determinism, were subsequently analyzed by Principal Component Analysis. The RQA descriptors showed that (i) within protein sequences is embedded specific information neither present in the codes nor in the amino acid composition and (ii) the most sensitive code for detecting ordered recurrent (deterministic) patterns of residues in protein sequences is the Miyazawa-Jernigan hydrophobicity scale. The most deterministic proteins in terms of autocorrelation properties of primary structures were found (i) to be involved in protein-protein and protein-DNA interactions and (ii) to display a significantly higher proportion of structural disorder with respect to the average data set. A study of the scaling behavior of the average determinism with the setting parameters of RQA (embedding dimension and radius) allows for the identification of patterns of minimal length (six residues) as possible markers of zones specifically prone to inter- and intramolecular interactions.

Archaeal S-Layers: Overview and Current State of the Art.

PubMed

Rodrigues-Oliveira, Thiago; Belmok, Aline; Vasconcellos, Deborah; Schuster, Bernhard; Kyaw, Cynthia M

2017-01-01

In contrast to bacteria, all archaea possess cell walls lacking peptidoglycan and a number of different cell envelope components have also been described. A paracrystalline protein surface layer, commonly referred to as S-layer, is present in nearly all archaea described to date. S-layers are composed of only one or two proteins and form different lattice structures. In this review, we summarize current understanding of archaeal S-layer proteins, discussing topics such as structure, lattice type distribution among archaeal phyla and glycosylation. The hexagonal lattice type is dominant within the phylum Euryarchaeota, while in the Crenarchaeota this feature is mainly associated with specific orders. S-layers exclusive to the Crenarchaeota have also been described, which are composed of two proteins. Information regarding S-layers in the remaining archaeal phyla is limited, mainly due to organism description through only culture-independent methods. Despite the numerous applied studies using bacterial S-layers, few reports have employed archaea as a study model. As such, archaeal S-layers represent an area for exploration in both basic and applied research.

Archaeal S-Layers: Overview and Current State of the Art

PubMed Central

Rodrigues-Oliveira, Thiago; Belmok, Aline; Vasconcellos, Deborah; Schuster, Bernhard; Kyaw, Cynthia M.

2017-01-01

In contrast to bacteria, all archaea possess cell walls lacking peptidoglycan and a number of different cell envelope components have also been described. A paracrystalline protein surface layer, commonly referred to as S-layer, is present in nearly all archaea described to date. S-layers are composed of only one or two proteins and form different lattice structures. In this review, we summarize current understanding of archaeal S-layer proteins, discussing topics such as structure, lattice type distribution among archaeal phyla and glycosylation. The hexagonal lattice type is dominant within the phylum Euryarchaeota, while in the Crenarchaeota this feature is mainly associated with specific orders. S-layers exclusive to the Crenarchaeota have also been described, which are composed of two proteins. Information regarding S-layers in the remaining archaeal phyla is limited, mainly due to organism description through only culture-independent methods. Despite the numerous applied studies using bacterial S-layers, few reports have employed archaea as a study model. As such, archaeal S-layers represent an area for exploration in both basic and applied research. PMID:29312266

Dynameomics: Data-driven methods and models for utilizing large-scale protein structure repositories for improving fragment-based loop prediction

PubMed Central

Rysavy, Steven J; Beck, David AC; Daggett, Valerie

2014-01-01

Protein function is intimately linked to protein structure and dynamics yet experimentally determined structures frequently omit regions within a protein due to indeterminate data, which is often due protein dynamics. We propose that atomistic molecular dynamics simulations provide a diverse sampling of biologically relevant structures for these missing segments (and beyond) to improve structural modeling and structure prediction. Here we make use of the Dynameomics data warehouse, which contains simulations of representatives of essentially all known protein folds. We developed novel computational methods to efficiently identify, rank and retrieve small peptide structures, or fragments, from this database. We also created a novel data model to analyze and compare large repositories of structural data, such as contained within the Protein Data Bank and the Dynameomics data warehouse. Our evaluation compares these structural repositories for improving loop predictions and analyzes the utility of our methods and models. Using a standard set of loop structures, containing 510 loops, 30 for each loop length from 4 to 20 residues, we find that the inclusion of Dynameomics structures in fragment-based methods improves the quality of the loop predictions without being dependent on sequence homology. Depending on loop length, ∼25–75% of the best predictions came from the Dynameomics set, resulting in lower main chain root-mean-square deviations for all fragment lengths using the combined fragment library. We also provide specific cases where Dynameomics fragments provide better predictions for NMR loop structures than fragments from crystal structures. Online access to these fragment libraries is available at http://www.dynameomics.org/fragments. PMID:25142412

Matricellular proteins in drug delivery: Therapeutic targets, active agents, and therapeutic localization.

PubMed

Sawyer, Andrew J; Kyriakides, Themis R

2016-02-01

Extracellular matrix is composed of a complex array of molecules that together provide structural and functional support to cells. These properties are mainly mediated by the activity of collagenous and elastic fibers, proteoglycans, and proteins such as fibronectin and laminin. ECM composition is tissue-specific and could include matricellular proteins whose primary role is to modulate cell-matrix interactions. In adults, matricellular proteins are primarily expressed during injury, inflammation and disease. Particularly, they are closely associated with the progression and prognosis of cardiovascular and fibrotic diseases, and cancer. This review aims to provide an overview of the potential use of matricellular proteins in drug delivery including the generation of therapeutic agents based on the properties and structures of these proteins as well as their utility as biomarkers for specific diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Revisiting the NMR structure of the ultrafast downhill folding protein gpW from bacteriophage λ.

PubMed

Sborgi, Lorenzo; Verma, Abhinav; Muñoz, Victor; de Alba, Eva

2011-01-01

GpW is a 68-residue protein from bacteriophage λ that participates in virus head morphogenesis. Previous NMR studies revealed a novel α+β fold for this protein. Recent experiments have shown that gpW folds in microseconds by crossing a marginal free energy barrier (i.e., downhill folding). These features make gpW a highly desirable target for further experimental and computational folding studies. As a step in that direction, we have re-determined the high-resolution structure of gpW by multidimensional NMR on a construct that eliminates the purification tags and unstructured C-terminal tail present in the prior study. In contrast to the previous work, we have obtained a full manual assignment and calculated the structure using only unambiguous distance restraints. This new structure confirms the α+β topology, but reveals important differences in tertiary packing. Namely, the two α-helices are rotated along their main axis to form a leucine zipper. The β-hairpin is orthogonal to the helical interface rather than parallel, displaying most tertiary contacts through strand 1. There also are differences in secondary structure: longer and less curved helices and a hairpin that now shows the typical right-hand twist. Molecular dynamics simulations starting from both gpW structures, and calculations with CS-Rosetta, all converge to our gpW structure. This confirms that the original structure has strange tertiary packing and strained secondary structure. A comparison of NMR datasets suggests that the problems were mainly caused by incomplete chemical shift assignments, mistakes in NOE assignment and the inclusion of ambiguous distance restraints during the automated procedure used in the original study. The new gpW corrects these problems, providing the appropriate structural reference for future work. Furthermore, our results are a cautionary tale against the inclusion of ambiguous experimental information in the determination of protein structures.

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

PubMed

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

2014-01-01

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

Cellular Retinoic Acid Binding Proteins: Genomic and Non-genomic Functions and their Regulation.

PubMed

Wei, Li-Na

Cellular retinoic acid binding proteins (CRABPs) are high-affinity retinoic acid (RA) binding proteins that mainly reside in the cytoplasm. In mammals, this family has two members, CRABPI and II, both highly conserved during evolution. The two proteins share a very similar structure that is characteristic of a "β-clam" motif built up from10-strands. The proteins are encoded by two different genes that share a very similar genomic structure. CRABPI is widely distributed and CRABPII has restricted expression in only certain tissues. The CrabpI gene is driven by a housekeeping promoter, but can be regulated by numerous factors, including thyroid hormones and RA, which engage a specific chromatin-remodeling complex containing either TRAP220 or RIP140 as coactivator and corepressor, respectively. The chromatin-remodeling complex binds the DR4 element in the CrabpI gene promoter to activate or repress this gene in different cellular backgrounds. The CrabpII gene promoter contains a TATA-box and is rapidly activated by RA through an RA response element. Biochemical and cell culture studies carried out in vitro show the two proteins have distinct biological functions. CRABPII mainly functions to deliver RA to the nuclear RA receptors for gene regulation, although recent studies suggest that CRABPII may also be involved in other cellular events, such as RNA stability. In contrast, biochemical and cell culture studies suggest that CRABPI functions mainly in the cytoplasm to modulate intracellular RA availability/concentration and to engage other signaling components such as ERK activity. However, these functional studies remain inconclusive because knocking out one or both genes in mice does not produce definitive phenotypes. Further studies are needed to unambiguously decipher the exact physiological activities of these two proteins.

Crystal Structure of a Four-Layer Aggregate of Engineered TMV CP Implies the Importance of Terminal Residues for Oligomer Assembly

PubMed Central

Li, Xiangyang; Song, Baoan; Chen, Xi; Wang, Zhenchao; Zeng, Mengjiao; Yu, Dandan; Hu, Deyu; Chen, Zhuo; Jin, Linhong; Yang, Song; Yang, Caiguang; Chen, Baoen

2013-01-01

Background Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination. Methodology/Principal Findings The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CP19). Overall, N-His-TMV CP19 protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP19, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP19 layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle. Conclusion The terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA. PMID:24223721

Conservative mutation Met8 --> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I.

PubMed Central

Zhukov, I.; Jaroszewski, L.; Bierzyński, A.

2000-01-01

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process. PMID:10716179

Preparation of mesoporous silica microparticles by sol-gel/emulsion route for protein release.

PubMed

Vlasenkova, Mariya I; Dolinina, Ekaterina S; Parfenyuk, Elena V

2018-04-06

Encapsulation of therapeutic proteins into particles from appropriate material can improve both stability and delivery of the drugs, and the obtained particles can serve as a platform for development of their new oral formulations. The main goal of this work was development of sol-gel/emulsion method for preparation of silica microcapsules capable of controlled release of encapsulated protein without loss of its native structure. For this purpose, the reported in literature direct sol-gel/W/O/W emulsion method of protein encapsulation was used with some modifications, because the original method did not allow to prepare silica microcapsules capable for protein release. The particles were synthesized using sodium silicate and tetraethoxysilane as silica precursors and different compositions of oil phase. In vitro kinetics of bovine serum albumin (BSA) release in buffer (pH 7.4) was studied by Fourier transform infrared (FTIR) and fluorescence spectrometry, respectively. Structural state of encapsulated BSA and after release was evaluated. It was found that the synthesis conditions influenced substantially the porous structure of the unloaded silica particles, release properties of the BSA-loaded silica particles and structural state of the encapsulated and released protein. The modified synthesis conditions made it possible to obtain the silica particles capable of controlled release of the protein during a week without loss of the protein native structure.

Change of the structure and the digestibility of myofibrillar proteins in Nanjing dry-cured duck during processing.

PubMed

Du, Xiaojing; Sun, Yangying; Pan, Daodong; Wang, Ying; Ou, Changrong; Cao, Jinxuan

2018-06-01

To investigate the change of bioavailability and structure of myofibrillar proteins during Nanjing dry-cured duck processing, carbonyl content, sulfhydryl (SH) group, disulfide (SS) group, sodium dodecyl sulfate polyacrylamide gel electrophoresis, surface hydrophobicity, secondary structures and in vitro digestibility were determined. During processing, carbonyl content and surface hydrophobicity increased; SH turned into SS group; α-helix turned into β-sheet and random coil fractions. Protein degradation occurred during dry-curing and drying-ripening stages. The in vitro digestibility of pepsin and pancreatic proteases increased during the salt curing stage and decreased during the drying-ripening stage. The increase of digestibility could be attributed to the mild oxidation, degradation and unfolding of proteins while the decrease of digestibility was related to the intensive oxidation and aggregation of proteins. Protein degradation was not a main factor of digestibility during the drying-ripening stage. Results demonstrated that the bioavailability loss of myofibrillar proteins in Nanjing dry-cured duck occurred during the stage of drying-ripening instead of curing. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

In silico characterization and analysis of RTBP1 and NgTRF1 protein through MD simulation and molecular docking - A comparative study.

PubMed

Mukherjee, Koel; Pandey, Dev Mani; Vidyarthi, Ambarish Saran

2015-02-06

Gaining access to sequence and structure information of telomere binding proteins helps in understanding the essential biological processes involve in conserved sequence specific interaction between DNA and the proteins. Rice telomere binding protein (RTBP1) and Nicotiana glutinosa telomere repeat binding factor (NgTRF1) are helix turn helix motif type of proteins that plays role in telomeric DNA protection and length regulation. Both the proteins share same type of domain but till now there is very less communication on the in silico studies of these complete proteins.Here we intend to do a comparative study between two proteins through modeling of the complete proteins, physiochemical characterization, MD simulation and DNA-protein docking. I-TASSER and CLC protein work bench was performed to find out the protein 3D structure as well as the different parameters to characterize the proteins. MD simulation was completed by GROMOS forcefield of GROMACS for 10 ns of time stretch. The simulated 3D structures were docked with template DNA (3D DNA modeled through 3D-DART) of TTTAGGG conserved sequence motif using HADDOCK web server.Digging up all the facts about the proteins it was reveled that around 120 amino acids in the tail part was showing a good sequence similarity between the proteins. Molecular modeling, sequence characterization and secondary structure prediction also indicates the similarity between the protein's structure and sequence. The result of MD simulation highlights on the RMSD, RMSF, Rg, PCA and Energy plots which also conveys the similar type of motional behavior between them. The best complex formation for both the proteins in docking result also indicates for the first interaction site which is mainly the helix3 region of the DNA binding domain. The overall computational analysis reveals that RTBP1 and NgTRF1 proteins display good amount of similarity in their physicochemical properties, structure, dynamics and binding mode.

In Silico Characterization and Analysis of RTBP1 and NgTRF1 Protein Through MD Simulation and Molecular Docking: A Comparative Study.

PubMed

Mukherjee, Koel; Pandey, Dev Mani; Vidyarthi, Ambarish Saran

2015-09-01

Gaining access to sequence and structure information of telomere-binding proteins helps in understanding the essential biological processes involve in conserved sequence-specific interaction between DNA and the proteins. Rice telomere-binding protein (RTBP1) and Nicotiana glutinosa telomere repeat binding factor (NgTRF1) are helix-turn-helix motif type of proteins that plays role in telomeric DNA protection and length regulation. Both the proteins share same type of domain, but till now there is very less communication on the in silico studies of these complete proteins. Here we intend to do a comparative study between two proteins through modeling of the complete proteins, physiochemical characterization, MD simulation and DNA-protein docking. I-TASSER and CLC protein work bench was performed to find out the protein 3D structure as well as the different parameters to characterize the proteins. MD simulation was completed by GROMOS forcefield of GROMACS for 10 ns of time stretch. The simulated 3D structures were docked with template DNA (3D DNA modeled through 3D-DART) of TTTAGGG conserved sequence motif using HADDOCK Web server. By digging up all the facts about the proteins, it was revealed that around 120 amino acids in the tail part were showing a good sequence similarity between the proteins. Molecular modeling, sequence characterization and secondary structure prediction also indicate the similarity between the protein's structure and sequence. The result of MD simulation highlights on the RMSD, RMSF, Rg, PCA and energy plots which also conveys the similar type of motional behavior between them. The best complex formation for both the proteins in docking result also indicates for the first interaction site which is mainly the helix3 region of the DNA-binding domain. The overall computational analysis reveals that RTBP1 and NgTRF1 proteins display good amount of similarity in their physicochemical properties, structure, dynamics and binding mode.

Using Variable-Length Aligned Fragment Pairs and an Improved Transition Function for Flexible Protein Structure Alignment.

PubMed

Cao, Hu; Lu, Yonggang

2017-01-01

With the rapid growth of known protein 3D structures in number, how to efficiently compare protein structures becomes an essential and challenging problem in computational structural biology. At present, many protein structure alignment methods have been developed. Among all these methods, flexible structure alignment methods are shown to be superior to rigid structure alignment methods in identifying structure similarities between proteins, which have gone through conformational changes. It is also found that the methods based on aligned fragment pairs (AFPs) have a special advantage over other approaches in balancing global structure similarities and local structure similarities. Accordingly, we propose a new flexible protein structure alignment method based on variable-length AFPs. Compared with other methods, the proposed method possesses three main advantages. First, it is based on variable-length AFPs. The length of each AFP is separately determined to maximally represent a local similar structure fragment, which reduces the number of AFPs. Second, it uses local coordinate systems, which simplify the computation at each step of the expansion of AFPs during the AFP identification. Third, it decreases the number of twists by rewarding the situation where nonconsecutive AFPs share the same transformation in the alignment, which is realized by dynamic programming with an improved transition function. The experimental data show that compared with FlexProt, FATCAT, and FlexSnap, the proposed method can achieve comparable results by introducing fewer twists. Meanwhile, it can generate results similar to those of the FATCAT method in much less running time due to the reduced number of AFPs.

Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes.

PubMed

Stepanenko, Olesya V; Verkhusha, Vladislav V; Kuznetsova, Irina M; Uversky, Vladimir N; Turoverov, K K

2008-08-01

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.

Functional Dynamics of PDZ Binding Domains: A Normal-Mode Analysis

PubMed Central

De Los Rios, Paolo; Cecconi, Fabio; Pretre, Anna; Dietler, Giovanni; Michielin, Olivier; Piazza, Francesco; Juanico, Brice

2005-01-01

Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domains are relatively small (80–120 residues) protein binding modules central in the organization of receptor clusters and in the association of cellular proteins. Their main function is to bind C-terminals of selected proteins that are recognized through specific amino acids in their carboxyl end. Binding is associated with a deformation of the PDZ native structure and is responsible for dynamical changes in regions not in direct contact with the target. We investigate how this deformation is related to the harmonic dynamics of the PDZ structure and show that one low-frequency collective normal mode, characterized by the concerted movements of different secondary structures, is involved in the binding process. Our results suggest that even minimal structural changes are responsible for communication between distant regions of the protein, in agreement with recent NMR experiments. Thus, PDZ domains are a very clear example of how collective normal modes are able to characterize the relation between function and dynamics of proteins, and to provide indications on the precursors of binding/unbinding events. PMID:15821164

A monomeric TIM-barrel structure from Pyrococcus furiosus is optimized for extreme temperatures.

PubMed

Repo, Heidi; Oeemig, Jesper S; Djupsjöbacka, Janica; Iwaï, Hideo; Heikinheimo, Pirkko

2012-11-01

The structure of phosphoribosyl anthranilate isomerase (TrpF) from the hyperthermophilic archaeon Pyrococcus furiosus (PfTrpF) has been determined at 1.75 Å resolution. The PfTrpF structure has a monomeric TIM-barrel fold which differs from the dimeric structures of two other known thermophilic TrpF proteins. A comparison of the PfTrpF structure with the two known bacterial thermophilic TrpF structures and the structure of a related mesophilic protein from Escherichia coli (EcTrpF) is presented. The thermophilic TrpF structures contain a higher proportion of ion pairs and charged residues compared with the mesophilic EcTrpF. These residues contribute to the closure of the central barrel and the stabilization of the barrel and the surrounding α-helices. In the monomeric PfTrpF conserved structural water molecules are mostly absent; instead, the structural waters are replaced by direct side-chain-main-chain interactions. As a consequence of these combined mechanisms, the P. furiosus enzyme is a thermodynamically stable and entropically optimized monomeric TIM-barrel enzyme which defines a good framework for further protein engineering for industrial applications.

Structure, Biology, and Therapeutic Application of Toxin-Antitoxin Systems in Pathogenic Bacteria.

PubMed

Lee, Ki-Young; Lee, Bong-Jin

2016-10-22

Bacterial toxin-antitoxin (TA) systems have received increasing attention for their diverse identities, structures, and functional implications in cell cycle arrest and survival against environmental stresses such as nutrient deficiency, antibiotic treatments, and immune system attacks. In this review, we describe the biological functions and the auto-regulatory mechanisms of six different types of TA systems, among which the type II TA system has been most extensively studied. The functions of type II toxins include mRNA/tRNA cleavage, gyrase/ribosome poison, and protein phosphorylation, which can be neutralized by their cognate antitoxins. We mainly explore the similar but divergent structures of type II TA proteins from 12 important pathogenic bacteria, including various aspects of protein-protein interactions. Accumulating knowledge about the structure-function correlation of TA systems from pathogenic bacteria has facilitated a novel strategy to develop antibiotic drugs that target specific pathogens. These molecules could increase the intrinsic activity of the toxin by artificially interfering with the intermolecular network of the TA systems.

A Brief Review of West Nile Virus Biology.

PubMed

Londono-Renteria, Berlin; Colpitts, Tonya M

2016-01-01

West Nile virus (WNV) is an arbovirus with increased global incidence in the last decade. It is also a major cause of human encephalitis in the USA. WNV is an arthropod-transmitted virus that mainly affects birds but humans become infected as incidental dead-end hosts which can cause outbreaks in naïve populations. The main vectors of WNV are mosquitoes of the genus Culex, which preferentially feed on birds. As in many other arboviruses, the characteristics that allow Flaviviruses like WNV to replicate and transmit to different hosts are encrypted in their genome, which also contains information for the production of structural and nonstructural proteins needed for host cell infection. WNV and other Flaviviruses have developed different strategies to establish infection, replication, and successful transmission. Most of these strategies include the diversion of the host's immune responses away from the virus. In this review, we describe the molecular structure and protein function of WNV with emphasis on protein involvement in the modulation of antiviral immune responses.

Quality assessment of protein model-structures based on structural and functional similarities

PubMed Central

2012-01-01

Background Experimental determination of protein 3D structures is expensive, time consuming and sometimes impossible. A gap between number of protein structures deposited in the World Wide Protein Data Bank and the number of sequenced proteins constantly broadens. Computational modeling is deemed to be one of the ways to deal with the problem. Although protein 3D structure prediction is a difficult task, many tools are available. These tools can model it from a sequence or partial structural information, e.g. contact maps. Consequently, biologists have the ability to generate automatically a putative 3D structure model of any protein. However, the main issue becomes evaluation of the model quality, which is one of the most important challenges of structural biology. Results GOBA - Gene Ontology-Based Assessment is a novel Protein Model Quality Assessment Program. It estimates the compatibility between a model-structure and its expected function. GOBA is based on the assumption that a high quality model is expected to be structurally similar to proteins functionally similar to the prediction target. Whereas DALI is used to measure structure similarity, protein functional similarity is quantified using standardized and hierarchical description of proteins provided by Gene Ontology combined with Wang's algorithm for calculating semantic similarity. Two approaches are proposed to express the quality of protein model-structures. One is a single model quality assessment method, the other is its modification, which provides a relative measure of model quality. Exhaustive evaluation is performed on data sets of model-structures submitted to the CASP8 and CASP9 contests. Conclusions The validation shows that the method is able to discriminate between good and bad model-structures. The best of tested GOBA scores achieved 0.74 and 0.8 as a mean Pearson correlation to the observed quality of models in our CASP8 and CASP9-based validation sets. GOBA also obtained the best result for two targets of CASP8, and one of CASP9, compared to the contest participants. Consequently, GOBA offers a novel single model quality assessment program that addresses the practical needs of biologists. In conjunction with other Model Quality Assessment Programs (MQAPs), it would prove useful for the evaluation of single protein models. PMID:22998498

DNA and proteins of the nuclear matrix are the main targets of benzo[a]pyrene's action in rat hepatocytes.

PubMed

Widłak, P; Rzeszowska-Wolny, J

1993-01-01

The binding of [14C]benzo[a]pyrene (B[a]P) to DNA and proteins in total nuclei and subnuclear fractions of cultured rat hepatocytes was compared. The main targets of B[a]P were non-histone high molecular weight proteins of the nuclear matrix and DNA sequences attached to this structure. Following 24 h exposure to B[a]P the amounts of adducts in the nuclear matrix DNA and proteins were twice as high as in total nuclei. After withdrawal of the carcinogen containing medium the level of B[a]P-induced adducts gradually decreased but always remained the highest in the nuclear matrix proteins. Removal of adducts from the nuclear matrix DNA was more efficient than from the other DNA fractions, and 72 h after exposure to the carcinogen the level of DNA adducts in this fraction was similar to that in total nuclei.

Stacking and T-shape competition in aromatic-aromatic amino acid interactions.

PubMed

Chelli, Riccardo; Gervasio, Francesco Luigi; Procacci, Piero; Schettino, Vincenzo

2002-05-29

The potential of mean force of interacting aromatic amino acids is calculated using molecular dynamics simulations. The free energy surface is determined in order to study stacking and T-shape competition for phenylalanine-phenylalanine (Phe-Phe), phenylalanine-tyrosine (Phe-Tyr), and tyrosine-tyrosine (Tyr-Tyr) complexes in vacuo, water, carbon tetrachloride, and methanol. Stacked structures are favored in all solvents with the exception of the Tyr-Tyr complex in carbon tetrachloride, where T-shaped structures are also important. The effect of anchoring the two alpha-carbons (C(alpha)) at selected distances is investigated. We find that short and large C(alpha)-C(alpha) distances favor stacked and T-shaped structures, respectively. We analyze a set of 2396 protein structures resolved experimentally. Comparison of theoretical free energies for the complexes to the experimental analogue shows that Tyr-Tyr interaction occurs mainly at the protein surface, while Phe-Tyr and Phe-Phe interactions are more frequent in the hydrophobic protein core. This is confirmed by the Voronoi polyhedron analysis on the database protein structures. As found from the free energy calculation, analysis of the protein database has shown that proximal and distal interacting aromatic residues are predominantly stacked and T-shaped, respectively.

Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin-like proteins.

PubMed

Pérez-Munive, Clara; Blumenthal, Sonal S D; de la Espina, Susana Moreno Díaz

2012-01-01

Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled-coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross-react with anti-intermediate filament and anti-lamin antibodies, form filaments 6-12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin-like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin-like proteins by co-immunoprecipitation and co-localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with Mr and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin-like proteins. Its similarities with some of the proteins described as onion lamin-like proteins suggest that they are highly related or perhaps the same proteins.

Prediction of Protein Structural Classes for Low-Similarity Sequences Based on Consensus Sequence and Segmented PSSM.

PubMed

Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

2015-01-01

Prediction of protein structural classes for low-similarity sequences is useful for understanding fold patterns, regulation, functions, and interactions of proteins. It is well known that feature extraction is significant to prediction of protein structural class and it mainly uses protein primary sequence, predicted secondary structure sequence, and position-specific scoring matrix (PSSM). Currently, prediction solely based on the PSSM has played a key role in improving the prediction accuracy. In this paper, we propose a novel method called CSP-SegPseP-SegACP by fusing consensus sequence (CS), segmented PsePSSM, and segmented autocovariance transformation (ACT) based on PSSM. Three widely used low-similarity datasets (1189, 25PDB, and 640) are adopted in this paper. Then a 700-dimensional (700D) feature vector is constructed and the dimension is decreased to 224D by using principal component analysis (PCA). To verify the performance of our method, rigorous jackknife cross-validation tests are performed on 1189, 25PDB, and 640 datasets. Comparison of our results with the existing PSSM-based methods demonstrates that our method achieves the favorable and competitive performance. This will offer an important complementary to other PSSM-based methods for prediction of protein structural classes for low-similarity sequences.

Structure of the ordered hydration of amino acids in proteins: analysis of crystal structures

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

Biedermannová, Lada, E-mail: lada.biedermannova@ibt.cas.cz; Schneider, Bohdan

2015-10-27

The hydration of protein crystal structures was studied at the level of individual amino acids. The dependence of the number of water molecules and their preferred spatial localization on various parameters, such as solvent accessibility, secondary structure and side-chain conformation, was determined. Crystallography provides unique information about the arrangement of water molecules near protein surfaces. Using a nonredundant set of 2818 protein crystal structures with a resolution of better than 1.8 Å, the extent and structure of the hydration shell of all 20 standard amino-acid residues were analyzed as function of the residue conformation, secondary structure and solvent accessibility. Themore » results show how hydration depends on the amino-acid conformation and the environment in which it occurs. After conformational clustering of individual residues, the density distribution of water molecules was compiled and the preferred hydration sites were determined as maxima in the pseudo-electron-density representation of water distributions. Many hydration sites interact with both main-chain and side-chain amino-acid atoms, and several occurrences of hydration sites with less canonical contacts, such as carbon–donor hydrogen bonds, OH–π interactions and off-plane interactions with aromatic heteroatoms, are also reported. Information about the location and relative importance of the empirically determined preferred hydration sites in proteins has applications in improving the current methods of hydration-site prediction in molecular replacement, ab initio protein structure prediction and the set-up of molecular-dynamics simulations.« 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

β-structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation.

PubMed

Dobrov, Evgeny N; Nikitin, Nikolai A; Trifonova, Ekaterina A; Parshina, Evgenia Yu; Makarov, Valentin V; Maksimov, George V; Karpova, Olga V; Atabekov, Joseph G

2014-01-01

Conversion of the rod-like tobacco mosaic virus (TMV) virions into "ball-like particles" by thermal denaturation at 90-98 °C had been described by R.G. Hart in 1956. We have reported recently that spherical particles (SPs) generated by thermal denaturation of TMV at 94-98 °C were highly stable, RNA-free, and water-insoluble. The SPs were uniform in shape but varied widely in size (53-800 nm), which depended on the virus concentration. Here, we describe some structural characteristics of SPs using circular dichroism, fluorescence spectroscopy, and Raman spectroscopy. It was found that the structure of SPs protein differs strongly from that of the native TMV and is characterized by coat protein subunits transition from mainly (about 50%) α-helical structure to a structure with low content of α-helices and a significant fraction of β-sheets. The SPs demonstrate strong reaction with thioflavin T suggesting the formation of amyloid-like structures.

Protein structure prediction with local adjust tabu search algorithm

PubMed Central

2014-01-01

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

Keratins and lipids in ethnic hair.

PubMed

Cruz, C F; Fernandes, M M; Gomes, A C; Coderch, L; Martí, M; Méndez, S; Gales, L; Azoia, N G; Shimanovich, U; Cavaco-Paulo, A

2013-06-01

Human hair has an important and undeniable relevance in society due to its important role in visual appearance and social communication. Hair is mainly composed of structural proteins, mainly keratin and keratin associated proteins and lipids. Herein, we report a comprehensive study of the content and distribution of the lipids among ethnic hair, African, Asian and Caucasian hair. More interestingly, we also report the study of the interaction between those two main components of hair, specifically, the influence of the hair internal lipids in the structure of the hair keratin. This was achieved by the use of a complete set of analytical tools, such as thin layer chromatography-flame ionization detector, X-ray analysis, molecular dynamics simulation and confocal microscopy. The experimental results indicated different amounts of lipids on ethnic hair compositions and higher percentage of hair internal lipids in African hair. In this type of hair, the axial diffraction of keratin was not observed in X-ray analysis, but after hair lipids removal, the keratin returned to its typical packing arrangement. In molecular dynamic simulation, lipids were shown to intercalate dimers of keratin, changing its structure. From those results, we assume that keratin structure may be influenced by higher concentration of lipids in African hair. © 2013 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Molecular modeling of the AhR structure and interactions can shed light on ligand-dependent activation and transformation mechanisms.

PubMed

Bonati, Laura; Corrada, Dario; Tagliabue, Sara Giani; Motta, Stefano

2017-02-01

Molecular modeling has given important contributions to elucidation of the main stages in the AhR signal transduction pathway. Despite the lack of experimentally determined structures of the AhR functional domains, information derived from homologous systems has been exploited for modeling their structure and interactions. Homology models of the AhR PASB domain have provided information on the binding cavity and contributed to elucidate species-specific differences in ligand binding. Molecular Docking simulations of the ligand binding process have given insights into differences in binding of diverse agonists, antagonists, and selective AhR modulators, and their application to virtual screening of large databases of compounds have allowed identification of novel AhR ligands. Recently available structural information on protein-protein and protein-DNA complexes of other bHLH-PAS systems has opened the way for modeling the AhR:ARNT dimer structure and investigating the mechanisms of AhR transformation and DNA binding. Future research directions should include simulation of the protein dynamics to obtain a more reliable description of intermolecular interactions involved in signal transmission.

Structural alignment of protein descriptors - a combinatorial model.

PubMed

Antczak, Maciej; Kasprzak, Marta; Lukasiak, Piotr; Blazewicz, Jacek

2016-09-17

Structural alignment of proteins is one of the most challenging problems in molecular biology. The tertiary structure of a protein strictly correlates with its function and computationally predicted structures are nowadays a main premise for understanding the latter. However, computationally derived 3D models often exhibit deviations from the native structure. A way to confirm a model is a comparison with other structures. The structural alignment of a pair of proteins can be defined with the use of a concept of protein descriptors. The protein descriptors are local substructures of protein molecules, which allow us to divide the original problem into a set of subproblems and, consequently, to propose a more efficient algorithmic solution. In the literature, one can find many applications of the descriptors concept that prove its usefulness for insight into protein 3D structures, but the proposed approaches are presented rather from the biological perspective than from the computational or algorithmic point of view. Efficient algorithms for identification and structural comparison of descriptors can become crucial components of methods for structural quality assessment as well as tertiary structure prediction. In this paper, we propose a new combinatorial model and new polynomial-time algorithms for the structural alignment of descriptors. The model is based on the maximum-size assignment problem, which we define here and prove that it can be solved in polynomial time. We demonstrate suitability of this approach by comparison with an exact backtracking algorithm. Besides a simplification coming from the combinatorial modeling, both on the conceptual and complexity level, we gain with this approach high quality of obtained results, in terms of 3D alignment accuracy and processing efficiency. All the proposed algorithms were developed and integrated in a computationally efficient tool descs-standalone, which allows the user to identify and structurally compare descriptors of biological molecules, such as proteins and RNAs. Both PDB (Protein Data Bank) and mmCIF (macromolecular Crystallographic Information File) formats are supported. The proposed tool is available as an open source project stored on GitHub ( https://github.com/mantczak/descs-standalone ).

X-ray crystallographic studies of RNase A variants engineered at the most destabilizing positions of the main hydrophobic core: further insight into protein stability.

PubMed

Kurpiewska, Katarzyna; Font, Josep; Ribó, Marc; Vilanova, Maria; Lewiński, Krzysztof

2009-11-15

To investigate the structural origin of decreased pressure and temperature stability, the crystal structure of bovine pancreatic ribonuclease A variants V47A, V54A, V57A, I81A, I106A, and V108A was solved at 1.4-2.0 A resolution and compared with the structure of wild-type protein. The introduced mutations had only minor influence on the global structure of ribonuclease A. The structural changes had individual character that depends on the localization of mutated residue, however, they seemed to expand from mutation site to the rest of the structure. Several different parameters have been evaluated to find correlation with decrease of free energy of unfolding DeltaDeltaG(T), and the most significant correlation was found for main cavity volume change. Analysis of the difference distance matrices revealed that the ribonuclease A molecule is organized into five relatively rigid subdomains with individual response to mutation. This behavior consistent with results of unfolding experiments is an intrinsic feature of ribonuclease A that might be surviving remnants of folding intermediates and reflects the dynamic nature of the molecule. 2009 Wiley-Liss, Inc.

Caseoperoxidase, mixed β-casein-SDS-hemin-imidazole complex: a nano artificial enzyme.

PubMed

Moosavi-Movahedi, Zainab; Gharibi, Hussein; Hadi-Alijanvand, Hamid; Akbarzadeh, Mohammad; Esmaili, Mansoore; Atri, Maliheh S; Sefidbakht, Yahya; Bohlooli, Mousa; Nazari, Khodadad; Javadian, Soheila; Hong, Jun; Saboury, Ali A; Sheibani, Nader; Moosavi-Movahedi, Ali A

2015-01-01

A novel peroxidase-like artificial enzyme, named "caseoperoxidase", was biomimetically designed using a nano artificial amino acid apo-protein hydrophobic pocket. This four-component nano artificial enzyme containing heme-imidazole-β-casein-SDS exhibited high activity growth and k(cat) performance toward the native horseradish peroxidase demonstrated by the steady state kinetics using UV-vis spectrophotometry. The hydrophobicity and secondary structure of the caseoperoxidase were studied by ANS fluorescence and circular dichroism spectroscopy. Camel β-casein (Cβ-casein) was selected as an appropriate apo-protein for the heme active site because of its innate flexibility and exalted hydrophobicity. This selection was confirmed by homology modeling method. Heme docking into the newly obtained Cβ-casein structure indicated one heme was mainly incorporated with Cβ-casein. The presence of a main electrostatic site for the active site in the Cβ-casein was also confirmed by experimental methods through Wyman binding potential and isothermal titration calorimetry. The existence of Cβ-casein protein in this biocatalyst lowered the suicide inactivation and provided a suitable protective role for the heme active-site. Additional experiments confirmed the retention of caseoperoxidase structure and function as an artificial enzyme.

The effect of high pressure on the functional properties of pork myofibrillar proteins.

PubMed

Grossi, Alberto; Olsen, Karsten; Bolumar, Tomas; Rinnan, Åsmund; Øgendal, Lars H; Orlien, Vibeke

2016-04-01

Complementary methodologies were used to analyse the pressure-induced modification and functionality of myofibrillar proteins from pork meat pressurised at 200, 400, 600, or 800 MPa (10 min, 5 or 20 °C). Pressure at 400 MPa was found to be the threshold for loss of solubility, and the structural proteins, myosin and actin, lost their native solubility due to aggregation. The results from the extraction of proteins with different reagents targeting the disruption of specific molecular interactions suggested that pressure-induced aggregation was caused mainly by hydrogen bonding during pressurisation and not hydrophobic interactions nor disulphide cross-links. Furthermore, the soluble proteins were exposed to remarkable structural changes already at 200 MPa and lost their native functionality. The modification of the proteins in pressurised meat affected the water binding sites of the myofibrillar proteins and, thereby, the interactions between proteins and water molecules, and distribution between myofibrillar and extra-myofibrillar compartments. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular dynamics simulations and CD spectroscopy reveal hydration-induced unfolding of the intrinsically disordered LEA proteins COR15A and COR15B from Arabidopsis thaliana.

PubMed

Navarro-Retamal, Carlos; Bremer, Anne; Alzate-Morales, Jans; Caballero, Julio; Hincha, Dirk K; González, Wendy; Thalhammer, Anja

2016-10-07

The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain α-helical structure during dehydration, which is reversible upon rehydration. To understand this unusual structural transition, both proteins were investigated by circular dichroism (CD) and molecular dynamics (MD) approaches. MD simulations showed unfolding of the proteins in water, in agreement with CD data obtained with both HIS-tagged and untagged recombinant proteins. Mainly intramolecular hydrogen bonds (H-bonds) formed by the protein backbone were replaced by H-bonds with water molecules. As COR15 proteins function in vivo as protectants in leaves partially dehydrated by freezing, unfolding was further assessed under crowded conditions. Glycerol reduced (40%) or prevented (100%) unfolding during MD simulations, in agreement with CD spectroscopy results. H-bonding analysis indicated that preferential exclusion of glycerol from the protein backbone increased stability of the folded state.

Structure-guided Protein Transition Modeling with a Probabilistic Roadmap Algorithm.

PubMed

Maximova, Tatiana; Plaku, Erion; Shehu, Amarda

2016-07-07

Proteins are macromolecules in perpetual motion, switching between structural states to modulate their function. A detailed characterization of the precise yet complex relationship between protein structure, dynamics, and function requires elucidating transitions between functionally-relevant states. Doing so challenges both wet and dry laboratories, as protein dynamics involves disparate temporal scales. In this paper we present a novel, sampling-based algorithm to compute transition paths. The algorithm exploits two main ideas. First, it leverages known structures to initialize its search and define a reduced conformation space for rapid sampling. This is key to address the insufficient sampling issue suffered by sampling-based algorithms. Second, the algorithm embeds samples in a nearest-neighbor graph where transition paths can be efficiently computed via queries. The algorithm adapts the probabilistic roadmap framework that is popular in robot motion planning. In addition to efficiently computing lowest-cost paths between any given structures, the algorithm allows investigating hypotheses regarding the order of experimentally-known structures in a transition event. This novel contribution is likely to open up new venues of research. Detailed analysis is presented on multiple-basin proteins of relevance to human disease. Multiscaling and the AMBER ff14SB force field are used to obtain energetically-credible paths at atomistic detail.

Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching.

PubMed

Harrison, Rane A; Lu, Jia; Carrasco, Martin; Hunter, John; Manandhar, Anuj; Gondi, Sudershan; Westover, Kenneth D; Engen, John R

2016-11-20

Structural dynamics of Ras proteins contributes to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on the movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras-targeting strategies, experimental methods to measure Ras dynamics are required. Here, we demonstrate the utility of hydrogen-deuterium exchange (HDX) mass spectrometry (MS) to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main-chain movement. HDX provides a means of evaluating Ras protein dynamics, which may be useful for understanding the mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Generation of a consensus protein domain dictionary

PubMed Central

Schaeffer, R. Dustin; Jonsson, Amanda L.; Simms, Andrew M.; Daggett, Valerie

2011-01-01

Motivation: The discovery of new protein folds is a relatively rare occurrence even as the rate of protein structure determination increases. This rarity reinforces the concept of folds as reusable units of structure and function shared by diverse proteins. If the folding mechanism of proteins is largely determined by their topology, then the folding pathways of members of existing folds could encompass the full set used by globular protein domains. Results: We have used recent versions of three common protein domain dictionaries (SCOP, CATH and Dali) to generate a consensus domain dictionary (CDD). Surprisingly, 40% of the metafolds in the CDD are not composed of autonomous structural domains, i.e. they are not plausible independent folding units. This finding has serious ramifications for bioinformatics studies mining these domain dictionaries for globular protein properties. However, our main purpose in deriving this CDD was to generate an updated CDD to choose targets for MD simulation as part of our dynameomics effort, which aims to simulate the native and unfolding pathways of representatives of all globular protein consensus folds (metafolds). Consequently, we also compiled a list of representative protein targets of each metafold in the CDD. Availability and implementation: This domain dictionary is available at www.dynameomics.org. Contact: daggett@u.washington.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:21068000

Computational protein design: a review

NASA Astrophysics Data System (ADS)

Coluzza, Ivan

2017-04-01

Proteins are one of the most versatile modular assembling systems in nature. Experimentally, more than 110 000 protein structures have been identified and more are deposited every day in the Protein Data Bank. Such an enormous structural variety is to a first approximation controlled by the sequence of amino acids along the peptide chain of each protein. Understanding how the structural and functional properties of the target can be encoded in this sequence is the main objective of protein design. Unfortunately, rational protein design remains one of the major challenges across the disciplines of biology, physics and chemistry. The implications of solving this problem are enormous and branch into materials science, drug design, evolution and even cryptography. For instance, in the field of drug design an effective computational method to design protein-based ligands for biological targets such as viruses, bacteria or tumour cells, could give a significant boost to the development of new therapies with reduced side effects. In materials science, self-assembly is a highly desired property and soon artificial proteins could represent a new class of designable self-assembling materials. The scope of this review is to describe the state of the art in computational protein design methods and give the reader an outline of what developments could be expected in the near future.

Biological and functional relevance of CASP predictions.

PubMed

Liu, Tianyun; Ish-Shalom, Shirbi; Torng, Wen; Lafita, Aleix; Bock, Christian; Mort, Matthew; Cooper, David N; Bliven, Spencer; Capitani, Guido; Mooney, Sean D; Altman, Russ B

2018-03-01

Our goal is to answer the question: compared with experimental structures, how useful are predicted models for functional annotation? We assessed the functional utility of predicted models by comparing the performances of a suite of methods for functional characterization on the predictions and the experimental structures. We identified 28 sites in 25 protein targets to perform functional assessment. These 28 sites included nine sites with known ligand binding (holo-sites), nine sites that are expected or suggested by experimental authors for small molecule binding (apo-sites), and Ten sites containing important motifs, loops, or key residues with important disease-associated mutations. We evaluated the utility of the predictions by comparing their microenvironments to the experimental structures. Overall structural quality correlates with functional utility. However, the best-ranked predictions (global) may not have the best functional quality (local). Our assessment provides an ability to discriminate between predictions with high structural quality. When assessing ligand-binding sites, most prediction methods have higher performance on apo-sites than holo-sites. Some servers show consistently high performance for certain types of functional sites. Finally, many functional sites are associated with protein-protein interaction. We also analyzed biologically relevant features from the protein assemblies of two targets where the active site spanned the protein-protein interface. For the assembly targets, we find that the features in the models are mainly determined by the choice of template. © 2017 The Authors Proteins: Structure, Function and Bioinformatics Published by Wiley Periodicals, Inc.

Extracellular matrix proteins in healthy and retained placentas, comparing hemochorial and synepitheliochorial placentas.

PubMed

Franczyk, M; Lopucki, M; Stachowicz, N; Morawska, D; Kankofer, M

2017-02-01

The placenta expresses structural and biologically active proteins. Their synthesis is mainly regulated by genomic or nongenomic signals and modulated by hormones. These protein profiles are altered during different stages of pregnancy. The biological properties of extracellular matrix (ECM) proteins were defined and described in a number of tissues including placenta. These properties enable them to be the main players in the processes of attachment or invasion into the endometrium during initial placenta formation and its timely separation after delivery and detachment. In this review, we focused on the role of ECM proteins during attachment of the placenta to the uterine wall, its timely separation, and the implications of this process on retained or pathologically attached placenta. Although the amount of published information in this area is relatively scant, some of the key proteins and processes are well defined. We focused on the available data detailing the ECM protein profiles of human (histologically thin; hemochorial) and bovine (histologically thick; epitheliochorial) placentas and compared the shared and unique ECM proteins that are relevant to placental attachment and separation. Copyright © 2016 Elsevier Ltd. All rights reserved.

A glycoconjugate of Haemophilus influenzae Type b capsular polysaccharide with tetanus toxoid protein: hydrodynamic properties mainly influenced by the carbohydrate.

PubMed

Abdelhameed, Ali Saber; Adams, Gary G; Morris, Gordon A; Almutairi, Fahad M; Duvivier, Pierre; Conrath, Karel; Harding, Stephen E

2016-02-26

Three important physical properties which may affect the performance of glycoconjugate vaccines against serious disease are molar mass (molecular weight), heterogeneity (polydispersity), and conformational flexibility in solution. The dilute solution behaviour of native and activated capsular polyribosylribitol (PRP) polysaccharides extracted from Haemophilus influenzae type b (Hib), and the corresponding glycoconjugate made by conjugating this with the tetanus toxoid (TT) protein have been characterized and compared using a combination of sedimentation equilibrium and sedimentation velocity in the analytical ultracentrifuge with viscometry. The weight average molar mass of the activated material was considerably reduced (Mw ~ 0.24 × 10(6) g.mol(-1)) compared to the native (Mw ~ 1.2 × 10(6) g.mol(-1)). Conjugation with the TT protein yielded large polydisperse structures (of Mw ~ 7.4 × 10(6) g.mol(-1)), but which retained the high degree of flexibility of the native and activated polysaccharide, with frictional ratio, intrinsic viscosity, sedimentation conformation zoning behaviour and persistence length all commensurate with highly flexible coil behaviour and unlike the previously characterised tetanus toxoid protein (slightly extended and hydrodynamically compact structure with an aspect ratio of ~3). This non-protein like behaviour clearly indicates that it is the carbohydrate component which mainly influences the physical behaviour of the glycoconjugate in solution.

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.

Reconstruction of the experimentally supported human protein interactome: what can we learn?

PubMed

Klapa, Maria I; Tsafou, Kalliopi; Theodoridis, Evangelos; Tsakalidis, Athanasios; Moschonas, Nicholas K

2013-10-02

Understanding the topology and dynamics of the human protein-protein interaction (PPI) network will significantly contribute to biomedical research, therefore its systematic reconstruction is required. Several meta-databases integrate source PPI datasets, but the protein node sets of their networks vary depending on the PPI data combined. Due to this inherent heterogeneity, the way in which the human PPI network expands via multiple dataset integration has not been comprehensively analyzed. We aim at assembling the human interactome in a global structured way and exploring it to gain insights of biological relevance. First, we defined the UniProtKB manually reviewed human "complete" proteome as the reference protein-node set and then we mined five major source PPI datasets for direct PPIs exclusively between the reference proteins. We updated the protein and publication identifiers and normalized all PPIs to the UniProt identifier level. The reconstructed interactome covers approximately 60% of the human proteome and has a scale-free structure. No apparent differentiating gene functional classification characteristics were identified for the unrepresented proteins. The source dataset integration augments the network mainly in PPIs. Polyubiquitin emerged as the highest-degree node, but the inclusion of most of its identified PPIs may be reconsidered. The high number (>300) of connections of the subsequent fifteen proteins correlates well with their essential biological role. According to the power-law network structure, the unrepresented proteins should mainly have up to four connections with equally poorly-connected interactors. Reconstructing the human interactome based on the a priori definition of the protein nodes enabled us to identify the currently included part of the human "complete" proteome, and discuss the role of the proteins within the network topology with respect to their function. As the network expansion has to comply with the scale-free theory, we suggest that the core of the human interactome has essentially emerged. Thus, it could be employed in systems biology and biomedical research, despite the considerable number of currently unrepresented proteins. The latter are probably involved in specialized physiological conditions, justifying the scarcity of related PPI information, and their identification can assist in designing relevant functional experiments and targeted text mining algorithms.

On the Development, Characterization, and Use of Protein Fluorescence and Infrared Spectroscopic Probes

NASA Astrophysics Data System (ADS)

Hilaire, Mary Rose

Proteins possess unique physical and chemical properties that allow them to carry out a wide variety of biological activities and functions. While it is generally understood that a protein's function is dictated by its structure and dynamics, arriving at a molecule-level understanding of the underlying structure-dynamics-function relationship still poses a challenging task in many cases. This is due, at least in part, to the fact that we lack the ability to take snapshots along the reaction coordinate of proteins with sufficient temporal and structural resolution. Therefore, to improve one's ability to acquire site-specific structural and/or environmental information of proteins via either infrared (IR) or fluorescence spectroscopy, the main focus of this thesis is to develop and characterize amino acid-based spectroscopic probes as well as to use such probes to study important biological questions. Specifically, we show that (1) p-cyanophenylalanine and selenomethionine constitute an efficient fluorophore-quencher pair, useful for characterizing protein conformational changes that occur on a short distance; (2) 4-cyanotryptophan is a novel blue fluorescent amino acid, applicable for biological imaging due to its unique photophysical properties; (3) the dielectric constant inside the hydrophobic interior of staphylococcal nuclease is about 10-15, significantly larger than previously assumed; and (4) a single mutation in a short segment of the protein transthyretin (i.e., 110-115) induces formation of amyloid fibrils consisting of both beta- and alpha-sheets, where the latter is a proposed structure in proteins, but has never been observed previously.

A protein-dependent side-chain rotamer library.

PubMed

Bhuyan, Md Shariful Islam; Gao, Xin

2011-12-14

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

The Use of Polymerized Genipin for the Stabilization of the Collagen Structure of Animal Hides

USDA-ARS?s Scientific Manuscript database

Animal hides are the major byproduct of meat industry and the collagen fibers is the main constituent. Crosslinkers play a key role in stabilizing the collagen structure for useful applications. Genipin is widely used as an ideal biological protein crosslinking agent due to its low toxicity compare...

Emerging facets of prokaryotic glycosylation

PubMed Central

Schäffer, Christina; Messner, Paul

2017-01-01

Glycosylation of proteins is one of the most prevalent post-translational modifications occurring in nature, with a wide repertoire of biological implications. Pathways for the main types of this modification, the N- and O-glycosylation, can be found in all three domains of life—the Eukarya, Bacteria and Archaea—thereby following common principles, which are valid also for lipopolysaccharides, lipooligosaccharides and glycopolymers. Thus, studies on any glycoconjugate can unravel novel facets of the still incompletely understood fundamentals of protein N- and O-glycosylation. While it is estimated that more than two-thirds of all eukaryotic proteins would be glycosylated, no such estimate is available for prokaryotic glycoproteins, whose understanding is lagging behind, mainly due to the enormous variability of their glycan structures and variations in the underlying glycosylation processes. Combining glycan structural information with bioinformatic, genetic, biochemical and enzymatic data has opened up an avenue for in-depth analyses of glycosylation processes as a basis for glycoengineering endeavours. Here, the common themes of glycosylation are conceptualised for the major classes of prokaryotic (i.e. bacterial and archaeal) glycoconjugates, with a special focus on glycosylated cell-surface proteins. We describe the current knowledge of biosynthesis and importance of these glycoconjugates in selected pathogenic and beneficial microbes. PMID:27566466

Ferritin family proteins and their use in bionanotechnology

PubMed Central

He, Didi; Marles-Wright, Jon

2015-01-01

Ferritin family proteins are found in all kingdoms of life and act to store iron within a protein cage and to protect the cell from oxidative damage caused by the Fenton reaction. The structural and biochemical features of the ferritins have been widely exploited in bionanotechnology applications: from the production of metal nanoparticles; as templates for semi-conductor production; and as scaffolds for vaccine design and drug delivery. In this review we first discuss the structural properties of the main ferritin family proteins, and describe how their organisation specifies their functions. Second, we describe materials science applications of ferritins that rely on their ability to sequester metal within their cavities. Finally, we explore the use of ferritin as a container for drug delivery and as a scaffold for the production of vaccines. PMID:25573765

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

PubMed

Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

2017-01-01

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Reduced Fragment Diversity for Alpha and Alpha-Beta Protein Structure Prediction using Rosetta.

PubMed

Abbass, Jad; Nebel, Jean-Christophe

2017-01-01

Protein structure prediction is considered a main challenge in computational biology. The biannual international competition, Critical Assessment of protein Structure Prediction (CASP), has shown in its eleventh experiment that free modelling target predictions are still beyond reliable accuracy, therefore, much effort should be made to improve ab initio methods. Arguably, Rosetta is considered as the most competitive method when it comes to targets with no homologues. Relying on fragments of length 9 and 3 from known structures, Rosetta creates putative structures by assembling candidate fragments. Generally, the structure with the lowest energy score, also known as first model, is chosen to be the "predicted one". A thorough study has been conducted on the role and diversity of 3-mers involved in Rosetta's model "refinement" phase. Usage of the standard number of 3-mers - i.e. 200 - has been shown to degrade alpha and alpha-beta protein conformations initially achieved by assembling 9-mers. Therefore, a new prediction pipeline is proposed for Rosetta where the "refinement" phase is customised according to a target's structural class prediction. Over 8% improvement in terms of first model structure accuracy is reported for alpha and alpha-beta classes when decreasing the number of 3- mers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

The dual role of fragments in fragment-assembly methods for de novo protein structure prediction

PubMed Central

Handl, Julia; Knowles, Joshua; Vernon, Robert; Baker, David; Lovell, Simon C.

2013-01-01

In fragment-assembly techniques for protein structure prediction, models of protein structure are assembled from fragments of known protein structures. This process is typically guided by a knowledge-based energy function and uses a heuristic optimization method. The fragments play two important roles in this process: they define the set of structural parameters available, and they also assume the role of the main variation operators that are used by the optimiser. Previous analysis has typically focused on the first of these roles. In particular, the relationship between local amino acid sequence and local protein structure has been studied by a range of authors. The correlation between the two has been shown to vary with the window length considered, and the results of these analyses have informed directly the choice of fragment length in state-of-the-art prediction techniques. Here, we focus on the second role of fragments and aim to determine the effect of fragment length from an optimization perspective. We use theoretical analyses to reveal how the size and structure of the search space changes as a function of insertion length. Furthermore, empirical analyses are used to explore additional ways in which the size of the fragment insertion influences the search both in a simulation model and for the fragment-assembly technique, Rosetta. PMID:22095594

Mapping the Geometric Evolution of Protein Folding Motor.

PubMed

Jerath, Gaurav; Hazam, Prakash Kishore; Shekhar, Shashi; Ramakrishnan, Vibin

2016-01-01

Polypeptide chain has an invariant main-chain and a variant side-chain sequence. How the side-chain sequence determines fold in terms of its chemical constitution has been scrutinized extensively and verified periodically. However, a focussed investigation on the directive effect of side-chain geometry may provide important insights supplementing existing algorithms in mapping the geometrical evolution of protein chains and its structural preferences. Geometrically, folding of protein structure may be envisaged as the evolution of its geometric variables: ϕ, and ψ dihedral angles of polypeptide main-chain directed by χ1, and χ2 of side chain. In this work, protein molecule is metaphorically modelled as a machine with 4 rotors ϕ, ψ, χ1 and χ2, with its evolution to the functional fold is directed by combinations of its rotor directions. We observe that differential rotor motions lead to different secondary structure formations and the combinatorial pattern is unique and consistent for particular secondary structure type. Further, we found that combination of rotor geometries of each amino acid is unique which partly explains how different amino acid sequence combinations have unique structural evolution and functional adaptation. Quantification of these amino acid rotor preferences, resulted in the generation of 3 substitution matrices, which later on plugged in the BLAST tool, for evaluating their efficiency in aligning sequences. We have employed BLOSUM62 and PAM30 as standard for primary evaluation. Generation of substitution matrices is a logical extension of the conceptual framework we attempted to build during the development of this work. Optimization of matrices following the conventional routines and possible application with biologically relevant data sets are beyond the scope of this manuscript, though it is a part of the larger project design.

Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.

PubMed Central

Slama-Schwok, A; Zakrzewska, K; Léger, G; Leroux, Y; Takahashi, M; Käs, E; Debey, P

2000-01-01

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation. PMID:10777751

Fluorescent Proteins as Biomarkers and Biosensors: Throwing Color Lights on Molecular and Cellular Processes

PubMed Central

Stepanenko, Olesya V.; Verkhusha, Vladislav V.; Kuznetsova, Irina M.; Uversky, Vladimir N.; Turoverov, K.K.

2010-01-01

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology. PMID:18691124

Structure solution of DNA-binding proteins and complexes with ARCIMBOLDO libraries

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

Pröpper, Kevin; Instituto de Biologia Molecular de Barcelona; Meindl, Kathrin

2014-06-01

The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described. Protein–DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein–DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms. An increasing number of high-quality DNA-binding protein structure determinations have been witnessed despite themore » fact that the crystallographic particularities of nucleic acids tend to pose specific challenges to methods primarily developed for proteins. Crystallographic structure solution of protein–DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The potential of the structure-solution program ARCIMBOLDO for the solution of protein–DNA complexes has therefore been assessed. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE. Whereas for typical proteins main-chain α-helices provide the ideal, almost ubiquitous, small fragments to start searches, in the case of DNA complexes the binding motifs and DNA double helix constitute suitable search fragments. The aim of this work is to provide an effective library of search fragments as well as to determine the optimal ARCIMBOLDO strategy for the solution of this class of structures.« less

How a mycoparasite employs g-protein signaling: using the example of trichoderma.

PubMed

Omann, Markus; Zeilinger, Susanne

2010-01-01

Mycoparasitic Trichoderma spp. act as potent biocontrol agents against a number of plant pathogenic fungi, whereupon the mycoparasitic attack includes host recognition followed by infection structure formation and secretion of lytic enzymes and antifungal metabolites leading to the host's death. Host-derived signals are suggested to be recognized by receptors located on the mycoparasite's cell surface eliciting an internal signal transduction cascade which results in the transcription of mycoparasitism-relevant genes. Heterotrimeric G proteins of fungi transmit signals originating from G-protein-coupled receptors mainly to the cAMP and the MAP kinase pathways resulting in regulation of downstream effectors. Components of the G-protein signaling machinery such as Gα subunits and G-protein-coupled receptors were recently shown to play crucial roles in Trichoderma mycoparasitism as they govern processes such as the production of extracellular cell wall lytic enzymes, the secretion of antifungal metabolites, and the formation of infection structures.

How a Mycoparasite Employs G-Protein Signaling: Using the Example of Trichoderma

PubMed Central

Omann, Markus; Zeilinger, Susanne

2010-01-01

Mycoparasitic Trichoderma spp. act as potent biocontrol agents against a number of plant pathogenic fungi, whereupon the mycoparasitic attack includes host recognition followed by infection structure formation and secretion of lytic enzymes and antifungal metabolites leading to the host's death. Host-derived signals are suggested to be recognized by receptors located on the mycoparasite's cell surface eliciting an internal signal transduction cascade which results in the transcription of mycoparasitism-relevant genes. Heterotrimeric G proteins of fungi transmit signals originating from G-protein-coupled receptors mainly to the cAMP and the MAP kinase pathways resulting in regulation of downstream effectors. Components of the G-protein signaling machinery such as Gα subunits and G-protein-coupled receptors were recently shown to play crucial roles in Trichoderma mycoparasitism as they govern processes such as the production of extracellular cell wall lytic enzymes, the secretion of antifungal metabolites, and the formation of infection structures. PMID:21637351

TAP score: torsion angle propensity normalization applied to local protein structure evaluation

PubMed Central

Tosatto, Silvio CE; Battistutta, Roberto

2007-01-01

Background Experimentally determined protein structures may contain errors and require validation. Conformational criteria based on the Ramachandran plot are mainly used to distinguish between distorted and adequately refined models. While the readily available criteria are sufficient to detect totally wrong structures, establishing the more subtle differences between plausible structures remains more challenging. Results A new criterion, called TAP score, measuring local sequence to structure fitness based on torsion angle propensities normalized against the global minimum and maximum is introduced. It is shown to be more accurate than previous methods at estimating the validity of a protein model in terms of commonly used experimental quality parameters on two test sets representing the full PDB database and a subset of obsolete PDB structures. Highly selective TAP thresholds are derived to recognize over 90% of the top experimental structures in the absence of experimental information. Both a web server and an executable version of the TAP score are available at . Conclusion A novel procedure for energy normalization (TAP) has significantly improved the possibility to recognize the best experimental structures. It will allow the user to more reliably isolate problematic structures in the context of automated experimental structure determination. PMID:17504537

Targeting of a Nicotiana plumbaginifolia H+ -ATPase to the plasma membrane is not by default and requires cytosolic structural determinants.

PubMed

Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

2004-07-01

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

Targeting of a Nicotiana plumbaginifolia H+-ATPase to the Plasma Membrane Is Not by Default and Requires Cytosolic Structural Determinants

PubMed Central

Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

2004-01-01

The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+-ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane–span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting. PMID:15208389

Exploring the aggregation free energy landscape of the amyloid-β protein (1–40)

PubMed Central

Zheng, Weihua; Tsai, Min-Yeh; Chen, Mingchen; Wolynes, Peter G.

2016-01-01

A predictive coarse-grained protein force field [associative memory, water-mediated, structure, and energy model for molecular dynamics (AWSEM)-MD] is used to study the energy landscapes and relative stabilities of amyloid-β protein (1–40) in the monomer and all of its oligomeric forms up to an octamer. We find that an isolated monomer is mainly disordered with a short α-helix formed at the central hydrophobic core region (L17-D23). A less stable hairpin structure, however, becomes increasingly more stable in oligomers, where hydrogen bonds can form between neighboring monomers. We explore the structure and stability of both prefibrillar oligomers that consist of mainly antiparallel β-sheets and fibrillar oligomers with only parallel β-sheets. Prefibrillar oligomers are polymorphic but typically take on a cylindrin-like shape composed of mostly antiparallel β-strands. At the concentration of the simulation, the aggregation free energy landscape is nearly downhill. We use umbrella sampling along a structural progress coordinate for interconversion between prefibrillar and fibrillar forms to identify a conversion pathway between these forms. The fibrillar oligomer only becomes favored over its prefibrillar counterpart in the pentamer where an interconversion bottleneck appears. The structural characterization of the pathway along with statistical mechanical perturbation theory allow us to evaluate the effects of concentration on the free energy landscape of aggregation as well as the effects of the Dutch and Arctic mutations associated with early onset of Alzheimer’s disease. PMID:27698130

Structure and non-structure of centrosomal proteins.

PubMed

Dos Santos, Helena G; Abia, David; Janowski, Robert; Mortuza, Gulnahar; Bertero, Michela G; Boutin, Maïlys; Guarín, Nayibe; Méndez-Giraldez, Raúl; Nuñez, Alfonso; Pedrero, Juan G; Redondo, Pilar; Sanz, María; Speroni, Silvia; Teichert, Florian; Bruix, Marta; Carazo, José M; Gonzalez, Cayetano; Reina, José; Valpuesta, José M; Vernos, Isabelle; Zabala, Juan C; Montoya, Guillermo; Coll, Miquel; Bastolla, Ugo; Serrano, Luis

2013-01-01

Here we perform a large-scale study of the structural properties and the expression of proteins that constitute the human Centrosome. Centrosomal proteins tend to be larger than generic human proteins (control set), since their genes contain in average more exons (20.3 versus 14.6). They are rich in predicted disordered regions, which cover 57% of their length, compared to 39% in the general human proteome. They also contain several regions that are dually predicted to be disordered and coiled-coil at the same time: 55 proteins (15%) contain disordered and coiled-coil fragments that cover more than 20% of their length. Helices prevail over strands in regions homologous to known structures (47% predicted helical residues against 17% predicted as strands), and even more in the whole centrosomal proteome (52% against 7%), while for control human proteins 34.5% of the residues are predicted as helical and 12.8% are predicted as strands. This difference is mainly due to residues predicted as disordered and helical (30% in centrosomal and 9.4% in control proteins), which may correspond to alpha-helix forming molecular recognition features (α-MoRFs). We performed expression assays for 120 full-length centrosomal proteins and 72 domain constructs that we have predicted to be globular. These full-length proteins are often insoluble: Only 39 out of 120 expressed proteins (32%) and 19 out of 72 domains (26%) were soluble. We built or retrieved structural models for 277 out of 361 human proteins whose centrosomal localization has been experimentally verified. We could not find any suitable structural template with more than 20% sequence identity for 84 centrosomal proteins (23%), for which around 74% of the residues are predicted to be disordered or coiled-coils. The three-dimensional models that we built are available at http://ub.cbm.uam.es/centrosome/models/index.php.

Drug Target Protein-Protein Interaction Networks: A Systematic Perspective

PubMed Central

2017-01-01

The identification and validation of drug targets are crucial in biomedical research and many studies have been conducted on analyzing drug target features for getting a better understanding on principles of their mechanisms. But most of them are based on either strong biological hypotheses or the chemical and physical properties of those targets separately. In this paper, we investigated three main ways to understand the functional biomolecules based on the topological features of drug targets. There are no significant differences between targets and common proteins in the protein-protein interactions network, indicating the drug targets are neither hub proteins which are dominant nor the bridge proteins. According to some special topological structures of the drug targets, there are significant differences between known targets and other proteins. Furthermore, the drug targets mainly belong to three typical communities based on their modularity. These topological features are helpful to understand how the drug targets work in the PPI network. Particularly, it is an alternative way to predict potential targets or extract nontargets to test a new drug target efficiently and economically. By this way, a drug target's homologue set containing 102 potential target proteins is predicted in the paper. PMID:28691014

Structure and properties of a complex of α-synuclein and a single-domain camelid antibody.

PubMed

De Genst, Erwin J; Guilliams, Tim; Wellens, Joke; O'Day, Elizabeth M; Waudby, Christopher A; Meehan, Sarah; Dumoulin, Mireille; Hsu, Shang-Te Danny; Cremades, Nunilo; Verschueren, Koen H G; Pardon, Els; Wyns, Lode; Steyaert, Jan; Christodoulou, John; Dobson, Christopher M

2010-09-17

The aggregation of the intrinsically disordered protein α-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of α-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to α-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric α-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of α-synuclein. The NbSyn2:α-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of α-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of α-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of α-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo. Copyright © 2010. Published by Elsevier Ltd.

Functional and evolutionary insight from the crystal structure of rubella virus protein E1.

PubMed

DuBois, Rebecca M; Vaney, Marie-Christine; Tortorici, M Alejandra; Kurdi, Rana Al; Barba-Spaeth, Giovanna; Krey, Thomas; Rey, Félix A

2013-01-24

Little is known about the three-dimensional organization of rubella virus, which causes a relatively mild measles-like disease in children but leads to serious congenital health problems when contracted in utero. Although rubella virus belongs to the same family as the mosquito-borne alphaviruses, in many respects it is more similar to other aerosol-transmitted human viruses such as the agents of measles and mumps. Although the use of the triple MMR (measles, mumps and rubella) live vaccine has limited its incidence in western countries, congenital rubella syndrome remains an important health problem in the developing world. Here we report the 1.8 Å resolution crystal structure of envelope glycoprotein E1, the main antigen and sole target of neutralizing antibodies against rubella virus. E1 is the main player during entry into target cells owing to its receptor-binding and membrane-fusion functions. The structure reveals the epitope and the neutralization mechanism of an important category of protecting antibodies against rubella infection. It also shows that rubella virus E1 is a class II fusion protein, which had hitherto only been structurally characterized for the arthropod-borne alphaviruses and flaviviruses. In addition, rubella virus E1 has an extensive membrane-fusion surface that includes a metal site, reminiscent of the T-cell immunoglobulin and mucin family of cellular proteins that bind phosphatidylserine lipids at the plasma membrane of cells undergoing apoptosis. Such features have not been seen in any fusion protein crystallized so far. Structural comparisons show that the class II fusion proteins from alphaviruses and flaviviruses, despite belonging to different virus families, are closer to each other than they are to rubella virus E1. This suggests that the constraints on arboviruses imposed by alternating cycles between vertebrates and arthropods resulted in more conservative evolution. By contrast, in the absence of this constraint, the strictly human rubella virus seems to have drifted considerably into a unique niche as sole member of the Rubivirus genus.

Membrane Transporters: Structure, Function and Targets for Drug Design

NASA Astrophysics Data System (ADS)

Ravna, Aina W.; Sager, Georg; Dahl, Svein G.; Sylte, Ingebrigt

Current therapeutic drugs act on four main types of molecular targets: enzymes, receptors, ion channels and transporters, among which a major part (60-70%) are membrane proteins. This review discusses the molecular structures and potential impact of membrane transporter proteins on new drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family. Transporters have a recognition site for a particular substrate, which may be used as a target for drugs inhibiting the transporter or acting as a false substrate. Three groups of transporters have particular interest as drug targets: the major facilitator superfamily, which includes almost 4000 different proteins transporting sugars, polyols, drugs, neurotransmitters, metabolites, amino acids, peptides, organic and inorganic anions and many other substrates; the ATP-binding cassette superfamily, which plays an important role in multidrug resistance in cancer chemotherapy; and the neurotransmitter:sodium symporter family, which includes the molecular targets for some of the most widely used psychotropic drugs. Recent technical advances have increased the number of known 3D structures of membrane transporters, and demonstrated that they form a divergent group of proteins with large conformational flexibility which facilitates transport of the substrate.

Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

PubMed

De Filippis, Vincenzo; Acquasaliente, Laura; Pontarollo, Giulia; Peterle, Daniele

2018-01-01

The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Automated side-chain model building and sequence assignment by template matching.

PubMed

Terwilliger, Thomas C

2003-01-01

An algorithm is described for automated building of side chains in an electron-density map once a main-chain model is built and for alignment of the protein sequence to the map. The procedure is based on a comparison of electron density at the expected side-chain positions with electron-density templates. The templates are constructed from average amino-acid side-chain densities in 574 refined protein structures. For each contiguous segment of main chain, a matrix with entries corresponding to an estimate of the probability that each of the 20 amino acids is located at each position of the main-chain model is obtained. The probability that this segment corresponds to each possible alignment with the sequence of the protein is estimated using a Bayesian approach and high-confidence matches are kept. Once side-chain identities are determined, the most probable rotamer for each side chain is built into the model. The automated procedure has been implemented in the RESOLVE software. Combined with automated main-chain model building, the procedure produces a preliminary model suitable for refinement and extension by an experienced crystallographer.

Mechanisms of amyloid formation revealed by solution NMR

PubMed Central

Karamanos, Theodoros K.; Kalverda, Arnout P.; Thompson, Gary S.; Radford, Sheena E.

2015-01-01

Amyloid fibrils are proteinaceous elongated aggregates involved in more than fifty human diseases. Recent advances in electron microscopy and solid state NMR have allowed the characterization of fibril structures to different extents of refinement. However, structural details about the mechanism of fibril formation remain relatively poorly defined. This is mainly due to the complex, heterogeneous and transient nature of the species responsible for assembly; properties that make them difficult to detect and characterize in structural detail using biophysical techniques. The ability of solution NMR spectroscopy to investigate exchange between multiple protein states, to characterize transient and low-population species, and to study high molecular weight assemblies, render NMR an invaluable technique for studies of amyloid assembly. In this article we review state-of-the-art solution NMR methods for investigations of: (a) protein dynamics that lead to the formation of aggregation-prone species; (b) amyloidogenic intrinsically disordered proteins; and (c) protein–protein interactions on pathway to fibril formation. Together, these topics highlight the power and potential of NMR to provide atomic level information about the molecular mechanisms of one of the most fascinating problems in structural biology. PMID:26282197

Structural Investigation of a Phosphorylation-Catalyzed, Isoaspartate-Free, Protein Succinimide: Crystallographic Structure of Post-Succinimide His15Asp Histidine-Containing Protein

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

Napper, Scott; Prasad, Lata; Delbaere, Louis T.J.

2008-09-08

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation. Although typically regarded as a form of protein damage, we hypothesize succinimides could represent an effective mechanism of phosphoaspartate autophosphatase activity, provided hydrolysis is limited to aspartate products. We previously reported the serendipitous creation of a protein, His15Asp histidine-containing protein (HPr), which undergoes phosphorylation-catalyzed formation of a succinimide whose hydrolysis is seemingly exclusive for aspartate formation. Here, through themore » high-resolution structure of postsuccinimide His15Asp HPr, we confirm the absence of isoaspartate residues and propose mechanisms for phosphorylation-catalyzed succinimide formation and its directed hydrolysis to aspartate. His15Asp HPr represents the first characterized protein example of an isoaspartate-free succinimide and lends credence to the hypothesis that intramolecular cyclization could represent a physiological mechanism of autophosphatase activity. Furthermore, this indicates that current strategies for succinimide evaluation, based on isoaspartate detection, underestimate the frequencies of these reactions. This is considerably significant for evaluation of protein stability and integrity.« less

Solid supports for extraction and preconcentration of proteins and peptides in microfluidic devices: A review.

PubMed

Dziomba, Szymon; Araya-Farias, Monica; Smadja, Claire; Taverna, Myriam; Carbonnier, Benjamin; Tran, N Thuy

2017-02-22

Determination of proteins and peptides is among the main challenges of today's bioanalytical chemistry. The application of microchip technology in this field is an exhaustively developed concept that aims to create integrated and fully automated analytical devices able to quantify or detect one or several proteins from a complex matrix. Selective extraction and preconcentration of targeted proteins and peptides especially from biological fluids is of the highest importance for a successful realization of these microsystems. Incorporation of solid structures or supports is a convenient solution employed to face these demands. This review presents a critical view on the latest achievements in sample processing techniques for protein determination using solid supports in microfluidics. The study covers the period from 2006 to 2015 and focuses mainly on the strategies based on microbeads, monolithic materials and membranes. Less common approaches are also briefly discussed. The reviewed literature suggests future trends which are discussed in the concluding remarks. Copyright © 2016 Elsevier B.V. All rights reserved.

Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

PubMed

Moore, M H; Gulbis, J M; Dodson, E J; Demple, B; Moody, P C

1994-04-01

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6-methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self-methylation of the active site cysteine renders the protein inactive. Here we report the X-ray structure of the 19 kDa C-terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.

Structural Insight into Amino Group-carrier Protein-mediated Lysine Biosynthesis

PubMed Central

Yoshida, Ayako; Tomita, Takeo; Fujimura, Tsutomu; Nishiyama, Chiharu; Kuzuyama, Tomohisa; Nishiyama, Makoto

2015-01-01

In the biosynthesis of lysine by Thermus thermophilus, the metabolite α-ketoglutarate is converted to the intermediate α-aminoadipate (AAA), which is protected by the 54-amino acid acidic protein LysW. In this study, we determined the crystal structure of LysZ from T. thermophilus (TtLysZ), an amino acid kinase that catalyzes the second step in the AAA to lysine conversion, which was in a complex with LysW at a resolution of 1.85 Å. A crystal analysis coupled with isothermal titration calorimetry of the TtLysZ mutants for TtLysW revealed tight interactions between LysZ and the globular and C-terminal extension domains of the LysW protein, which were mainly attributed to electrostatic forces. These results provided structural evidence for LysW acting as a protecting molecule for the α-amino group of AAA and also as a carrier protein to guarantee better recognition by biosynthetic enzymes for the efficient biosynthesis of lysine. PMID:25392000

Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody

PubMed Central

Korotkov, Konstantin V.; Pardon, Els

2009-01-01

Summary Secretins are among the largest bacterial outer membrane proteins known. Here we report the crystal structure of the periplasmic N-terminal domain of GspD (peri-GspD) from the type 2 secretion system (T2SS) secretin in complex with a “nanobody”, the VHH domain of a “heavy-chain” camelid antibody. Two different crystal forms contained the same compact peri-GspD:nanobody heterotetramer. The nanobody contacts peri-GspD mainly via CDR3 and framework residues. The peri-GspD structure reveals three subdomains with the second and third subdomains exhibiting the KH-fold which also occurs in ring-forming proteins of the type 3 secretion system. The first subdomain of GspD is related to domains in phage tail proteins and outer membrane TonB-dependent receptors. A dodecameric peri-GspD model is proposed in which a solvent-accessible β-strand of the first subdomain interacts with secreted proteins and/or T2SS partner proteins by β-strand complementation. PMID:19217396

A reduced amino acid alphabet for understanding and designing protein adaptation to mutation.

PubMed

Etchebest, C; Benros, C; Bornot, A; Camproux, A-C; de Brevern, A G

2007-11-01

Protein sequence world is considerably larger than structure world. In consequence, numerous non-related sequences may adopt similar 3D folds and different kinds of amino acids may thus be found in similar 3D structures. By grouping together the 20 amino acids into a smaller number of representative residues with similar features, sequence world simplification may be achieved. This clustering hence defines a reduced amino acid alphabet (reduced AAA). Numerous works have shown that protein 3D structures are composed of a limited number of building blocks, defining a structural alphabet. We previously identified such an alphabet composed of 16 representative structural motifs (5-residues length) called Protein Blocks (PBs). This alphabet permits to translate the structure (3D) in sequence of PBs (1D). Based on these two concepts, reduced AAA and PBs, we analyzed the distributions of the different kinds of amino acids and their equivalences in the structural context. Different reduced sets were considered. Recurrent amino acid associations were found in all the local structures while other were specific of some local structures (PBs) (e.g Cysteine, Histidine, Threonine and Serine for the alpha-helix Ncap). Some similar associations are found in other reduced AAAs, e.g Ile with Val, or hydrophobic aromatic residues Trp with Phe and Tyr. We put into evidence interesting alternative associations. This highlights the dependence on the information considered (sequence or structure). This approach, equivalent to a substitution matrix, could be useful for designing protein sequence with different features (for instance adaptation to environment) while preserving mainly the 3D fold.

Structure prediction, expression, and antigenicity of c-terminal of GRP78.

PubMed

Aghamollaei, Hossein; Mousavi Gargari, Seyed Latif; Ghanei, Mostafa; Rasaee, Mohamad Javad; Amani, Jafar; Bakherad, Hamid; Farnoosh, Gholamreza

2017-01-01

Glucose-regulated protein 78 (GRP78) is a typical endoplasmic reticulum luminal chaperone having a main role in the activation of the unfolded protein response. Because of hypoxia and nutrient deprivation in the tumor microenvironment, expression of GRP78 in these cells becomes higher than the native cells, which makes it a suitable candidate for cancer targeting. Suppression of survival signals by antibody production against C-terminal domain of GR78 (CGRP) can induce apoptosis of cancer cells. The aim of this study was in silico analysis, recombinant production, and characterization of CGRP in Escherichia coli. Structural prediction of CGRP by bioinformatics tools was done and the construct containing optimized sequence was transferred to E. coli T7 shuffle. Expression was induced by isopropyl-β-d-thiogalactoside, and recombinant protein was purified by Ni-NTA agarose resin. The content of secondary structures was obtained by circular dichroism (CD) spectrum. CGRP immunogenicity was evaluated from the immunized mouse sera. SDS-PAGE analysis showed CGRP expression in E. coli. CD spectrum also confirmed prediction of structures by bioinformatics tools. The enzyme-linked immunosorbent assay using sera from immunized mice revealed CGRP as a good immunogen. The results obtained in this study showed that the structure of truncated CGRP is very similar to its structure in the whole protein context. This protein can be used in cancer researches. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Protein Secondary Structures (alpha-helix and beta-sheet) at a Cellular Levle and Protein Fractions in Relation to Rumen Degradation Behaviours of Protein: A New Approach

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

Yu,P.

2007-01-01

Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein, and such an understanding of the structure of the whole protein is often vital to understanding its digestive behaviour and nutritive value in animals. The main protein secondary structures are the {alpha}-helix and {beta}-sheet. The percentage of these two structures in protein secondary structures influences protein nutritive value, quality and digestive behaviour. A high percentage of {beta}-sheet structure may partly cause a low access to gastrointestinal digestive enzymes, which results in a low protein value. The objectives of the present studymore » were to use advanced synchrotron-based Fourier transform IR (S-FTIR) microspectroscopy as a new approach to reveal the molecular chemistry of the protein secondary structures of feed tissues affected by heat-processing within intact tissue at a cellular level, and to quantify protein secondary structures using multicomponent peak modelling Gaussian and Lorentzian methods, in relation to protein digestive behaviours and nutritive value in the rumen, which was determined using the Cornell Net Carbohydrate Protein System. The synchrotron-based molecular chemistry research experiment was performed at the National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy. The results showed that, with S-FTIR microspectroscopy, the molecular chemistry, ultrastructural chemical make-up and nutritive characteristics could be revealed at a high ultraspatial resolution ({approx}10 {mu}m). S-FTIR microspectroscopy revealed that the secondary structure of protein differed between raw and roasted golden flaxseeds in terms of the percentages and ratio of {alpha}-helixes and {beta}-sheets in the mid-IR range at the cellular level. By using multicomponent peak modelling, the results show that the roasting reduced (P <0.05) the percentage of {alpha}-helixes (from 47.1% to 36.1%: S-FTIR absorption intensity), increased the percentage of {beta}-sheets (from 37.2% to 49.8%: S-FTIR absorption intensity) and reduced the {alpha}-helix to {beta}-sheet ratio (from 0.3 to 0.7) in the golden flaxseeds, which indicated a negative effect of the roasting on protein values, utilisation and bioavailability. These results were proved by the Cornell Net Carbohydrate Protein System in situ animal trial, which also revealed that roasting increased the amount of protein bound to lignin, and well as of the Maillard reaction protein (both of which are poorly used by ruminants), and increased the level of indigestible and undegradable protein in ruminants. The present results demonstrate the potential of highly spatially resolved synchrotron-based infrared microspectroscopy to locate 'pure' protein in feed tissues, and reveal protein secondary structures and digestive behaviour, making a significant step forward in and an important contribution to protein nutritional research. Further study is needed to determine the sensitivities of protein secondary structures to various heat-processing conditions, and to quantify the relationship between protein secondary structures and the nutrient availability and digestive behaviour of various protein sources. Information from the present study arising from the synchrotron-based IR probing of the protein secondary structures of protein sources at the cellular level will be valuable as a guide to maintaining protein quality and predicting digestive behaviours.« less

Expression, refolding and bio-structural analysis of a tetravalent recombinant dengue envelope domain III protein for serological diagnosis.

PubMed

Combe, Maxime; Lacoux, Xavier; Martinez, Jérôme; Méjan, Odile; Luciani, Françoise; Daniel, Soizic

2017-05-01

Dengue is a mosquito-borne disease caused by four genetically and serologically related viruses that affect several millions of people. Envelope domain III (EDIII) of the viral envelope protein contains dengue virus (DENV) type-specific and DENV complex-reactive antigenic sites. Here, we describe the expression in Escherichia coli, the refolding and bio-structural analysis of envelope domain III of the four dengue serotypes as a tetravalent dengue protein (EDIIIT2), generating an attractive diagnostic candidate. In vitro refolding of denatured EDIIIT2 was performed by successive dialysis with decreasing concentrations of chaotropic reagent and in the presence of oxidized glutathione. The efficiency of refolding was demonstrated by protein mobility shifting and fluorescent visualization of labeled cysteine in non-reducing SDS-PAGE. The identity and the fully oxidized state of the protein were verified by mass spectrometry. Analysis of the structure by fluorescence, differential scanning calorimetry and circular dichroism showed a well-formed structural conformation mainly composed of β-strands. A label-free immunoassay based on biolayer interferometry technology was subsequently used to evaluate antigenic properties of folded EDIIIT2 protein using a panel of dengue IgM positive and negative human sera. Our data collectively support the use of an oxidatively refolded EDIIIT2 recombinant chimeric protein as a promising antigen in the serological diagnosis of dengue virus infections. Copyright © 2017 Elsevier Inc. All rights reserved.

The E. coli thioredoxin folding mechanism: the key role of the C-terminal helix.

PubMed

Vazquez, Diego S; Sánchez, Ignacio E; Garrote, Ana; Sica, Mauricio P; Santos, Javier

2015-02-01

In this work, the unfolding mechanism of oxidized Escherichia coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability. Copyright © 2014 Elsevier B.V. All rights reserved.

Structure and evolution of plant centromeres.

PubMed

Nagaki, Kiyotaka; Walling, Jason; Hirsch, Cory; Jiang, Jiming; Murata, Minoru

2009-01-01

Investigations of centromeric DNA and proteins and centromere structures in plants have lagged behind those conducted with yeasts and animals; however, many attractive results have been obtained from plants during this decade. In particular, intensive investigations have been conducted in Arabidopsis and Gramineae species. We will review our understanding of centromeric components, centromere structures, and the evolution of these attributes of centromeres among plants using data mainly from Arabidopsis and Gramineae species.

Using Symmetry to Design Self-Assembling Protein Cages and Nanomaterials on the Mid-Nanometer Scale

NASA Astrophysics Data System (ADS)

Yeates, Todd

Self-assembling molecular structures having diverse cellular functions are widespread in nature. Some of the largest and most sophisticated types are built from many copies of the same or similar protein molecules arranged following principles of symmetry. A long-standing engineering goal has been to design novel protein molecules to self-assemble into geometrically specific structures similar to the extraordinary structures that have evolved in Nature. Practical routes to this goal have been developed by using ideas in symmetry to articulate the minimum design requirements for achieving various types of symmetric architectures, including cages, extended two-dimensional layers, and three-dimensional crystalline materials. The key requirement is that two distinct self-associating interfaces, each conferring one element of rotational symmetry, have to be engineered into the protein molecule (or molecules), following particular geometric specifications. The main principle is that combining two separate symmetry elements into a single molecular entity produces a molecule that necessarily assembles into an architecture dictated by a symmetry group that is the product of the two simpler contributing symmetries. Recent experiments have demonstrated success using a variety of symmetry-based strategies. Strategic variations are emerging that differ from each other with respect to biophysical features such as flexibility vs rigidity in the assembled structures, and with respect to design aspects such as whether the protein interfaces are inherited from natural oligomeric proteins or are designed de novo by advanced computational methods. The success of these strategies has been proven by determining crystal structures of several giant, self-assembling protein cages and clusters (10-25 nm in diameter), created by design. The ability to create sophisticated supramolecular structures from designed protein subunits opens the way to broad applications in synthetic biology and nanotechnology.

Synergistic interactions of lipids and myelin basic protein

NASA Astrophysics Data System (ADS)

Hu, Yufang; Doudevski, Ivo; Wood, Denise; Moscarello, Mario; Husted, Cynthia; Genain, Claude; Zasadzinski, Joseph A.; Israelachvili, Jacob

2004-09-01

This report describes force measurements and atomic force microscope imaging of lipid-protein interactions that determine the structure of a model membrane system that closely mimics the myelin sheath. Our results suggest that noncovalent, mainly electrostatic and hydrophobic, interactions are responsible for the multilamellar structure and stability of myelin. We find that myelin basic protein acts as a lipid coupler between two apposed bilayers and as a lipid "hole-filler," effectively preventing defect holes from developing. From our protein-mediated-adhesion and force-distance measurements, we develop a simple quantitative model that gives a reasonably accurate picture of the molecular mechanism and adhesion of bilayer-bridging proteins by means of noncovalent interactions. The results and model indicate that optimum myelin adhesion and stability depend on the difference between, rather than the product of, the opposite charges on the lipid bilayers and myelin basic protein, as well as on the repulsive forces associated with membrane fluidity, and that small changes in any of these parameters away from the synergistically optimum values can lead to large changes in the adhesion or even its total elimination. Our results also show that the often-asked question of which membrane species, the lipids or the proteins, are the "important ones" may be misplaced. Both components work synergistically to provide the adhesion and overall structure. A better appreciation of the mechanism of this synergy may allow for a better understanding of stacked and especially myelin membrane structures and may lead to better treatments for demyelinating diseases such as multiple sclerosis. lipid-protein interactions | myelin membrane structure | membrane adhesion | membrane regeneration/healing | demyelinating diseases

Domain swapping dissection in Thermotoga maritima arginine binding protein: How structural flexibility may compensate destabilization.

PubMed

Smaldone, Giovanni; Berisio, Rita; Balasco, Nicole; D'Auria, Sabato; Vitagliano, Luigi; Ruggiero, Alessia

2018-05-31

Thermotoga maritima Arginine Binding Protein (TmArgBP) is a valuable candidate for arginine biosensing in diagnostics. This protein is endowed with unusual structural properties that include an extraordinary thermal/chemical stability, a domain swapped structure that undergoes large tertiary and quaternary structural transition, and the ability to form non-canonical oligomeric species. As the intrinsic stability of TmArgBP allows for extensive protein manipulations, we here dissected its structure in two parts: its main body deprived of the swapping fragment (TmArgBP 20-233 ) and the C-terminal peptide corresponding to the helical swapping element. Both elements have been characterized independently or in combination using a repertoire of biophysical/structural techniques. Present investigations clearly indicate that TmArgBP 20-233 represents a better scaffold for arginine sensing compared to the wild-type protein. Moreover, our data demonstrate that the ligand-free and the ligand-bound forms respond very differently to this helix deletion. This drastic perturbation has an important impact on the ligand-bound form of TmArgBP 20-233 stability whereas it barely affects its ligand-free state. The crystallographic structures of these forms provide a rationale to this puzzling observation. Indeed, the arginine-bound state is very rigid and virtually unchanged upon protein truncation. On the other hand, the flexible ligand-free TmArgBP 20-233 is able to adopt a novel state as a consequence of the helix deletion. Therefore, the flexibility of the ligand-free form endows this state with a remarkable robustness upon severe perturbations. In this scenario, TmArgBP dissection highlights an intriguing connection between destabilizing/stabilizing effects and the overall flexibility that could operate also in other proteins. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

PubMed

Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores; Alvarez-Salas, Luis Marat

2014-10-01

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles

PubMed Central

Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores

2014-01-01

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3′end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection. PMID:25111024

Virtual Interactomics of Proteins from Biochemical Standpoint

PubMed Central

Kubrycht, Jaroslav; Sigler, Karel; Souček, Pavel

2012-01-01

Virtual interactomics represents a rapidly developing scientific area on the boundary line of bioinformatics and interactomics. Protein-related virtual interactomics then comprises instrumental tools for prediction, simulation, and networking of the majority of interactions important for structural and individual reproduction, differentiation, recognition, signaling, regulation, and metabolic pathways of cells and organisms. Here, we describe the main areas of virtual protein interactomics, that is, structurally based comparative analysis and prediction of functionally important interacting sites, mimotope-assisted and combined epitope prediction, molecular (protein) docking studies, and investigation of protein interaction networks. Detailed information about some interesting methodological approaches and online accessible programs or databases is displayed in our tables. Considerable part of the text deals with the searches for common conserved or functionally convergent protein regions and subgraphs of conserved interaction networks, new outstanding trends and clinically interesting results. In agreement with the presented data and relationships, virtual interactomic tools improve our scientific knowledge, help us to formulate working hypotheses, and they frequently also mediate variously important in silico simulations. PMID:22928109

Protective activity of green tea against free radical- and glucose-mediated protein damage.

PubMed

Nakagawa, Takako; Yokozawa, Takako; Terasawa, Katsutoshi; Shu, Seiji; Juneja, Lekh Raj

2002-04-10

Protein oxidation and glycation are posttranslational modifications that are implicated in the pathological development of many age-related disease processes. This study investigated the effects of green tea extract, and a green tea tannin mixture and its components, on protein damage induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (a free radical generator) and glucose in in vitro assay systems. We found that green tea extract can effectively protect against protein damage, and showed that its action is mainly due to tannin. In addition, it was shown that the chemical structures of tannin components are also involved in this activity, suggesting that the presence of the gallate group at the 3 position plays the most important role in the protective activity against protein oxidation and glycation, and that there is also a contribution by the hydroxyl group at the 5' position in the B ring and the sterical structure. These findings demonstrate the mechanisms of the usefulness of green tea in protein oxidation- and glycation-associated diseases.

Computer Modeling of the Structure and Spectra of Fluorescent Proteins

PubMed Central

Grigorenko, B.L.; Savitsky, A.P.

2009-01-01

Fluorescent proteins from the family of green fluorescent proteins are intensively used as biomarkers in living systems. The chromophore group based on the hydroxybenzylidene-imidazoline molecule, which is formed in nature from three amino-acid residues inside the protein globule and well shielded from external media, is responsible for light absorption and fluorescence. Along with the intense experimental studies of the properties of fluorescent proteins and their chromophores by biochemical, X-ray, and spectroscopic tools, in recent years, computer modeling has been used to characterize their properties and spectra. We present in this review the most interesting results of the molecular modeling of the structural parameters and optical and vibrational spectra of the chromophorecontaining domains of fluorescent proteins by methods of quantum chemistry, molecular dynamics, and combined quantum-mechanical-molecular-mechanical approaches. The main emphasis is on the correlation of theoretical and experimental data and on the predictive power of modeling, which may be useful for creating new, efficient biomarkers. PMID:22649601

RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair

PubMed Central

Maltseva, Ekaterina A.

2018-01-01

Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA. PMID:29320546

RPA and XPA interaction with DNA structures mimicking intermediates of the late stages in nucleotide excision repair.

PubMed

Krasikova, Yuliya S; Rechkunova, Nadejda I; Maltseva, Ekaterina A; Lavrik, Olga I

2018-01-01

Replication protein A (RPA) and the xeroderma pigmentosum group A (XPA) protein are indispensable for both pathways of nucleotide excision repair (NER). Here we analyze the interaction of RPA and XPA with DNA containing a flap and different size gaps that imitate intermediates of the late NER stages. Using gel mobility shift assays, we found that RPA affinity for DNA decreased when DNA contained both extended gap and similar sized flap in comparison with gapped-DNA structure. Moreover, crosslinking experiments with the flap-gap DNA revealed that RPA interacts mainly with the ssDNA platform within the long gap and contacts flap in DNA with a short gap. XPA exhibits higher affinity for bubble-DNA structures than to flap-gap-containing DNA. Protein titration analysis showed that formation of the RPA-XPA-DNA ternary complex depends on the protein concentration ratio and these proteins can function as independent players or in tandem. Using fluorescently-labelled RPA, direct interaction of this protein with XPA was detected and characterized quantitatively. The data obtained allow us to suggest that XPA can be involved in the post-incision NER stages via its interaction with RPA.

The C-terminal priming domain is strongly associated with the main body of bacteriophage ϕ6 RNA-dependent RNA polymerase.

PubMed

Sarin, L Peter; Wright, Sam; Chen, Qing; Degerth, Linda H; Stuart, David I; Grimes, Jonathan M; Bamford, Dennis H; Poranen, Minna M

2012-10-10

Double-stranded RNA viruses encode a single protein species containing RNA-dependent RNA polymerase (RdRP) motifs. This protein is responsible for RNA transcription and replication. The architecture of viral RdRPs resembles that of a cupped right hand with fingers, palm and thumb domains. Those using de novo initiation have a flexible structural elaboration that constitutes the priming platform. Here we investigate the properties of the C-terminal priming domain of bacteriophage ϕ6 to get insights into the role of an extended loop connecting this domain to the main body of the polymerase. Proteolyzed ϕ6 RdRP that possesses a nick in the hinge region of this loop was better suited for de novo initiation. The clipped C-terminus remained associated with the main body of the polymerase via the anchor helix. The structurally flexible hinge region appeared to be involved in the control of priming platform movement. Moreover, we detected abortive initiation products for a bacteriophage RdRP. Copyright © 2012 Elsevier Inc. All rights reserved.

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

PubMed Central

Sima, Michal; Novotny, Marian; Pravda, Lukas; Sumova, Petra; Rohousova, Iva; Volf, Petr

2016-01-01

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies. PMID:27812196

Structural studies of bee melittin

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

Eisenberg, D.; Terwilliger, T.C.; Tsui, F.

The question of how proteins refold in passing from an aqueous phase to an amphipathic environment such as a membrane is beig addressed by a structural study of bee melittin. Melittin is the toxic, main protein of bee venom, and has been shown by others to integrate into natural and synthetic membranes and to lyse a variety of cells. This function is presumably related to its unusual sequence. Except for charges at the N-terminus and at lysine 7, the first 20 residues are largely apolar. In contrast, the last six residues contain four charges and two polar residues.

Action of plant proteinase inhibitors on enzymes of physiopathological importance.

PubMed

Oliva, Maria Luiza V; Sampaio, Misako U

2009-09-01

Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.

[Advances in Parvovirus Non-structural Protein NS1 Induced Apoptosis].

PubMed

Tu, Mengyu; Liu, Fei; Chen, Shun; Wang, Mingshu; Cheng, Anchun

2015-11-01

Until now, more than seventeen parvovirus have been reported which can infect mammals and poultries. The infected cells appeared different properties of apoptosis and death, present a typical cytopathic effect. NS1 is a major nonstructural protein of parvovirus, with a conservative structure and function, which plays an important role in the viral life cycle. In addition to the influence on viral replication, the NS1 also participates in apoptosis induced by viruses. Parvovirus induced apoptosis which is mainly mediated by mitochondrial pathway, this review summarized the latest research progresses of parvovirus induced apoptosis.

Identification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and Demonstration that it Interacts with ICP8, the Major DNA Binding Protein of Herpes Simplex Virus

DTIC Science & Technology

1992-10-20

Identification of ORFs HSV DNA binding proteins • 1 3 3 5 7 7 11 17 18 22 reps and its role in HSV replication 23 Biochemical properties . . 23...Figure 1 . 2. 3 • 4. 5. 6. 7. 8. Structural model of the herpesvirus virion Schematic diagram of HSV pathogenesis . Diagram of the main...vaccinia virus- 13. Autoradiogram of an immunoblot of HSV - 1 -infected cell proteins harvested at various times postinfec- 85 tioD probed with anti-UL42

What is gluten?

PubMed

Biesiekierski, Jessica R

2017-03-01

Gluten is the main storage protein of wheat grains. Gluten is a complex mixture of hundreds of related but distinct proteins, mainly gliadin and glutenin. Similar storage proteins exist as secalin in rye, hordein in barley, and avenins in oats and are collectively referred to as "gluten." The objective was to discuss the biochemical and functional properties of the gluten proteins, including structure, sources, and dietary intakes. Literature was reviewed from food science and nutrition journals. The gluten protein networks vary because of different components and sizes, and variability caused by genotype, growing conditions, and technological processes. The structures and interactions of this matrix contribute to the unique properties of gluten. The resulting functions are essential to determining the dough quality of bread and other baked products. Gluten is heat stable and has the capacity to act as a binding and extending agent and is commonly used as an additive in processed foods for improved texture, moisture retention, and flavor. Gliadin contains peptide sequences that are highly resistant to gastric, pancreatic, and intestinal proteolytic digestion in the gastrointestinal tract. The average daily gluten intake in a Western diet is thought to be 5-20 g/day and has been implicated in several disorders. Gluten containing grains (wheat, rye, barley, and oats) are important staple foods. Gluten is among the most complex protein networks and plays a key role in determining the rheological dough properties. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Toxicological effects of benzo(a)pyrene, DDT and their mixture on the green mussel Perna viridis revealed by proteomic and metabolomic approaches.

PubMed

Song, Qinqin; Chen, Hao; Li, Yuhu; Zhou, Hailong; Han, Qian; Diao, Xiaoping

2016-02-01

Benzo(a)pyrene (BaP) and dichlorodiphenyltrichloroethane (DDT) are persistent organic pollutants and environmental estrogens (EEs) with known toxicity towards the green mussel, Perna viridis. In this study, the toxic effects of BaP (10 µg/L) and DDT (10 µg/L) and their mixture were assessed in green mussel gills with proteomic and metabolomic approaches. Metabolic responses indicated that BaP mainly caused disturbance in osmotic regulation by significantly decrease in branched chain amino acids, dimethylamine and dimethylglycine in gills of male green mussels after exposure for 7 days. DDT mainly caused disturbance in osmotic regulation and energy metabolism by differential alteration of betaine, dimethylamine, dimethylglycine, amino acids, and succinate in gills of male green mussels. However, the mixture of BaP and DDT didn't show obvious metabolite changes. Proteomic analysis showed different protein expression profiles between different treatment groups, which demonstrated that BaP, DDT and their mixture may have different modes of action. Proteomic responses revealed that BaP induced cell apoptosis, disturbance in protein digestion and energy metabolism in gills of green mussels, whereas DDT exposure altered proteins that were associated with oxidative stress, cytoskeleton and cell structure, protein digestion and energy metabolism. However, the mixture of BaP and DDT affected proteins related to the oxidative stress, cytoskeleton and cell structure, protein biosynthesis and modification, energy metabolism, growth and apoptosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme

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

Matte, Allan; Grosse, Stephan; Bergeron, Hélène

The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavoring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a -barrel structure and two -helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally relatedmore » proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the -barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.« less

Discrete Haar transform and protein structure.

PubMed

Morosetti, S

1997-12-01

The discrete Haar transform of the sequence of the backbone dihedral angles (phi and psi) was performed over a set of X-ray protein structures of high resolution from the Brookhaven Protein Data Bank. Afterwards, the new dihedral angles were calculated by the inverse transform, using a growing number of Haar functions, from the lower to the higher degree. New structures were obtained using these dihedral angles, with standard values for bond lengths and angles, and with omega = 0 degree. The reconstructed structures were compared with the experimental ones, and analyzed by visual inspection and statistical analysis. When half of the Haar coefficients were used, all the reconstructed structures were not yet collapsed to a tertiary folding, but they showed yet realized most of the secondary motifs. These results indicate a substantial separation of structural information in the space of Haar transform, with the secondary structural information mainly present in the Haar coefficients of lower degrees, and the tertiary one present in the higher degree coefficients. Because of this separation, the representation of the folded structures in the space of Haar transform seems a promising candidate to encompass the problem of premature convergence in genetic algorithms.

The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro

PubMed Central

Serfiotis-Mitsa, Dimitra; Herbert, Andrew P.; Roberts, Gareth A.; Soares, Dinesh C.; White, John H.; Blakely, Garry W.; Uhrín, Dušan; Dryden, David T. F.

2010-01-01

Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic. PMID:20007596

Evolutionary distance from human homologs reflects allergenicity of animal food proteins.

PubMed

Jenkins, John A; Breiteneder, Heimo; Mills, E N Clare

2007-12-01

In silico analysis of allergens can identify putative relationships among protein sequence, structure, and allergenic properties. Such systematic analysis reveals that most plant food allergens belong to a restricted number of protein superfamilies, with pollen allergens behaving similarly. We have investigated the structural relationships of animal food allergens and their evolutionary relatedness to human homologs to define how closely a protein must resemble a human counterpart to lose its allergenic potential. Profile-based sequence homology methods were used to classify animal food allergens into Pfam families, and in silico analyses of their evolutionary and structural relationships were performed. Animal food allergens could be classified into 3 main families--tropomyosins, EF-hand proteins, and caseins--along with 14 minor families each composed of 1 to 3 allergens. The evolutionary relationships of each of these allergen superfamilies showed that in general, proteins with a sequence identity to a human homolog above approximately 62% were rarely allergenic. Single substitutions in otherwise highly conserved regions containing IgE epitopes in EF-hand parvalbumins may modulate allergenicity. These data support the premise that certain protein structures are more allergenic than others. Contrasting with plant food allergens, animal allergens, such as the highly conserved tropomyosins, challenge the capability of the human immune system to discriminate between foreign and self-proteins. Such immune responses run close to becoming autoimmune responses. Exploiting the closeness between animal allergens and their human homologs in the development of recombinant allergens for immunotherapy will need to consider the potential for developing unanticipated autoimmune responses.

High hydrophobic amino acid exposure is responsible of the neurotoxic effects induced by E200K or D202N disease-related mutations of the human prion protein.

PubMed

Corsaro, Alessandro; Thellung, Stefano; Bucciarelli, Tonino; Scotti, Luca; Chiovitti, Katia; Villa, Valentina; D'Arrigo, Cristina; Aceto, Antonio; Florio, Tullio

2011-03-01

Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a β-rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in α-helix, hPrP90-231 E200K is spontaneously refolded in a β-structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides. Copyright © 2010 Elsevier Ltd. All rights reserved.

Structure-Based Phylogenetic Analysis of the Lipocalin Superfamily.

PubMed

Lakshmi, Balasubramanian; Mishra, Madhulika; Srinivasan, Narayanaswamy; Archunan, Govindaraju

2015-01-01

Lipocalins constitute a superfamily of extracellular proteins that are found in all three kingdoms of life. Although very divergent in their sequences and functions, they show remarkable similarity in 3-D structures. Lipocalins bind and transport small hydrophobic molecules. Earlier sequence-based phylogenetic studies of lipocalins highlighted that they have a long evolutionary history. However the molecular and structural basis of their functional diversity is not completely understood. The main objective of the present study is to understand functional diversity of the lipocalins using a structure-based phylogenetic approach. The present study with 39 protein domains from the lipocalin superfamily suggests that the clusters of lipocalins obtained by structure-based phylogeny correspond well with the functional diversity. The detailed analysis on each of the clusters and sub-clusters reveals that the 39 lipocalin domains cluster based on their mode of ligand binding though the clustering was performed on the basis of gross domain structure. The outliers in the phylogenetic tree are often from single member families. Also structure-based phylogenetic approach has provided pointers to assign putative function for the domains of unknown function in lipocalin family. The approach employed in the present study can be used in the future for the functional identification of new lipocalin proteins and may be extended to other protein families where members show poor sequence similarity but high structural similarity.

Integrative Structure Determination of Protein Assemblies by Satisfaction of Spatial Restraints

NASA Astrophysics Data System (ADS)

Alber, Frank; Chait, Brian T.; Rout, Michael P.; Sali, Andrej

To understand the cell, we need to determine the structures of macromolecular assemblies, many of which consist of tens to hundreds of components. A great variety of experimental data can be used to characterize the assemblies at several levels of resolution, from atomic structures to component configurations. To maximize completeness, resolution, accuracy, precision and efficiency of the structure determination, a computational approach is needed that can use spatial information from a variety of experimental methods. We propose such an approach, defined by its three main components: a hierarchical representation of the assembly, a scoring function consisting of spatial restraints derived from experimental data, and an optimization method that generates structures consistent with the data. We illustrate the approach by determining the configuration of the 456 proteins in the nuclear pore complex from Baker's yeast.

Phylogeny-dominant classification of J-proteins in Arabidopsis thaliana and Brassica oleracea.

PubMed

Zhang, Bin; Qiu, Han-Lin; Qu, Dong-Hai; Ruan, Ying; Chen, Dong-Hong

2018-04-05

Hsp40s or DnaJ/J-proteins are evolutionarily conserved in all organisms as co-chaperones of molecular chaperone HSP70s that mainly participate in maintaining cellular protein homeostasis, such as protein folding, assembly, stabilization, and translocation under normal conditions as well as refolding and degradation under environmental stresses. It has been reported that Arabidopsis J-proteins are classified into four classes (types A-D) according to domain organization, but their phylogenetic relationships are unknown. Here, we identified 129 J-proteins in the world-wide popular vegetable Brassica oleracea, a close relative of the model plant Arabidopsis, and also revised the information of Arabidopsis J-proteins based on the latest online bioresources. According to phylogenetic analysis with domain organization and gene structure as references, the J-proteins from Arabidopsis and B. oleracea were classified into 15 main clades (I-XV) separated by a number of undefined small branches with remote relationship. Based on the number of members, they respectively belong to multigene clades, oligo-gene clades, and mono-gene clades. The J-protein genes from different clades may function together or separately to constitute a complicated regulatory network. This study provides a constructive viewpoint for J-protein classification and an informative platform for further functional dissection and resistant genes discovery related to genetic improvement of crop plants.

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

Aggregation of gluten proteins in model dough after fibre polysaccharide addition.

PubMed

Nawrocka, Agnieszka; Szymańska-Chargot, Monika; Miś, Antoni; Wilczewska, Agnieszka Z; Markiewicz, Karolina H

2017-09-15

FT-Raman spectroscopy, thermogravimetry and differential scanning calorimetry were used to study changes in structure of gluten proteins and their thermal properties influenced by four dietary fibre polysaccharides (microcrystalline cellulose, inulin, apple pectin and citrus pectin) during development of a model dough. The flour reconstituted from wheat starch and wheat gluten was mixed with the polysaccharides in five concentrations: 3%, 6%, 9%, 12% and 18%. The obtained results showed that all polysaccharides induced similar changes in secondary structure of gluten proteins concerning formation of aggregates (1604cm -1 ), H-bonded parallel- and antiparallel-β-sheets (1690cm -1 ) and H-bonded β-turns (1664cm -1 ). These changes concerned mainly glutenins since β-structures are characteristic for them. The observed structural changes confirmed hypothesis about partial dehydration of gluten network after polysaccharides addition. The gluten aggregation and dehydration processes were also reflected in the DSC results, while the TGA ones showed that gluten network remained thermally stable after polysaccharides addition. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cholesterol oxidase: ultrahigh-resolution crystal structure and multipolar atom model-based analysis.

PubMed

Zarychta, Bartosz; Lyubimov, Artem; Ahmed, Maqsood; Munshi, Parthapratim; Guillot, Benoît; Vrielink, Alice; Jelsch, Christian

2015-04-01

Examination of protein structure at the subatomic level is required to improve the understanding of enzymatic function. For this purpose, X-ray diffraction data have been collected at 100 K from cholesterol oxidase crystals using synchrotron radiation to an optical resolution of 0.94 Å. After refinement using the spherical atom model, nonmodelled bonding peaks were detected in the Fourier residual electron density on some of the individual bonds. Well defined bond density was observed in the peptide plane after averaging maps on the residues with the lowest thermal motion. The multipolar electron density of the protein-cofactor complex was modelled by transfer of the ELMAM2 charge-density database, and the topology of the intermolecular interactions between the protein and the flavin adenine dinucleotide (FAD) cofactor was subsequently investigated. Taking advantage of the high resolution of the structure, the stereochemistry of main-chain bond lengths and of C=O···H-N hydrogen bonds was analyzed with respect to the different secondary-structure elements.

Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation.

PubMed

Post, K; Pitschke, M; Schäfer, O; Wille, H; Appel, T R; Kirsch, D; Mehlhorn, I; Serban, H; Prusiner, S B; Riesner, D

1998-11-01

The N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP, were solubilized in 0.2% SDS, and the transitions induced by changing the conditions from 0.2% SDS to physiological conditions, i.e. removing SDS, were characterized with respect to solubility, resistance to proteolysis, secondary structure and multimerization. Circular dichroism, electron microscopy and fluorescence correlation spectroscopy were used to study the structural transitions of PrP. Within one minute the alpha-helical structure of PrP was transformed into one that was enriched in beta-sheets and consisted mainly of dimers. Larger oligomers were found after 20 minutes and larger multimers exhibiting resistance to proteolysis were found after several hours. It was concluded that the monomeric alpha-helical conformation was stable in SDS or when attached to the membrane; however, the state of lowest free energy in aqueous solution at neutral pH seems to be the multimeric, beta-sheet enriched conformation.

Contributions to the study of the mechanisms of photodynamic cross-linking of proteins

NASA Astrophysics Data System (ADS)

Shen, Hui-Rong

The illumination of proteins in solution and in cells in the presence of photosensitizers may lead to the inter- and/or intramolecular crosslinking of the proteins (photosensitized or photodynamic crosslinking). This phenomenon appears to be involved in the photohemolysis of red cells, cataract development, skin photoaging, photodynamic therapy for cancers, laser welding of tissues, biomaterial modification, and other biological situations. Although the processes involved in the photocrosslinking of proteins have been extensively studied, the mechanisms involved are still largely unknown. The main objectives of the studies reported in this dissertation were to investigate the detailed mechanisms involved in the photocrosslinking of proteins and to determine the chemical nature of the crosslinks formed. The first part of this study was devoted to the verification of the roles of His, Lys and Tyr in the photodynamic crosslinking of proteins. The crosslinking reaction was modeled using tailor-made water-soluble synthetic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing epsilon-aminocaproic acid side chains terminating in His, Lys or tyrosinamide residues photosensitized by rose bengal (RB) and flavin mononucleotide (FMN). RB typically produces singlet oxygen, whereas FMN produces both singlet oxygen and radicals. His-His and His-Lys crosslinks were formed with RB as the sensitizer. RB-sensitization did not crosslink Tyr residues, whereas FMN coupled two Tyr residues via a radical pathway. Protection of the His and/or Lys residues in ribonuclease A (RNase A) significantly inhibited the extent of intermolecular crosslinking, and confirmed the key roles played by His and Lys in crosslinking reactions. The second part of this study involved the elucidation of the detailed reaction mechanisms and the chemical structures of His-His and Tyr-Tyr crosslinks. N-benzoyl-histidine (Bz-His) and N-acetyl-tyrosine (Ac-Tyr) were used to model the photosensitized crosslinking of proteins involving His and Tyr residues. Photocrosslinking of Bz-His was performed in phosphate buffer at pH 7.4 with immobilized RB beads as sensitizer. The main dimerized product was isolated and characterized. Its chemical structure was established by MS and NMR methods. Ac-Tyr was photocrosslinked with FMN as the sensitizer at pH 6.0; oxygen was necessary. Three main crosslinked dimers were obtained. Their chemical structures were determined by MS and NMR data.

Differential expression of odorant-binding proteins in the mandibular glands of the honey bee according to caste and age.

PubMed

Iovinella, Immacolata; Dani, Francesca Romana; Niccolini, Alberto; Sagona, Simona; Michelucci, Elena; Gazzano, Angelo; Turillazzi, Stefano; Felicioli, Antonio; Pelosi, Paolo

2011-08-05

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) mediate both perception and release of chemical stimuli in insects. The genome of the honey bee contains 21 genes encoding OBPs and 6 encoding CSPs. Using a proteomic approach, we have investigated the expression of OBPs and CSPs in the mandibular glands of adult honey bees in relation to caste and age. OBP13 is mostly expressed in young individuals and in virgin queens, while OBP21 is abundant in older bees and is prevalent in mated queens. OBP14, which had been found in larvae, is produced in hive workers' glands. Quite unexpectedly, the mandibular glands of drones also contain OBPs, mainly OBP18 and OBP21. We have expressed three of the most represented OBPs and studied their binding properties. OBP13 binds with good specificity oleic acid and some structurally related compounds, OBP14 is better tuned to monoterpenoid structures, while OBP21 binds the main components of queen mandibular pheromone as well as farnesol, a compound used as a trail pheromone in the honey bee and other hymenopterans. The high expression of different OBPs in the mandibular glands suggests that such proteins could be involved in solubilization and release of semiochemicals.

Reconstruction of the experimentally supported human protein interactome: what can we learn?

PubMed Central

2013-01-01

Background Understanding the topology and dynamics of the human protein-protein interaction (PPI) network will significantly contribute to biomedical research, therefore its systematic reconstruction is required. Several meta-databases integrate source PPI datasets, but the protein node sets of their networks vary depending on the PPI data combined. Due to this inherent heterogeneity, the way in which the human PPI network expands via multiple dataset integration has not been comprehensively analyzed. We aim at assembling the human interactome in a global structured way and exploring it to gain insights of biological relevance. Results First, we defined the UniProtKB manually reviewed human “complete” proteome as the reference protein-node set and then we mined five major source PPI datasets for direct PPIs exclusively between the reference proteins. We updated the protein and publication identifiers and normalized all PPIs to the UniProt identifier level. The reconstructed interactome covers approximately 60% of the human proteome and has a scale-free structure. No apparent differentiating gene functional classification characteristics were identified for the unrepresented proteins. The source dataset integration augments the network mainly in PPIs. Polyubiquitin emerged as the highest-degree node, but the inclusion of most of its identified PPIs may be reconsidered. The high number (>300) of connections of the subsequent fifteen proteins correlates well with their essential biological role. According to the power-law network structure, the unrepresented proteins should mainly have up to four connections with equally poorly-connected interactors. Conclusions Reconstructing the human interactome based on the a priori definition of the protein nodes enabled us to identify the currently included part of the human “complete” proteome, and discuss the role of the proteins within the network topology with respect to their function. As the network expansion has to comply with the scale-free theory, we suggest that the core of the human interactome has essentially emerged. Thus, it could be employed in systems biology and biomedical research, despite the considerable number of currently unrepresented proteins. The latter are probably involved in specialized physiological conditions, justifying the scarcity of related PPI information, and their identification can assist in designing relevant functional experiments and targeted text mining algorithms. PMID:24088582

A Systems Biology-Based Approach to Uncovering Molecular Mechanisms Underlying Effects of Traditional Chinese Medicine Qingdai in Chronic Myelogenous Leukemia, Involving Integration of Network Pharmacology and Molecular Docking Technology.

PubMed

Zhou, Chao; Liu, LiJuan; Zhuang, Jing; Wei, JunYu; Zhang, TingTing; Gao, ChunDi; Liu, Cun; Li, HuaYao; Si, HongZong; Sun, ChangGang

2018-06-23

BACKGROUND The method of multiple targets overall control is increasingly used to predict the main active ingredient and potential target group of Chinese traditional medicines and to determine the mechanisms involved in their curative effects. Qingdai is the main traditional Chinese medicine used in the treatment of chronic myelogenous leukemia (CML), but the complex active ingredients and antitumor targets in treatment of CML have not been clearly defined in previous studies. MATERIAL AND METHODS We constructed a protein-protein interaction network diagram of CML with 638 nodes (proteins) and 1830 edges, based on the biological function of chronic myelocytic leukemia by use of Cytoscape, and we determined 19 key gene nodes in the CML molecule by network topological properties analysis in a data bank. Then, we used the Surflex-dock plugin in SYBYL7.3 docking and acquired the protein crystal structures of key genes involved in CML from the chemical composition of the traditional Chinese medicine Qingdai with key proteins in CML networks. RESULTS According to the score and the spatial structure, the pharmacodynamically active ingredients of Qingdai are Isdirubin, Isoindigo, N-phenyl-2-naphthylamine, and Isatin, among which Isdirubin is the most important. We further screened the most effective activity key protein structures of CML to find the best pharmacodynamically active ingredients of Qingdai, according to the binding interactions of the inhibitors at the catalytic site performed in best docking combinations. CONCLUSIONS The results suggest that Isdirubin plays a role in resistance to CML by altering the expressions of PIK3CA, MYC, JAK2, and TP53 target proteins. Network pharmacology and molecular docking technology can be used to search for possible reactive molecules in traditional chinese medicines (TCM) and to elucidate their molecular mechanisms.

Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form.

PubMed

Royuela, Enrique; Sánchez-Fauquier, Alicia

2010-01-01

The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus-cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26 Delta C), followed by purification by NTA-Ni(2+) agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies.

Electronic polarization stabilizes tertiary structure prediction of HP-36.

PubMed

Duan, Li L; Zhu, Tong; Zhang, Qing G; Tang, Bo; Zhang, John Z H

2014-04-01

Molecular dynamic (MD) simulations with both implicit and explicit solvent models have been carried out to study the folding dynamics of HP-36 protein. Starting from the extended conformation, the secondary structure of all three helices in HP-36 was formed in about 50 ns and remained stable in the remaining simulation. However, the formation of the tertiary structure was difficult. Although some intermediates were close to the native structure, the overall conformation was not stable. Further analysis revealed that the large structure fluctuation of loop and hydrophobic core regions was devoted mostly to the instability of the structure during MD simulation. The backbone root-mean-square deviation (RMSD) of the loop and hydrophobic core regions showed strong correlation with the backbone RMSD of the whole protein. The free energy landscape indicated that the distribution of main chain torsions in loop and turn regions was far away from the native state. Starting from an intermediate structure extracted from the initial AMBER simulation, HP-36 was found to generally fold to the native state under the dynamically adjusted polarized protein-specific charge (DPPC) simulation, while the peptide did not fold into the native structure when AMBER force filed was used. The two best folded structures were extracted and taken into further simulations in water employing AMBER03 charge and DPPC for 25 ns. Result showed that introducing polarization effect into interacting potential could stabilize the near-native protein structure.

Generation of 3D templates of active sites of proteins with rigid prosthetic groups.

PubMed

Nebel, Jean-Christophe

2006-05-15

With the increasing availability of protein structures, the generation of biologically meaningful 3D patterns from the simultaneous alignment of several protein structures is an exciting prospect: active sites could be better understood, protein functions and protein 3D structures could be predicted more accurately. Although patterns can already be generated at the fold and topological levels, no system produces high-resolution 3D patterns including atom and cavity positions. To address this challenge, our research focuses on generating patterns from proteins with rigid prosthetic groups. Since these groups are key elements of protein active sites, the generated 3D patterns are expected to be biologically meaningful. In this paper, we present a new approach which allows the generation of 3D patterns from proteins with rigid prosthetic groups. Using 237 protein chains representing proteins containing porphyrin rings, our method was validated by comparing 3D templates generated from homologues with the 3D structure of the proteins they model. Atom positions were predicted reliably: 93% of them had an accuracy of 1.00 A or less. Moreover, similar results were obtained regarding chemical group and cavity positions. Results also suggested our system could contribute to the validation of 3D protein models. Finally, a 3D template was generated for the active site of human cytochrome P450 CYP17, the 3D structure of which is unknown. Its analysis showed that it is biologically meaningful: our method detected the main patterns of the cytochrome P450 superfamily and the motifs linked to catalytic reactions. The 3D template also suggested the position of a residue, which could be involved in a hydrogen bond with CYP17 substrates and the shape and location of a cavity. Comparisons with independently generated 3D models comforted these hypotheses. Alignment software (Nestor3D) is available at http://www.kingston.ac.uk/~ku33185/Nestor3D.html

A structural perspective of the flavivirus life cycle.

PubMed

Mukhopadhyay, Suchetana; Kuhn, Richard J; Rossmann, Michael G

2005-01-01

Dengue, Japanese encephalitis, West Nile and yellow fever belong to the Flavivirus genus, which is a member of the Flaviviridae family. They are human pathogens that cause large epidemics and tens of thousands of deaths annually in many parts of the world. The structural organization of these viruses and their associated structural proteins has provided insight into the molecular transitions that occur during the viral life cycle, such as assembly, budding, maturation and fusion. This review focuses mainly on structural studies of dengue virus.

The structure of people's hair.

PubMed

Yang, Fei-Chi; Zhang, Yuchen; Rheinstädter, Maikel C

2014-01-01

Hair is a filamentous biomaterial consisting mainly of proteins in particular keratin. The structure of human hair is well known: the medulla is a loosely packed, disordered region near the centre of the hair surrounded by the cortex, which contains the major part of the fibre mass, mainly consisting of keratin proteins and structural lipids. The cortex is surrounded by the cuticle, a layer of dead, overlapping cells forming a protective layer around the hair. The corresponding structures have been studied extensively using a variety of different techniques, such as light, electron and atomic force microscopes, and also X-ray diffraction. We were interested in the question how much the molecular hair structure differs from person to person, between male and female hair, hair of different appearances such as colour and waviness. We included hair from parent and child, identical and fraternal twins in the study to see if genetically similar hair would show similar structural features. The molecular structure of the hair samples was studied using high-resolution X-ray diffraction, which covers length scales from molecules up to the organization of secondary structures. Signals due to the coiled-coil phase of α-helical keratin proteins, intermediate keratin filaments in the cortex and from the lipid layers in the cell membrane complex were observed in the specimen of all individuals, with very small deviations. Despite the relatively small number of individuals (12) included in this study, some conclusions can be drawn. While the general features were observed in all individuals and the corresponding molecular structures were almost identical, additional signals were observed in some specimen and assigned to different types of lipids in the cell membrane complex. Genetics seem to play a role in this composition as identical patterns were observed in hair from father and daughter and identical twins, however, not for fraternal twins. Identification and characterization of these features is an important step towards the detection of abnormalities in the molecular structure of hair as a potential diagnostic tool for certain diseases.

Charge-density analysis of a protein structure at subatomic resolution: the human aldose reductase case.

PubMed

Guillot, Benoît; Jelsch, Christian; Podjarny, Alberto; Lecomte, Claude

2008-05-01

The valence electron density of the protein human aldose reductase was analyzed at 0.66 angstroms resolution. The methodological developments in the software MoPro to adapt standard charge-density techniques from small molecules to macromolecular structures are described. The deformation electron density visible in initial residual Fourier difference maps was significantly enhanced after high-order refinement. The protein structure was refined after transfer of the experimental library multipolar atom model (ELMAM). The effects on the crystallographic statistics, on the atomic thermal displacement parameters and on the structure stereochemistry are analyzed. Constrained refinements of the transferred valence populations Pval and multipoles Plm were performed against the X-ray diffraction data on a selected substructure of the protein with low thermal motion. The resulting charge densities are of good quality, especially for chemical groups with many copies present in the polypeptide chain. To check the effect of the starting point on the result of the constrained multipolar refinement, the same charge-density refinement strategy was applied but using an initial neutral spherical atom model, i.e. without transfer from the ELMAM library. The best starting point for a protein multipolar refinement is the structure with the electron density transferred from the database. This can be assessed by the crystallographic statistical indices, including Rfree, and the quality of the static deformation electron-density maps, notably on the oxygen electron lone pairs. The analysis of the main-chain bond lengths suggests that stereochemical dictionaries would benefit from a revision based on recently determined unrestrained atomic resolution protein structures.

Chemical and structural biology of protein lysine deacetylases

PubMed Central

YOSHIDA, Minoru; KUDO, Norio; KOSONO, Saori; ITO, Akihiro

2017-01-01

Histone acetylation is a reversible posttranslational modification that plays a fundamental role in regulating eukaryotic gene expression and chromatin structure/function. Key enzymes for removing acetyl groups from histones are metal (zinc)-dependent and NAD+-dependent histone deacetylases (HDACs). The molecular function of HDACs have been extensively characterized by various approaches including chemical, molecular, and structural biology, which demonstrated that HDACs regulate cell proliferation, differentiation, and metabolic homeostasis, and that their alterations are deeply involved in various human disorders including cancer. Notably, drug discovery efforts have achieved success in developing HDAC-targeting therapeutics for treatment of several cancers. However, recent advancements in proteomics technology have revealed much broader aspects of HDACs beyond gene expression control. Not only histones but also a large number of cellular proteins are subject to acetylation by histone acetyltransferases (HATs) and deacetylation by HDACs. Furthermore, some of their structures can flexibly accept and hydrolyze other acyl groups on protein lysine residues. This review mainly focuses on structural aspects of HDAC enzymatic activity regulated by interaction with substrates, co-factors, small molecule inhibitors, and activators. PMID:28496053

Introduction to bioinformatics.

PubMed

Can, Tolga

2014-01-01

Bioinformatics is an interdisciplinary field mainly involving molecular biology and genetics, computer science, mathematics, and statistics. Data intensive, large-scale biological problems are addressed from a computational point of view. The most common problems are modeling biological processes at the molecular level and making inferences from collected data. A bioinformatics solution usually involves the following steps: Collect statistics from biological data. Build a computational model. Solve a computational modeling problem. Test and evaluate a computational algorithm. This chapter gives a brief introduction to bioinformatics by first providing an introduction to biological terminology and then discussing some classical bioinformatics problems organized by the types of data sources. Sequence analysis is the analysis of DNA and protein sequences for clues regarding function and includes subproblems such as identification of homologs, multiple sequence alignment, searching sequence patterns, and evolutionary analyses. Protein structures are three-dimensional data and the associated problems are structure prediction (secondary and tertiary), analysis of protein structures for clues regarding function, and structural alignment. Gene expression data is usually represented as matrices and analysis of microarray data mostly involves statistics analysis, classification, and clustering approaches. Biological networks such as gene regulatory networks, metabolic pathways, and protein-protein interaction networks are usually modeled as graphs and graph theoretic approaches are used to solve associated problems such as construction and analysis of large-scale networks.

Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects.

PubMed

Banyuls, N; Hernández-Rodríguez, C S; Van Rie, J; Ferré, J

2018-05-15

Vip3 vegetative insecticidal proteins from Bacillus thuringiensis are an important tool for crop protection against caterpillar pests in IPM strategies. While there is wide consensus on their general mode of action, the details of their mode of action are not completely elucidated and their structure remains unknown. In this work the alanine scanning technique was performed on 558 out of the total of 788 amino acids of the Vip3Af1 protein. From the 558 residue substitutions, 19 impaired protein expression and other 19 substitutions severely compromised the insecticidal activity against Spodoptera frugiperda. The latter 19 substitutions mainly clustered in two regions of the protein sequence (amino acids 167-272 and amino acids 689-741). Most of these substitutions also decreased the activity to Agrotis segetum. The characterisation of the sensitivity to proteases of the mutant proteins displaying decreased insecticidal activity revealed 6 different band patterns as evaluated by SDS-PAGE. The study of the intrinsic fluorescence of most selected mutants revealed only slight shifts in the emission peak, likely indicating only minor changes in the tertiary structure. An in silico modelled 3D structure of Vip3Af1 is proposed for the first time.

Effect of polymer molecular weight on chitosan-protein interaction.

PubMed

Bekale, L; Agudelo, D; Tajmir-Riahi, H A

2015-01-01

We present a comprehensive study of the interactions between chitosan nanoparticles (15, 100 and 200 kDa with the same degree of deacetylation 90%) and two model proteins, i.e., bovine (BSA) and human serum albumins (HSA), with the aim of correlating chitosan molecular weight (Mw) and the binding affinity of these naturally occurring polymers to protein. The effect of chitosan on the protein secondary structure and the influence of protein complexation on the shape of chitosan nanoparticles are discussed. A combination of multiple spectroscopic methods, transmission electron microscopy (TEM) and thermodynamic analysis were used to assess the polymer-protein complex formation. Results revealed that the three chitosan nanoparticles interact with BSA to form chitosan-BSA complexes, mainly through hydrophobic contacts with the affinity order: 200>100>15 kDa. However, HSA-chitosan complexation is mainly via electrostatic interactions with the stability order: 100>200>15 kDa. Furthermore, the association between polymer and protein causes a partial protein conformational change by a major reduction of α-helix from 63% (free BSA) to 57% (chitosan-BSA) and 57% (free HSA) to 51% (chitosan-HSA). Finally, TEM micrographs clearly revealed that the binding of serum albumins with chitosan nanoparticles induces a significant change in protein morphology and the shape of the polymer. Copyright © 2014 Elsevier B.V. All rights reserved.

Ferritin family proteins and their use in bionanotechnology.

PubMed

He, Didi; Marles-Wright, Jon

2015-12-25

Ferritin family proteins are found in all kingdoms of life and act to store iron within a protein cage and to protect the cell from oxidative damage caused by the Fenton reaction. The structural and biochemical features of the ferritins have been widely exploited in bionanotechnology applications: from the production of metal nanoparticles; as templates for semi-conductor production; and as scaffolds for vaccine design and drug delivery. In this review we first discuss the structural properties of the main ferritin family proteins, and describe how their organisation specifies their functions. Second, we describe materials science applications of ferritins that rely on their ability to sequester metal within their cavities. Finally, we explore the use of ferritin as a container for drug delivery and as a scaffold for the production of vaccines. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

ClusCo: clustering and comparison of protein models.

PubMed

Jamroz, Michal; Kolinski, Andrzej

2013-02-22

The development, optimization and validation of protein modeling methods require efficient tools for structural comparison. Frequently, a large number of models need to be compared with the target native structure. The main reason for the development of Clusco software was to create a high-throughput tool for all-versus-all comparison, because calculating similarity matrix is the one of the bottlenecks in the protein modeling pipeline. Clusco is fast and easy-to-use software for high-throughput comparison of protein models with different similarity measures (cRMSD, dRMSD, GDT_TS, TM-Score, MaxSub, Contact Map Overlap) and clustering of the comparison results with standard methods: K-means Clustering or Hierarchical Agglomerative Clustering. The application was highly optimized and written in C/C++, including the code for parallel execution on CPU and GPU, which resulted in a significant speedup over similar clustering and scoring computation programs.

Prediction of Ordered Water Molecules in Protein Binding Sites from Molecular Dynamics Simulations: The Impact of Ligand Binding on Hydration Networks.

PubMed

Rudling, Axel; Orro, Adolfo; Carlsson, Jens

2018-02-26

Water plays a major role in ligand binding and is attracting increasing attention in structure-based drug design. Water molecules can make large contributions to binding affinity by bridging protein-ligand interactions or by being displaced upon complex formation, but these phenomena are challenging to model at the molecular level. Herein, networks of ordered water molecules in protein binding sites were analyzed by clustering of molecular dynamics (MD) simulation trajectories. Locations of ordered waters (hydration sites) were first identified from simulations of high resolution crystal structures of 13 protein-ligand complexes. The MD-derived hydration sites reproduced 73% of the binding site water molecules observed in the crystal structures. If the simulations were repeated without the cocrystallized ligands, a majority (58%) of the crystal waters in the binding sites were still predicted. In addition, comparison of the hydration sites obtained from simulations carried out in the absence of ligands to those identified for the complexes revealed that the networks of ordered water molecules were preserved to a large extent, suggesting that the locations of waters in a protein-ligand interface are mainly dictated by the protein. Analysis of >1000 crystal structures showed that hydration sites bridged protein-ligand interactions in complexes with different ligands, and those with high MD-derived occupancies were more likely to correspond to experimentally observed ordered water molecules. The results demonstrate that ordered water molecules relevant for modeling of protein-ligand complexes can be identified from MD simulations. Our findings could contribute to development of improved methods for structure-based virtual screening and lead optimization.

WS-SNPs&GO: a web server for predicting the deleterious effect of human protein variants using functional annotation

PubMed Central

2013-01-01

Background SNPs&GO is a method for the prediction of deleterious Single Amino acid Polymorphisms (SAPs) using protein functional annotation. In this work, we present the web server implementation of SNPs&GO (WS-SNPs&GO). The server is based on Support Vector Machines (SVM) and for a given protein, its input comprises: the sequence and/or its three-dimensional structure (when available), a set of target variations and its functional Gene Ontology (GO) terms. The output of the server provides, for each protein variation, the probabilities to be associated to human diseases. Results The server consists of two main components, including updated versions of the sequence-based SNPs&GO (recently scored as one of the best algorithms for predicting deleterious SAPs) and of the structure-based SNPs&GO3d programs. Sequence and structure based algorithms are extensively tested on a large set of annotated variations extracted from the SwissVar database. Selecting a balanced dataset with more than 38,000 SAPs, the sequence-based approach achieves 81% overall accuracy, 0.61 correlation coefficient and an Area Under the Curve (AUC) of the Receiver Operating Characteristic (ROC) curve of 0.88. For the subset of ~6,600 variations mapped on protein structures available at the Protein Data Bank (PDB), the structure-based method scores with 84% overall accuracy, 0.68 correlation coefficient, and 0.91 AUC. When tested on a new blind set of variations, the results of the server are 79% and 83% overall accuracy for the sequence-based and structure-based inputs, respectively. Conclusions WS-SNPs&GO is a valuable tool that includes in a unique framework information derived from protein sequence, structure, evolutionary profile, and protein function. WS-SNPs&GO is freely available at http://snps.biofold.org/snps-and-go. PMID:23819482

GRID: a high-resolution protein structure refinement algorithm.

PubMed

Chitsaz, Mohsen; Mayo, Stephen L

2013-03-05

The energy-based refinement of protein structures generated by fold prediction algorithms to atomic-level accuracy remains a major challenge in structural biology. Energy-based refinement is mainly dependent on two components: (1) sufficiently accurate force fields, and (2) efficient conformational space search algorithms. Focusing on the latter, we developed a high-resolution refinement algorithm called GRID. It takes a three-dimensional protein structure as input and, using an all-atom force field, attempts to improve the energy of the structure by systematically perturbing backbone dihedrals and side-chain rotamer conformations. We compare GRID to Backrub, a stochastic algorithm that has been shown to predict a significant fraction of the conformational changes that occur with point mutations. We applied GRID and Backrub to 10 high-resolution (≤ 2.8 Å) crystal structures from the Protein Data Bank and measured the energy improvements obtained and the computation times required to achieve them. GRID resulted in energy improvements that were significantly better than those attained by Backrub while expending about the same amount of computational resources. GRID resulted in relaxed structures that had slightly higher backbone RMSDs compared to Backrub relative to the starting crystal structures. The average RMSD was 0.25 ± 0.02 Å for GRID versus 0.14 ± 0.04 Å for Backrub. These relatively minor deviations indicate that both algorithms generate structures that retain their original topologies, as expected given the nature of the algorithms. Copyright © 2012 Wiley Periodicals, Inc.

Structural and functional analyses of genes encoding VQ proteins in apple.

PubMed

Dong, Qinglong; Zhao, Shuang; Duan, Dingyue; Tian, Yi; Wang, Yanpeng; Mao, Ke; Zhou, Zongshan; Ma, Fengwang

2018-07-01

Recent studies with Arabidopsis and soybean have shown that a class of valine-glutamine (VQ) motif-containing proteins interacts with some WRKY transcription factors. However, little is known about the evolution, structures, and functions of those proteins in apple. Here, we examined their features and identified 49 apple VQ genes. Our evolutional analysis revealed that the proteins could be clustered into nine groups together with their homologues in 33 species. Historically, the main characteristics of proteins in Groups I, V, VI, VII, IX, and X were thought to have been generated before the monocot-dicot split, whereas those in Groups II, III + IV, and VIII were generated after that split. In the structural analysis, apple MdVQ proteins appeared to bind only with Group I and IIc MdWRKY proteins. Meanwhile, MdVQ1, MdVQ10, MdVQ15, and MdVQ36 interacted with multiple MdVQ proteins to form heterodimers but MdVQ15 formed a homodimer. The functional analysis indicated that overexpression of some apple MdVQs in Arabidopsis and tobacco plants effected their vegetative and reproductive growth. These results provide important information about the characteristics of apple MdVQ genes and can serve as a solid foundation for further studies about the role of WRKY-VQ interactions in regulating apple developmental and defense mechanisms. Copyright © 2018 Elsevier B.V. All rights reserved.

The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations.

PubMed

Levy, Ariel R; Turgeman, Meital; Gevorkyan-Aiapetov, Lada; Ruthstein, Sharon

2017-08-01

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer. © 2017 The Protein Society.

The intriguing plant nuclear lamina.

PubMed

Ciska, Malgorzata; Moreno Díaz de la Espina, Susana

2014-01-01

The nuclear lamina is a complex protein mesh attached to the inner nuclear membrane (INM), which is also associated with nuclear pore complexes. It provides mechanical support to the nucleus and nuclear envelope, and as well as facilitating the connection of the nucleoskeleton to the cytoskeleton, it is also involved in chromatin organization, gene regulation, and signaling. In metazoans, the nuclear lamina consists of a polymeric layer of lamins and other interacting proteins responsible for its association with the INM and chromatin. In plants, field emission scanning electron microscopy of nuclei, and thin section transmission electron microscopy of isolated nucleoskeletons, reveals the lamina to have a similar structure to that of metazoans. Moreover, although plants lack lamin genes and the genes encoding most lamin-binding proteins, the main functions of the lamina are fulfilled in plants. Hence, it would appear that the plant lamina is not based on lamins and that other proteins substitute for lamins in plant cells. The nuclear matrix constituent proteins are the best characterized structural proteins in the plant lamina. Although these proteins do not display strong sequence similarity to lamins, their predicted secondary structure and sub-nuclear distribution, as well as their influence on nuclear size and shape, and on heterochromatin organization, suggest they could be functional lamin analogs. In this review we shall summarize what is currently known about the organization and composition of the plant nuclear lamina and its interacting complexes, and we will discuss the activity of this structure in the plant cell and its nucleus.

Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems*

PubMed Central

Durand, Eric; Zoued, Abdelrahim; Spinelli, Silvia; Watson, Paul J. H.; Aschtgen, Marie-Stéphanie; Journet, Laure; Cambillau, Christian; Cascales, Eric

2012-01-01

The Type VI secretion system (T6SS) is a macromolecular system distributed in Gram-negative bacteria, responsible for the secretion of effector proteins into target cells. The T6SS has a broad versatility as it can target both eukaryotic and prokaryotic cells. It is therefore involved in host pathogenesis or killing neighboring bacterial cells to colonize a new niche. At the architecture level, the T6SS core apparatus is composed of 13 proteins, which assemble in two subcomplexes. One of these subcomplexes, composed of subunits that share structural similarities with bacteriophage tail and baseplate components, is anchored to the cell envelope by the membrane subcomplex. This latter is constituted of at least three proteins, TssL, TssM, and TssJ. The crystal structure of the TssJ outer membrane lipoprotein and its interaction with the inner membrane TssM protein have been recently reported. TssL and TssM share sequence homology and characteristics with two components of the Type IVb secretion system (T4bSS), IcmH/DotU and IcmF, respectively. In this study, we report the crystal structure of the cytoplasmic domain of the TssL inner membrane protein from the enteroaggregative Escherichia coli Sci-1 T6SS. It folds as a hook-like structure composed of two three-helix bundles. Two TssL molecules associate to form a functional complex. Although the TssL trans-membrane segment is the main determinant of self-interaction, contacts between the cytoplasmic domains are required for TssL function. Based on sequence homology and secondary structure prediction, we propose that the TssL structure is the prototype for the members of the TssL and IcmH/DotU families. PMID:22371492

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

PubMed Central

Kim, Hak Jun; Lee, Jun Hyuck; Hur, Young Baek; Lee, Chang Woo; Park, Sun-Ha; Koo, Bon-Won

2017-01-01

Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis (TH), ice recrystallization inhibition (IRI), and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type. PMID:28134801

Isolation, characterization, and aggregation of a structured bacterial matrix precursor.

PubMed

Chai, Liraz; Romero, Diego; Kayatekin, Can; Akabayov, Barak; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2013-06-14

Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some β-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.

Structural properties of matrix metalloproteinases.

PubMed

Bode, W; Fernandez-Catalan, C; Tschesche, H; Grams, F; Nagase, H; Maskos, K

1999-04-01

Matrix metalloproteinases (MMPs) are involved in extracellular matrix degradation. Their proteolytic activity must be precisely regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumour growth and metastasis. Knowledge of the tertiary structures of the proteins involved is crucial for understanding their functional properties and interference with associated dysfunctions. Within the last few years, several three-dimensional MMP and MMP-TIMP structures became available, showing the domain organization, polypeptide fold and main specificity determinants. Complexes of the catalytic MMP domains with various synthetic inhibitors enabled the structure-based design and improvement of high-affinity ligands, which might be elaborated into drugs. A multitude of reviews surveying work done on all aspects of MMPs have appeared in recent years, but none of them has focused on the three-dimensional structures. This review was written to close the gap.

Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

PubMed

Panigrahi, Rashmi; Lemieux, M Joanne

2015-01-01

Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

Defining the loop structures in proteins based on composite β-turn mimics.

PubMed

Dhar, Jesmita; Chakrabarti, Pinak

2015-06-01

Asx- and ω-turns are β-turn mimics, which replace the conventional main-chain hydrogen bonds seen in the latter by those involving the side chains, and both involve three residues. In this paper we analyzed the cases where these turns occur together--side by side, with or without any gap, overlapping and in any order. These composite turns (of length 3-15 residues), occurring at ∼1 per 100 residues, may constitute the full length of many loops, and when the residues in the two component turns overlap or are adjacent to each other, the composite may take well-defined shape. It is thus possible for non-regular regions in protein structure to form local structural motifs, akin to the regular geometrical features exhibited by secondary structures. Composites having the order ω-turns followed by Asx-turns can constitute N-terminal helix capping motif. Ternary composite turns (made up of ω-, Asx- and ST-turns), some with characteristic shape, have also been identified. Delineation of composite turns would help in characterizing loops in protein structures, which often have functional roles. Some sequence patterns seen in composites can be used for their incorporation in protein design. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

PDB explorer -- a web based algorithm for protein annotation viewer and 3D visualization.

PubMed

Nayarisseri, Anuraj; Shardiwal, Rakesh Kumar; Yadav, Mukesh; Kanungo, Neha; Singh, Pooja; Shah, Pratik; Ahmed, Sheaza

2014-12-01

The PDB file format, is a text format characterizing the three dimensional structures of macro molecules available in the Protein Data Bank (PDB). Determined protein structure are found in coalition with other molecules or ions such as nucleic acids, water, ions, Drug molecules and so on, which therefore can be described in the PDB format and have been deposited in PDB database. PDB is a machine generated file, it's not human readable format, to read this file we need any computational tool to understand it. The objective of our present study is to develop a free online software for retrieval, visualization and reading of annotation of a protein 3D structure which is available in PDB database. Main aim is to create PDB file in human readable format, i.e., the information in PDB file is converted in readable sentences. It displays all possible information from a PDB file including 3D structure of that file. Programming languages and scripting languages like Perl, CSS, Javascript, Ajax, and HTML have been used for the development of PDB Explorer. The PDB Explorer directly parses the PDB file, calling methods for parsed element secondary structure element, atoms, coordinates etc. PDB Explorer is freely available at http://www.pdbexplorer.eminentbio.com/home with no requirement of log-in.

Predicting turns in proteins with a unified model.

PubMed

Song, Qi; Li, Tonghua; Cong, Peisheng; Sun, Jiangming; Li, Dapeng; Tang, Shengnan

2012-01-01

Turns are a critical element of the structure of a protein; turns play a crucial role in loops, folds, and interactions. Current prediction methods are well developed for the prediction of individual turn types, including α-turn, β-turn, and γ-turn, etc. However, for further protein structure and function prediction it is necessary to develop a uniform model that can accurately predict all types of turns simultaneously. In this study, we present a novel approach, TurnP, which offers the ability to investigate all the turns in a protein based on a unified model. The main characteristics of TurnP are: (i) using newly exploited features of structural evolution information (secondary structure and shape string of protein) based on structure homologies, (ii) considering all types of turns in a unified model, and (iii) practical capability of accurate prediction of all turns simultaneously for a query. TurnP utilizes predicted secondary structures and predicted shape strings, both of which have greater accuracy, based on innovative technologies which were both developed by our group. Then, sequence and structural evolution features, which are profile of sequence, profile of secondary structures and profile of shape strings are generated by sequence and structure alignment. When TurnP was validated on a non-redundant dataset (4,107 entries) by five-fold cross-validation, we achieved an accuracy of 88.8% and a sensitivity of 71.8%, which exceeded the most state-of-the-art predictors of certain type of turn. Newly determined sequences, the EVA and CASP9 datasets were used as independent tests and the results we achieved were outstanding for turn predictions and confirmed the good performance of TurnP for practical applications.

Predicting Turns in Proteins with a Unified Model

PubMed Central

Song, Qi; Li, Tonghua; Cong, Peisheng; Sun, Jiangming; Li, Dapeng; Tang, Shengnan

2012-01-01

Motivation Turns are a critical element of the structure of a protein; turns play a crucial role in loops, folds, and interactions. Current prediction methods are well developed for the prediction of individual turn types, including α-turn, β-turn, and γ-turn, etc. However, for further protein structure and function prediction it is necessary to develop a uniform model that can accurately predict all types of turns simultaneously. Results In this study, we present a novel approach, TurnP, which offers the ability to investigate all the turns in a protein based on a unified model. The main characteristics of TurnP are: (i) using newly exploited features of structural evolution information (secondary structure and shape string of protein) based on structure homologies, (ii) considering all types of turns in a unified model, and (iii) practical capability of accurate prediction of all turns simultaneously for a query. TurnP utilizes predicted secondary structures and predicted shape strings, both of which have greater accuracy, based on innovative technologies which were both developed by our group. Then, sequence and structural evolution features, which are profile of sequence, profile of secondary structures and profile of shape strings are generated by sequence and structure alignment. When TurnP was validated on a non-redundant dataset (4,107 entries) by five-fold cross-validation, we achieved an accuracy of 88.8% and a sensitivity of 71.8%, which exceeded the most state-of-the-art predictors of certain type of turn. Newly determined sequences, the EVA and CASP9 datasets were used as independent tests and the results we achieved were outstanding for turn predictions and confirmed the good performance of TurnP for practical applications. PMID:23144872

Identification of residue pairing in interacting β-strands from a predicted residue contact map.

PubMed

Mao, Wenzhi; Wang, Tong; Zhang, Wenxuan; Gong, Haipeng

2018-04-19

Despite the rapid progress of protein residue contact prediction, predicted residue contact maps frequently contain many errors. However, information of residue pairing in β strands could be extracted from a noisy contact map, due to the presence of characteristic contact patterns in β-β interactions. This information may benefit the tertiary structure prediction of mainly β proteins. In this work, we propose a novel ridge-detection-based β-β contact predictor to identify residue pairing in β strands from any predicted residue contact map. Our algorithm RDb 2 C adopts ridge detection, a well-developed technique in computer image processing, to capture consecutive residue contacts, and then utilizes a novel multi-stage random forest framework to integrate the ridge information and additional features for prediction. Starting from the predicted contact map of CCMpred, RDb 2 C remarkably outperforms all state-of-the-art methods on two conventional test sets of β proteins (BetaSheet916 and BetaSheet1452), and achieves F1-scores of ~ 62% and ~ 76% at the residue level and strand level, respectively. Taking the prediction of the more advanced RaptorX-Contact as input, RDb 2 C achieves impressively higher performance, with F1-scores reaching ~ 76% and ~ 86% at the residue level and strand level, respectively. In a test of structural modeling using the top 1 L predicted contacts as constraints, for 61 mainly β proteins, the average TM-score achieves 0.442 when using the raw RaptorX-Contact prediction, but increases to 0.506 when using the improved prediction by RDb 2 C. Our method can significantly improve the prediction of β-β contacts from any predicted residue contact maps. Prediction results of our algorithm could be directly applied to effectively facilitate the practical structure prediction of mainly β proteins. All source data and codes are available at http://166.111.152.91/Downloads.html or the GitHub address of https://github.com/wzmao/RDb2C .

Identification and Characterization of Novel Chitin-Binding Proteins from the Larval Cuticle of Silkworm, Bombyx mori.

PubMed

Dong, Zhaoming; Zhang, Weiwei; Zhang, Yan; Zhang, Xiaolu; Zhao, Ping; Xia, Qingyou

2016-05-06

Cuticle is mainly made of chitin filaments embedded in a matrix of cuticular proteins (CPs). Cuticular chitins have minor differences, whereas CPs are widely variable with respect to their sequences and structures. To understand the molecular basis underlying the mechanical properties of cuticle, it is necessary to know which CPs interact with chitin and how they are assembled into the cuticle structure. In the present study, a chitin-binding assay was performed followed by liquid chromatography-tandem mass spectrometry to identify the extracted proteins from the larval cuticle of silkworm, Bombyx mori. There were 463 proteins identified from the silkworm larval cuticle, 200 of which were recovered in the chitin-binding fraction. A total of 103 proteins were annotated as CPs, which were classified into 11 CP families based on their conserved motifs, including CPR, CPAP, CPT, CPF and CPFL, CPCFC, chitin_bind 3, BmCPH2 homologues, BmCPH9 homologues, BmCPG1 homologues, BmCPG20 homologues, and BmCPG21 homologues. A total of five CP families were newly identified in the chitin-binding fraction, thereby providing new information and insight into the composition, structure, and function of the silkworm larval cuticle.

A Hydrophobic Gold Surface Triggers Misfolding and Aggregation of the Amyloidogenic Josephin Domain in Monomeric Form, While Leaving the Oligomers Unaffected

PubMed Central

Apicella, Alessandra; Soncini, Monica; Deriu, Marco Agostino; Natalello, Antonino; Bonanomi, Marcella; Dellasega, David; Tortora, Paolo; Regonesi, Maria Elena; Casari, Carlo Spartaco

2013-01-01

Protein misfolding and aggregation in intracellular and extracellular spaces is regarded as a main marker of the presence of degenerative disorders such as amyloidoses. To elucidate the mechanisms of protein misfolding, the interaction of proteins with inorganic surfaces is of particular relevance, since surfaces displaying different wettability properties may represent model systems of the cell membrane. Here, we unveil the role of surface hydrophobicity/hydrophilicity in the misfolding of the Josephin domain (JD), a globular-shaped domain of ataxin-3, the protein responsible for the spinocerebellar ataxia type 3. By means of a combined experimental and theoretical approach based on atomic force microscopy, Fourier transform infrared spectroscopy and molecular dynamics simulations, we reveal changes in JD morphology and secondary structure elicited by the interaction with the hydrophobic gold substrate, but not by the hydrophilic mica. Our results demonstrate that the interaction with the gold surface triggers misfolding of the JD when it is in native-like configuration, while no structural modification is observed after the protein has undergone oligomerization. This raises the possibility that biological membranes would be unable to affect amyloid oligomeric structures and toxicity. PMID:23527026

Unique crystal structure of a novel surfactant protein from the foam nest of the frog Leptodactylus vastus.

PubMed

Cavalcante Hissa, Denise; Arruda Bezerra, Gustavo; Birner-Gruenberger, Ruth; Paulino Silva, Luciano; Usón, Isabel; Gruber, Karl; Maciel Melo, Vânia Maria

2014-02-10

Breeding by releasing eggs into stable biofoams ("foam nests") is a peculiar reproduction mode within anurans, fish, and tunicates; not much is known regarding the biochemistry or molecular mechanisms involved. Lv-ranaspumin (Lv-RSN-1) is the predominant protein from the foam nest of the frog Leptodactylus vastus. This protein shows natural surfactant activity, which is assumed to be crucial for stabilizing foam nests. We elucidated the amino acid sequence of Lv-RSN-1 by de novo sequencing with mass-spectrometry and determined the high-resolution X-ray structure of the protein. It has a unique fold mainly composed of a bundle of 11 α-helices and two small antiparallel β-strands. Lv-RSN-1 has a surface rich in hydrophilic residues and a lipophilic cavity in the region of the antiparallel β-sheet. It possesses intrinsic surface-active properties, reducing the surface tension of water from 73 to 61 mN m(-1) (15 μg mL(-1)). Lv-RSN-1 belongs to a new class of surfactants proteins for which little has been reported regarding structure or function. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Distribution of genotype network sizes in sequence-to-structure genotype-phenotype maps.

PubMed

Manrubia, Susanna; Cuesta, José A

2017-04-01

An essential quantity to ensure evolvability of populations is the navigability of the genotype space. Navigability, understood as the ease with which alternative phenotypes are reached, relies on the existence of sufficiently large and mutually attainable genotype networks. The size of genotype networks (e.g. the number of RNA sequences folding into a particular secondary structure or the number of DNA sequences coding for the same protein structure) is astronomically large in all functional molecules investigated: an exhaustive experimental or computational study of all RNA folds or all protein structures becomes impossible even for moderately long sequences. Here, we analytically derive the distribution of genotype network sizes for a hierarchy of models which successively incorporate features of increasingly realistic sequence-to-structure genotype-phenotype maps. The main feature of these models relies on the characterization of each phenotype through a prototypical sequence whose sites admit a variable fraction of letters of the alphabet. Our models interpolate between two limit distributions: a power-law distribution, when the ordering of sites in the prototypical sequence is strongly constrained, and a lognormal distribution, as suggested for RNA, when different orderings of the same set of sites yield different phenotypes. Our main result is the qualitative and quantitative identification of those features of sequence-to-structure maps that lead to different distributions of genotype network sizes. © 2017 The Author(s).

Plant Lectins: Wheat Defense Strategy Against Hessian Fly

USDA-ARS?s Scientific Manuscript database

Plants produce a variety of defense proteins, including lectins in response to attack by phytophagous insects. Ultrastructural studies reveal that binding to insect gut structures and resistance to proteolytic degradation by insect digestive enzymes are the two main prerequisites for the lectins to...

APPRIS 2017: principal isoforms for multiple gene sets

PubMed Central

Rodriguez-Rivas, Juan; Di Domenico, Tomás; Vázquez, Jesús; Valencia, Alfonso

2018-01-01

Abstract The APPRIS database (http://appris-tools.org) uses protein structural and functional features and information from cross-species conservation to annotate splice isoforms in protein-coding genes. APPRIS selects a single protein isoform, the ‘principal’ isoform, as the reference for each gene based on these annotations. A single main splice isoform reflects the biological reality for most protein coding genes and APPRIS principal isoforms are the best predictors of these main proteins isoforms. Here, we present the updates to the database, new developments that include the addition of three new species (chimpanzee, Drosophila melangaster and Caenorhabditis elegans), the expansion of APPRIS to cover the RefSeq gene set and the UniProtKB proteome for six species and refinements in the core methods that make up the annotation pipeline. In addition APPRIS now provides a measure of reliability for individual principal isoforms and updates with each release of the GENCODE/Ensembl and RefSeq reference sets. The individual GENCODE/Ensembl, RefSeq and UniProtKB reference gene sets for six organisms have been merged to produce common sets of splice variants. PMID:29069475

Predicting β-Turns in Protein Using Kernel Logistic Regression

PubMed Central

Elbashir, Murtada Khalafallah; Sheng, Yu; Wang, Jianxin; Wu, FangXiang; Li, Min

2013-01-01

A β-turn is a secondary protein structure type that plays a significant role in protein configuration and function. On average 25% of amino acids in protein structures are located in β-turns. It is very important to develope an accurate and efficient method for β-turns prediction. Most of the current successful β-turns prediction methods use support vector machines (SVMs) or neural networks (NNs). The kernel logistic regression (KLR) is a powerful classification technique that has been applied successfully in many classification problems. However, it is often not found in β-turns classification, mainly because it is computationally expensive. In this paper, we used KLR to obtain sparse β-turns prediction in short evolution time. Secondary structure information and position-specific scoring matrices (PSSMs) are utilized as input features. We achieved Q total of 80.7% and MCC of 50% on BT426 dataset. These results show that KLR method with the right algorithm can yield performance equivalent to or even better than NNs and SVMs in β-turns prediction. In addition, KLR yields probabilistic outcome and has a well-defined extension to multiclass case. PMID:23509793

Predicting β-turns in protein using kernel logistic regression.

PubMed

Elbashir, Murtada Khalafallah; Sheng, Yu; Wang, Jianxin; Wu, Fangxiang; Li, Min

2013-01-01

A β-turn is a secondary protein structure type that plays a significant role in protein configuration and function. On average 25% of amino acids in protein structures are located in β-turns. It is very important to develope an accurate and efficient method for β-turns prediction. Most of the current successful β-turns prediction methods use support vector machines (SVMs) or neural networks (NNs). The kernel logistic regression (KLR) is a powerful classification technique that has been applied successfully in many classification problems. However, it is often not found in β-turns classification, mainly because it is computationally expensive. In this paper, we used KLR to obtain sparse β-turns prediction in short evolution time. Secondary structure information and position-specific scoring matrices (PSSMs) are utilized as input features. We achieved Q total of 80.7% and MCC of 50% on BT426 dataset. These results show that KLR method with the right algorithm can yield performance equivalent to or even better than NNs and SVMs in β-turns prediction. In addition, KLR yields probabilistic outcome and has a well-defined extension to multiclass case.

Inferring the Skeletal Muscle Developmental Changes of Grazing and Barn-Fed Goats from Gene Expression Data.

PubMed

Huang, Jinyu; Jiao, Jinzhen; Tan, Zhi-Liang; He, Zhixiong; Beauchemin, Karen A; Forster, Robert; Han, Xue-Feng; Tang, Shao-Xun; Kang, Jinghe; Zhou, Chuanshe

2016-09-14

Thirty-six Xiangdong black goats were used to investigate age-related mRNA and protein expression levels of some genes related to skeletal muscle structural proteins, MRFs and MEF2 family, and skeletal muscle fiber type and composition during skeletal muscle growth under grazing (G) and barn-fed (BF) feeding systems. Goats were slaughtered at six time points selected to reflect developmental changes of skeletal muscle during nonrumination (days 0, 7, and 14), transition (day 42), and rumination phases (days 56 and 70). It was observed that the number of type IIx in the longissimus dorsi was increased quickly while numbers of type IIa and IIb decreased slightly, indicating that these genes were coordinated during the rapid growth and development stages of skeletal muscle. No gene expression was affected (P > 0.05) by feeding system except Myf5 and Myf6. Protein expressions of MYOZ3 and MEF2C were affected (P < 0.05) by age, whereas PGC-1α was linearly decreased in the G group, and only MYOZ3 protein was affected (P < 0.001) by feeding system. Moreover, it was found that PGC-1α and MEF2C proteins may interact with each other in promoting muscle growth. The current results indicate that (1) skeletal muscle growth during days 0-70 after birth is mainly myofiber hypertrophy and differentiation, (2) weaning affects the expression of relevant genes of skeletal muscle structural proteins, skeletal muscle growth, and skeletal muscle fiber type and composition, and (3) nutrition or feeding regimen mainly influences the expression of skeletal muscle growth genes.

Key role of the N-terminus of chicken annexin A5 in vesicle aggregation.

PubMed

Turnay, Javier; Guzmán-Aránguez, Ana; Lecona, Emilio; Barrasa, Juan I; Olmo, Nieves; Lizarbe, Ma Antonia

2009-05-01

Annexins are calcium-dependent phospholipid-binding proteins involved in calcium signaling and intracellular membrane trafficking among other functions. Vesicle aggregation is a crucial event to make possible the membrane remodeling but this process is energetically unfavorable, and phospholipid membranes do not aggregate and fuse spontaneously. This issue can be circumvented by the presence of different agents such as divalent cations and/or proteins, among them some annexins. Although human annexin A5 lacks the ability to aggregate vesicles, here we demonstrate that its highly similar chicken ortholog induces aggregation of vesicles containing acidic phospholipids even at low protein and/or calcium concentration by establishment of protein dimers. Our experiments show that the ability to aggregate vesicles mainly resides in the N-terminus as truncation of the N-terminus of chicken annexin A5 significantly decreases this process and replacement of the N-terminus of human annexin A5 by that of chicken switches on aggregation; in both cases, there are no changes in the overall protein structure and only minor changes in phospholipid binding. Electrostatic repulsions between negatively charged residues in the concave face of the molecule, mainly in the N-terminus, seem to be responsible for the impairment of dimer formation in human annexin A5. Taking into account that chicken annexin A5 presents a high sequence and structural similarity with mammalian annexins absent in birds, as annexins A3 and A4, some of the physiological functions exerted by these proteins may be carried out by chicken annexin A5, even those that could require calcium-dependent membrane aggregation.

Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins

PubMed Central

Debelyy, Mykhaylo O.; Waridel, Patrice; Quadroni, Manfredo; Conzelmann, Andreas

2017-01-01

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p). PMID:29073188

Comparative modeling without implicit sequence alignments.

PubMed

Kolinski, Andrzej; Gront, Dominik

2007-10-01

The number of known protein sequences is about thousand times larger than the number of experimentally solved 3D structures. For more than half of the protein sequences a close or distant structural analog could be identified. The key starting point in a classical comparative modeling is to generate the best possible sequence alignment with a template or templates. With decreasing sequence similarity, the number of errors in the alignments increases and these errors are the main causes of the decreasing accuracy of the molecular models generated. Here we propose a new approach to comparative modeling, which does not require the implicit alignment - the model building phase explores geometric, evolutionary and physical properties of a template (or templates). The proposed method requires prior identification of a template, although the initial sequence alignment is ignored. The model is built using a very efficient reduced representation search engine CABS to find the best possible superposition of the query protein onto the template represented as a 3D multi-featured scaffold. The criteria used include: sequence similarity, predicted secondary structure consistency, local geometric features and hydrophobicity profile. For more difficult cases, the new method qualitatively outperforms existing schemes of comparative modeling. The algorithm unifies de novo modeling, 3D threading and sequence-based methods. The main idea is general and could be easily combined with other efficient modeling tools as Rosetta, UNRES and others.

Aurelia aurita (Cnidaria) Oocytes' Contact Plate Structure and Development

PubMed Central

Adonin, Leonid S.; Shaposhnikova, Tatyana G.; Podgornaya, Olga

2012-01-01

One of the A. aurita medusa main mesoglea polypeptides, mesoglein, has been described previously. Mesoglein belongs to ZP-domain protein family and therefore we focused on A.aurita oogenesis. Antibodies against mesoglein (AB RA47) stain the plate in the place where germinal epithelium contacts oocyte on the paraffin sections. According to its position, we named the structure found the “contact plate”. Our main instrument was AB against mesoglein. ZP-domain occupies about half of the whole amino acid sequence of the mesoglein. Immunoblot after SDS-PAGE and AU-PAGE reveals two charged and high Mr bands among the female gonad germinal epithelium polypeptides. One of the gonads' polypeptides Mr corresponds to that of mesogleal cells, the other ones' Mr is higher. The morphological description of contact plate formation is the subject of the current work. Two types of AB RA47 positive granules were observed during progressive oogenesis stages. Granules form the contact plate in mature oocyte. Contact plate of A.aurita oocyte marks its animal pole and resembles Zona Pellucida by the following features: (1) it attracts spermatozoids; (2) the material of the contact plate is synthesized by oocyte and stored in granules; (3) these granules and the contact plate itself contain ZP domain protein(s); (4) contact plate is an extracellular structure made up of fiber bundles similar to those of conventional Zona Pellucida. PMID:23185235

Self-similar assemblies of globular whey proteins at the air-water interface: effect of the structure.

PubMed

Mahmoudi, Najet; Gaillard, Cédric; Boué, François; Axelos, Monique A V; Riaublanc, Alain

2010-05-01

We investigated the structure of heat-induced assemblies of whey globular proteins using small angle neutron scattering (SANS), static and dynamic light scattering (SLS and DLS), and cryogenic transmission electron microscopy (Cryo-TEM). Whey protein molecules self-assemble in fractal aggregates with a structure density depending on the electrostatic interactions. We determined the static and dynamic properties of interfacial layer formed by the protein assemblies, upon adsorption and spreading at the air-water interface using surface film balance and interfacial dilatational rheology. Upon spreading, all whey protein systems show a power-law scaling behavior of the surface pressure versus concentration in the semi-dilute surface concentration regime, with an exponent ranging from 5.5 to 9 depending on the electrostatic interactions and the aggregation state. The dilatational modulus derived from surface pressure isotherms shows a main peak at 6-8 mN/m, generally considered to be the onset of a conformational change in the monolayer, and a second peak or a shoulder at 15 mN/m. Long-time adsorption kinetics give similar results for both the native whey proteins and the corresponding self-similar assemblies, with a systematic effect of the ionic strength. Copyright 2010 Elsevier Inc. All rights reserved.

Structure-wise discrimination of cytosine, thymine, and uracil by proteins in terms of their nonbonded interactions.

PubMed

Usha, S; Selvaraj, S

2014-01-01

The molecular recognition and discrimination of very similar ligand moieties by proteins are important subjects in protein-ligand interaction studies. Specificity in the recognition of molecules is determined by the arrangement of protein and ligand atoms in space. The three pyrimidine bases, viz. cytosine, thymine, and uracil, are structurally similar, but the proteins that bind to them are able to discriminate them and form interactions. Since nonbonded interactions are responsible for molecular recognition processes in biological systems, our work attempts to understand some of the underlying principles of such recognition of pyrimidine molecular structures by proteins. The preferences of the amino acid residues to contact the pyrimidine bases in terms of nonbonded interactions; amino acid residue-ligand atom preferences; main chain and side chain atom contributions of amino acid residues; and solvent-accessible surface area of ligand atoms when forming complexes are analyzed. Our analysis shows that the amino acid residues, tyrosine and phenyl alanine, are highly involved in the pyrimidine interactions. Arginine prefers contacts with the cytosine base. The similarities and differences that exist between the interactions of the amino acid residues with each of the three pyrimidine base atoms in our analysis provide insights that can be exploited in designing specific inhibitors competitive to the ligands.

Memprot: a program to model the detergent corona around a membrane protein based on SEC–SAXS data

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

Pérez, Javier, E-mail: javier.perez@synchrotron-soleil.fr; Koutsioubas, Alexandros; Synchrotron SOLEIL, L’Orme des Merisiers, BP 48, Saint-Aubin, 91192 Gif-sur-Yvette

Systematic SAXS simulations have been analysed over a wide range of parameters in order to better understand the detergent corona around a membrane protein. The application of small-angle X-ray scattering (SAXS) to structural investigations of transmembrane proteins in detergent solution has been hampered by two main inherent hurdles. On the one hand, the formation of a detergent corona around the hydrophobic region of the protein strongly modifies the scattering curve of the protein. On the other hand, free micelles of detergent without a precisely known concentration coexist with the protein–detergent complex in solution, therefore adding an uncontrolled signal. To gainmore » robust structural information on such systems from SAXS data, in previous work, advantage was taken of the online combination of size-exclusion chromatography (SEC) and SAXS, and the detergent corona around aquaporin-0, a membrane protein of known structure, could be modelled. A precise geometrical model of the corona, shaped as an elliptical torus, was determined. Here, in order to better understand the correlations between the corona model parameters and to discuss the uniqueness of the model, this work was revisited by analyzing systematic SAXS simulations over a wide range of parameters of the torus.« less

Hydrogen/deuterium exchange in mass spectrometry.

PubMed

Kostyukevich, Yury; Acter, Thamina; Zherebker, Alexander; Ahmed, Arif; Kim, Sunghwan; Nikolaev, Eugene

2018-03-30

The isotopic exchange approach is in use since the first observation of such reactions in 1933 by Lewis. This approach allows the investigation of the pathways of chemical and biochemical reactions, determination of structure, composition, and conformation of molecules. Mass spectrometry has now become one of the most important analytical tools for the monitoring of the isotopic exchange reactions. Investigation of conformational dynamics of proteins, quantitative measurements, obtaining chemical, and structural information about individual compounds of the complex natural mixtures are mainly based on the use of isotope exchange in combination with high resolution mass spectrometry. The most important reaction is the Hydrogen/Deuterium exchange, which is mainly performed in the solution. Recently we have developed the approach allowing performing of the Hydrogen/Deuterium reaction on-line directly in the ionization source under atmospheric pressure. Such approach simplifies the sample preparation and can accelerate the exchange reaction so that certain hydrogens that are considered as non-labile will also participate in the exchange. The use of in-ionization source H/D exchange in modern mass spectrometry for structural elucidation of molecules serves as the basic theme in this review. We will focus on the mechanisms of the isotopic exchange reactions and on the application of in-ESI, in-APCI, and in-APPI source Hydrogen/Deuterium exchange for the investigation of petroleum, natural organic matter, oligosaccharides, and proteins including protein-protein complexes. The simple scenario for adaptation of H/D exchange reactions into mass spectrometric method is also highlighted along with a couple of examples collected from previous studies. © 2018 Wiley Periodicals, Inc.

[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

PubMed

Dong, Yun-qiao; Ma, Wen-li; Gu, Jin-bao; Zheng, Wen-ling

2009-12-01

To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.

Protein Binding and Astringent Taste of a Polymeric Procyanidin, 1,2,3,4,6-Penta-O-galloyl-β-D-glucopyranose, Castalagin and Grandinin

PubMed Central

Hofmann, Thomas; Glabasnia, Arne; Schwarz, Bernd; Wisman, Kimberly N.; Gangwer, Kelly A.; Hagerman, Ann E.

2008-01-01

The objective of the present investigation was to examine oral astringency and protein binding activity of four structurally well-defined tannins, namely procyanidin (epicatechin16(4→8)catechin), pentagalloyl glucose (1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose), castalagin, and grandinin, representing the three main structural categories of tannins, the proanthocyanidins, the gallotannins, and the ellagitannins. Astringency threshold and dose response were determined by the half-tongue test using a trained human panel. Protein binding stoichiometry and relative affinity were determined using radioiodinated bovine serum albumin in precipitation or competitive binding assays. Procyanidin and pentagalloyl glucose were perceived as highly astringent compounds and had relatively steep dose response curves but castalagin and grandinin had a lower mass threshold for detection. In vitro, procyanidin was the most effective protein precipitating agent, and grandinin the least. Increasing the temperature increased protein precipitation by the hydrolysable tannins, especially grandinin. All four polyphenols had higher relative affinity for proline-rich proteins than for bovine serum albumin. PMID:17147439

PaLaCe: A Coarse-Grain Protein Model for Studying Mechanical Properties.

PubMed

Pasi, Marco; Lavery, Richard; Ceres, Nicoletta

2013-01-08

We present a coarse-grain protein model PaLaCe (Pasi-Lavery-Ceres) that has been developed principally to allow fast computational studies of protein mechanics and to clarify the links between mechanics and function. PaLaCe uses a two-tier protein representation with one to three pseudoatoms representing each amino acid for the main nonbonded interactions, combined with atomic-scale peptide groups and some side chain atoms to allow the explicit representation of backbone hydrogen bonds and to simplify the treatment of bonded interactions. The PaLaCe force field is composed of physics-based terms, parametrized using Boltzmann inversion of conformational probability distributions derived from a protein structure data set, and iteratively refined to reproduce the experimental distributions. PaLaCe has been implemented in the MMTK simulation package and can be used for energy minimization, normal mode calculations, and molecular or stochastic dynamics. We present simulations with PaLaCe that test its ability to maintain stable structures for folded proteins, reproduce their dynamic fluctuations, and correctly model large-scale, force-induced conformational changes.

Structures and functions of proteins and nucleic acids in protein biosynthesis

NASA Astrophysics Data System (ADS)

Miyazawa, Tatsuo; Yokoyama, Shigeyuki

Infrared and Raman spectroscopy is useful for studying helical conformations of polypeptides, which are determined by molecular structure parameters. Nuclear magnetic resonance spectroscopy, as well as X-ray analysis, is now established to be important for conformation studies of proteins and nucleic acids in solution. This article is mainly concerned with the conformational aspect and function regulation in protein biosynthesis. The strict recognition of transfer ribonucleic acid (tRNA) by aminoacyl-tRNA synthetase (ARS) is achieved by multi-step mutual adaptation. The conformations of ARS-bound amino acids have been elucidated by transferred nuclear Overhauser effect analysis. Aminoacyl-tRNA takes the 3‧-isomeric form in the polypeptide chain elongation cycle. The regulation of codon recognition by post-transcriptional modification is achieved by conversion of the conformational characteristic of the anticodon of tRNA. The cytidine → lysidine modification of the anticodon of minor isoleucine tRNA concurrently converts the amino acid specificity and the codon specificity. As novel protein engineering, a basic strategy has been established for in vivo biosynthesis of proteins that are substituted with unnatural amino acids (alloproteins).

DASS: efficient discovery and p-value calculation of substructures in unordered data.

PubMed

Hollunder, Jens; Friedel, Maik; Beyer, Andreas; Workman, Christopher T; Wilhelm, Thomas

2007-01-01

Pattern identification in biological sequence data is one of the main objectives of bioinformatics research. However, few methods are available for detecting patterns (substructures) in unordered datasets. Data mining algorithms mainly developed outside the realm of bioinformatics have been adapted for that purpose, but typically do not determine the statistical significance of the identified patterns. Moreover, these algorithms do not exploit the often modular structure of biological data. We present the algorithm DASS (Discovery of All Significant Substructures) that first identifies all substructures in unordered data (DASS(Sub)) in a manner that is especially efficient for modular data. In addition, DASS calculates the statistical significance of the identified substructures, for sets with at most one element of each type (DASS(P(set))), or for sets with multiple occurrence of elements (DASS(P(mset))). The power and versatility of DASS is demonstrated by four examples: combinations of protein domains in multi-domain proteins, combinations of proteins in protein complexes (protein subcomplexes), combinations of transcription factor target sites in promoter regions and evolutionarily conserved protein interaction subnetworks. The program code and additional data are available at http://www.fli-leibniz.de/tsb/DASS

The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs

PubMed Central

Arieti, Fabiana; Gabus, Caroline; Tambalo, Margherita; Huet, Tiphaine; Round, Adam; Thore, Stéphane

2014-01-01

The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. We have determined the crystal structure of the region of the human SPEN homolog that contains these RRMs—the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 Å resolution. SHARP is a co-regulator of the nuclear receptors. We demonstrate that two of the three RRMs, namely RRM3 and RRM4, interact via a highly conserved interface. Furthermore, we show that the RRM3–RRM4 block is the main platform mediating the stable association with the H12–H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. We determine that SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP–RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein functions. PMID:24748666

Regulatory Peptides in Plants.

PubMed

Vanyushin, B F; Ashapkin, V V; Aleksandrushkina, N I

2017-02-01

Many different peptides regulating cell differentiation, growth, and development are found in plants. Peptides participate in regulation of plant ontogenesis starting from pollination, pollen tube growth, and the very early stages of embryogenesis, including formation of embryo and endosperm. They direct differentiation of meristematic stem cells, formation of tissues and individual organs, take part in regulation of aging, fruit maturation, and abscission of plant parts associated with apoptosis. Biological activity of peptides is observed at very low concentrations, and it has mainly signal nature and hormonal character. "Mature" peptides appear mainly due to processing of protein precursors with (or without) additional enzymatic modifications. Plant peptides differ in origin, structure, and functional properties. Their specific action is due to binding with respective receptors and interactions with various proteins and other factors. Peptides can also regulate physiological functions by direct peptide-protein interactions. Peptide action is coordinated with the action of known phytohormones (auxins, cytokinins, and others); thus, peptides control phytohormonal signal pathways.

Myoepithelial cells are the main component in pleomorphic adenomas?

PubMed

Ponce Bravo, Santa; Ledesma Montes, Constantino; López Becerril, Uriel; Morales Sánchez, Israel

2007-03-01

The aim of this study was to quantify by immunohistochemistry the number of myoepithelial cells (MyECs) in pleomorphic adenomas (PAs). We retrieved the paraffin cubes of 27 PAs, new slides were done and they were stained with anti-S100 protein antibody. The amount of S-100 protein positive cells was quantified, their morphology was recorded and comparison among MyEC number with age, gender and involved gland were also done. With S-100 protein, MyECs in normal salivary gland tissue were seen surrounding the ductual structures only. In the analysed PAs a mean of 27.4% of the neoplastic cells were positive to the antibody. With the exception of one PA, in all the analysed cases the plasmacytoid cells were the most commonly identified cells (48,6%). Results of this study suggest that MyECs do not constitute the main cellular component of the neoplastic compartment in PAs and corroborate the previously reported evidence by different authors, who studying the PAs suggested that MyECs does not comprise the main cellular neoplastic component of these entities.

The proteins of the grape (Vitis vinifera L.) seed endosperm: fractionation and identification of the major components.

PubMed

Gazzola, Diana; Vincenzi, Simone; Gastaldon, Luca; Tolin, Serena; Pasini, Gabriella; Curioni, Andrea

2014-07-15

In the present study, grape (Vitis vinifera L.) seed endosperm proteins were characterized after sequential fractionation, according to a modified Osborne procedure. The salt-soluble fraction (albumins and globulins) comprised the majority (58.4%) of the total extracted protein. The protein fractions analysed by SDS-PAGE showed similar bands, indicating different solubility of the same protein components. SDS-PAGE in non-reducing and reducing conditions revealed the polypeptide composition of the protein bands. The main polypeptides, which were similar in all the grape varieties analysed, were identified by LC-MS/MS as homologous to the 11S globulin-like seed storage proteins of other plant species, while a monomeric 43 kDa protein presented high homology with the 7S globulins of legume seeds. The results provide new insights about the identity, structure and polypeptide composition of the grape seed storage proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars.

PubMed

Popowska, Magdalena; Osińska, Magdalena; Rzeczkowska, Magdalena

2012-04-01

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.

Identification and sequence analyses of novel lipase encoding novel thermophillic bacilli isolated from Armenian geothermal springs.

PubMed

Shahinyan, Grigor; Margaryan, Armine; Panosyan, Hovik; Trchounian, Armen

2017-05-02

Among the huge diversity of thermophilic bacteria mainly bacilli have been reported as active thermostable lipase producers. Geothermal springs serve as the main source for isolation of thermostable lipase producing bacilli. Thermostable lipolytic enzymes, functioning in the harsh conditions, have promising applications in processing of organic chemicals, detergent formulation, synthesis of biosurfactants, pharmaceutical processing etc. In order to study the distribution of lipase-producing thermophilic bacilli and their specific lipase protein primary structures, three lipase producers from different genera were isolated from mesothermal (27.5-70 °C) springs distributed on the territory of Armenia and Nagorno Karabakh. Based on phenotypic characteristics and 16S rRNA gene sequencing the isolates were identified as Geobacillus sp., Bacillus licheniformis and Anoxibacillus flavithermus strains. The lipase genes of isolates were sequenced by using initially designed primer sets. Multiple alignments generated from primary structures of the lipase proteins and annotated lipase protein sequences, conserved regions analysis and amino acid composition have illustrated the similarity (98-99%) of the lipases with true lipases (family I) and GDSL esterase family (family II). A conserved sequence block that determines the thermostability has been identified in the multiple alignments of the lipase proteins. The results are spreading light on the lipase producing bacilli distribution in geothermal springs in Armenia and Nagorno Karabakh. Newly isolated bacilli strains could be prospective source for thermostable lipases and their genes.

Improvement of glucose and lipid metabolism via mung bean protein consumption: clinical trials of GLUCODIA™ isolated mung bean protein in the USA and Canada.

PubMed

Kohno, Mitsutaka; Sugano, Hideo; Shigihara, Yuhko; Shiraishi, Yoshiaki; Motoyama, Takayasu

2018-01-01

The aim of the present study was to confirm the effects of a commercially available mung bean protein isolate (GLUCODIA™) on glucose and lipid metabolism. The main component of GLUCODIA™ is 8S globulin, which constitutes 80 % of the total protein. The overall structure of this protein closely resembles soyabean β-conglycinin, which accounts for 20 % of total soya protein (soya protein isolate; SPI). Many physiological beneficial effects of β-conglycinin have been reported. GLUCODIA™ is expected to produce beneficial effects with fewer intakes than SPI. We conducted two independent double-blind, placebo-controlled clinical studies. In the first (preliminary dose decision trial) study, mung bean protein was shown to exert physiological beneficial effects when 3·0 g were ingested per d. In the second (main clinical trial) study, mung bean protein isolate did not lower plasma glucose levels, although the mean insulin level decreased with consumption of mung bean protein. The homeostatic model assessment of insulin resistance (HOMA-IR) values significantly decreased with mung bean protein. The mean TAG level significantly decreased with consumption of mung bean protein isolate. A significant increase in serum adiponectin levels and improvement in liver function enzymes were observed. These findings suggest that GLUCODIA™ could be useful in the prevention of insulin resistance and visceral fat accumulation, which are known to trigger the metabolic syndrome, and in the prevention of liver function decline.

Single-molecule protein sequencing through fingerprinting: computational assessment

NASA Astrophysics Data System (ADS)

Yao, Yao; Docter, Margreet; van Ginkel, Jetty; de Ridder, Dick; Joo, Chirlmin

2015-10-01

Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

PubMed

Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

2008-02-15

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. Copyright 2007 Wiley-Liss, Inc.

pyDockWEB: a web server for rigid-body protein-protein docking using electrostatics and desolvation scoring.

PubMed

Jiménez-García, Brian; Pons, Carles; Fernández-Recio, Juan

2013-07-01

pyDockWEB is a web server for the rigid-body docking prediction of protein-protein complex structures using a new version of the pyDock scoring algorithm. We use here a new custom parallel FTDock implementation, with adjusted grid size for optimal FFT calculations, and a new version of pyDock, which dramatically speeds up calculations while keeping the same predictive accuracy. Given the 3D coordinates of two interacting proteins, pyDockWEB returns the best docking orientations as scored mainly by electrostatics and desolvation energy. The server does not require registration by the user and is freely accessible for academics at http://life.bsc.es/servlet/pydock. Supplementary data are available at Bioinformatics online.

Screening and expression of selected taxonomically conserved and unique hypothetical proteins in Burkholderia pseudomallei K96243

NASA Astrophysics Data System (ADS)

Akhir, Nor Azurah Mat; Nadzirin, Nurul; Mohamed, Rahmah; Firdaus-Raih, Mohd

2015-09-01

Hypothetical proteins of bacterial pathogens represent a large numbers of novel biological mechanisms which could belong to essential pathways in the bacteria. They lack functional characterizations mainly due to the inability of sequence homology based methods to detect functional relationships in the absence of detectable sequence similarity. The dataset derived from this study showed 550 candidates conserved in genomes that has pathogenicity information and only present in the Burkholderiales order. The dataset has been narrowed down to taxonomic clusters. Ten proteins were selected for ORF amplification, seven of them were successfully amplified, and only four proteins were successfully expressed. These proteins will be great candidates in determining the true function via structural biology.

Crystal Structure of Allophycocyanin from Marine Cyanobacterium Phormidium sp. A09DM

PubMed Central

Gupta, Gagan Deep; Madamwar, Datta

2015-01-01

Isolated phycobilisome (PBS) sub-assemblies have been widely subjected to X-ray crystallography analysis to obtain greater insights into the structure-function relationship of this light harvesting complex. Allophycocyanin (APC) is the phycobiliprotein always found in the PBS core complex. Phycocyanobilin (PCB) chromophores, covalently bound to conserved Cys residues of α- and β- subunits of APC, are responsible for solar energy absorption from phycocyanin and for transfer to photosynthetic apparatus. In the known APC structures, heterodimers of α- and β- subunits (known as αβ monomers) assemble as trimer or hexamer. We here for the first time report the crystal structure of APC isolated from a marine cyanobacterium (Phormidium sp. A09DM). The crystal structure has been refined against all the observed data to the resolution of 2.51 Å to Rwork (Rfree) of 0.158 (0.229) with good stereochemistry of the atomic model. The Phormidium protein exists as a trimer of αβ monomers in solution and in crystal lattice. The overall tertiary structures of α- and β- subunits, and trimeric quaternary fold of the Phormidium protein resemble the other known APC structures. Also, configuration and conformation of the two covalently bound PCB chromophores in the marine APC are same as those observed in fresh water cyanobacteria and marine red algae. More hydrophobic residues, however, constitute the environment of the chromophore bound to α-subunit of the Phormidium protein, owing mainly to amino acid substitutions in the marine protein. PMID:25923120

SFESA: a web server for pairwise alignment refinement by secondary structure shifts.

PubMed

Tong, Jing; Pei, Jimin; Grishin, Nick V

2015-09-03

Protein sequence alignment is essential for a variety of tasks such as homology modeling and active site prediction. Alignment errors remain the main cause of low-quality structure models. A bioinformatics tool to refine alignments is needed to make protein alignments more accurate. We developed the SFESA web server to refine pairwise protein sequence alignments. Compared to the previous version of SFESA, which required a set of 3D coordinates for a protein, the new server will search a sequence database for the closest homolog with an available 3D structure to be used as a template. For each alignment block defined by secondary structure elements in the template, SFESA evaluates alignment variants generated by local shifts and selects the best-scoring alignment variant. A scoring function that combines the sequence score of profile-profile comparison and the structure score of template-derived contact energy is used for evaluation of alignments. PROMALS pairwise alignments refined by SFESA are more accurate than those produced by current advanced alignment methods such as HHpred and CNFpred. In addition, SFESA also improves alignments generated by other software. SFESA is a web-based tool for alignment refinement, designed for researchers to compute, refine, and evaluate pairwise alignments with a combined sequence and structure scoring of alignment blocks. To our knowledge, the SFESA web server is the only tool that refines alignments by evaluating local shifts of secondary structure elements. The SFESA web server is available at http://prodata.swmed.edu/sfesa.

Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

PubMed Central

He, Didi; Hughes, Sam; Vanden-Hehir, Sally; Georgiev, Atanas; Altenbach, Kirsten; Tarrant, Emma; Mackay, C Logan; Waldron, Kevin J; Clarke, David J; Marles-Wright, Jon

2016-01-01

Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins. DOI: http://dx.doi.org/10.7554/eLife.18972.001 PMID:27529188

Baculovirus-mediated expression of GPCRs in insect cells.

PubMed

Saarenpää, Tuulia; Jaakola, Veli-Pekka; Goldman, Adrian

2015-01-01

G-protein-coupled receptors (GPCRs) are a large family of seven transmembrane proteins that influence a considerable number of cellular events. For this reason, they are one of the most studied receptor types for their pharmacological and structural properties. Solving the structure of several GPCR receptor types has been possible using almost all expression systems, including Escherichia coli, yeast, mammalian, and insect cells. So far, however, most of the GPCR structures solved have been done using the baculovirus insect cell expression system. The reason for this is mainly due to cost-effectiveness, posttranslational modification efficiency, and overall effortless maintenance. The system has evolved so much that variables starting from vector type, purification tags, cell line, and growth conditions can be varied and optimized countless ways to suit the needs of new constructs. Here, we present the array of techniques that enable the rapid and efficient optimization of expression steps for maximal protein quality and quantity, including our emendations. © 2015 Elsevier Inc. All rights reserved.

Structure, Biology, and Therapeutic Application of Toxin–Antitoxin Systems in Pathogenic Bacteria

PubMed Central

Lee, Ki-Young; Lee, Bong-Jin

2016-01-01

Bacterial toxin–antitoxin (TA) systems have received increasing attention for their diverse identities, structures, and functional implications in cell cycle arrest and survival against environmental stresses such as nutrient deficiency, antibiotic treatments, and immune system attacks. In this review, we describe the biological functions and the auto-regulatory mechanisms of six different types of TA systems, among which the type II TA system has been most extensively studied. The functions of type II toxins include mRNA/tRNA cleavage, gyrase/ribosome poison, and protein phosphorylation, which can be neutralized by their cognate antitoxins. We mainly explore the similar but divergent structures of type II TA proteins from 12 important pathogenic bacteria, including various aspects of protein–protein interactions. Accumulating knowledge about the structure–function correlation of TA systems from pathogenic bacteria has facilitated a novel strategy to develop antibiotic drugs that target specific pathogens. These molecules could increase the intrinsic activity of the toxin by artificially interfering with the intermolecular network of the TA systems. PMID:27782085

Soft Computing Methods for Disulfide Connectivity Prediction.

PubMed

Márquez-Chamorro, Alfonso E; Aguilar-Ruiz, Jesús S

2015-01-01

The problem of protein structure prediction (PSP) is one of the main challenges in structural bioinformatics. To tackle this problem, PSP can be divided into several subproblems. One of these subproblems is the prediction of disulfide bonds. The disulfide connectivity prediction problem consists in identifying which nonadjacent cysteines would be cross-linked from all possible candidates. Determining the disulfide bond connectivity between the cysteines of a protein is desirable as a previous step of the 3D PSP, as the protein conformational search space is highly reduced. The most representative soft computing approaches for the disulfide bonds connectivity prediction problem of the last decade are summarized in this paper. Certain aspects, such as the different methodologies based on soft computing approaches (artificial neural network or support vector machine) or features of the algorithms, are used for the classification of these methods.

Large-scale analysis of intrinsic disorder flavors and associated functions in the protein sequence universe.

PubMed

Necci, Marco; Piovesan, Damiano; Tosatto, Silvio C E

2016-12-01

Intrinsic disorder (ID) in proteins has been extensively described for the last decade; a large-scale classification of ID in proteins is mostly missing. Here, we provide an extensive analysis of ID in the protein universe on the UniProt database derived from sequence-based predictions in MobiDB. Almost half the sequences contain an ID region of at least five residues. About 9% of proteins have a long ID region of over 20 residues which are more abundant in Eukaryotic organisms and most frequently cover less than 20% of the sequence. A small subset of about 67,000 (out of over 80 million) proteins is fully disordered and mostly found in Viruses. Most proteins have only one ID, with short ID evenly distributed along the sequence and long ID overrepresented in the center. The charged residue composition of Das and Pappu was used to classify ID proteins by structural propensities and corresponding functional enrichment. Swollen Coils seem to be used mainly as structural components and in biosynthesis in both Prokaryotes and Eukaryotes. In Bacteria, they are confined in the nucleoid and in Viruses provide DNA binding function. Coils & Hairpins seem to be specialized in ribosome binding and methylation activities. Globules & Tadpoles bind antigens in Eukaryotes but are involved in killing other organisms and cytolysis in Bacteria. The Undefined class is used by Bacteria to bind toxic substances and mediate transport and movement between and within organisms in Viruses. Fully disordered proteins behave similarly, but are enriched for glycine residues and extracellular structures. © 2016 The Protein Society.

A Factor Graph Approach to Automated GO Annotation

PubMed Central

Spetale, Flavio E.; Tapia, Elizabeth; Krsticevic, Flavia; Roda, Fernando; Bulacio, Pilar

2016-01-01

As volume of genomic data grows, computational methods become essential for providing a first glimpse onto gene annotations. Automated Gene Ontology (GO) annotation methods based on hierarchical ensemble classification techniques are particularly interesting when interpretability of annotation results is a main concern. In these methods, raw GO-term predictions computed by base binary classifiers are leveraged by checking the consistency of predefined GO relationships. Both formal leveraging strategies, with main focus on annotation precision, and heuristic alternatives, with main focus on scalability issues, have been described in literature. In this contribution, a factor graph approach to the hierarchical ensemble formulation of the automated GO annotation problem is presented. In this formal framework, a core factor graph is first built based on the GO structure and then enriched to take into account the noisy nature of GO-term predictions. Hence, starting from raw GO-term predictions, an iterative message passing algorithm between nodes of the factor graph is used to compute marginal probabilities of target GO-terms. Evaluations on Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster protein sequences from the GO Molecular Function domain showed significant improvements over competing approaches, even when protein sequences were naively characterized by their physicochemical and secondary structure properties or when loose noisy annotation datasets were considered. Based on these promising results and using Arabidopsis thaliana annotation data, we extend our approach to the identification of most promising molecular function annotations for a set of proteins of unknown function in Solanum lycopersicum. PMID:26771463

A Factor Graph Approach to Automated GO Annotation.

PubMed

Spetale, Flavio E; Tapia, Elizabeth; Krsticevic, Flavia; Roda, Fernando; Bulacio, Pilar

2016-01-01

As volume of genomic data grows, computational methods become essential for providing a first glimpse onto gene annotations. Automated Gene Ontology (GO) annotation methods based on hierarchical ensemble classification techniques are particularly interesting when interpretability of annotation results is a main concern. In these methods, raw GO-term predictions computed by base binary classifiers are leveraged by checking the consistency of predefined GO relationships. Both formal leveraging strategies, with main focus on annotation precision, and heuristic alternatives, with main focus on scalability issues, have been described in literature. In this contribution, a factor graph approach to the hierarchical ensemble formulation of the automated GO annotation problem is presented. In this formal framework, a core factor graph is first built based on the GO structure and then enriched to take into account the noisy nature of GO-term predictions. Hence, starting from raw GO-term predictions, an iterative message passing algorithm between nodes of the factor graph is used to compute marginal probabilities of target GO-terms. Evaluations on Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster protein sequences from the GO Molecular Function domain showed significant improvements over competing approaches, even when protein sequences were naively characterized by their physicochemical and secondary structure properties or when loose noisy annotation datasets were considered. Based on these promising results and using Arabidopsis thaliana annotation data, we extend our approach to the identification of most promising molecular function annotations for a set of proteins of unknown function in Solanum lycopersicum.

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.

Clusters of isoleucine, leucine, and valine side chains define cores of stability in high-energy states of globular proteins: Sequence determinants of structure and stability.

PubMed

Kathuria, Sagar V; Chan, Yvonne H; Nobrega, R Paul; Özen, Ayşegül; Matthews, C Robert

2016-03-01

Measurements of protection against exchange of main chain amide hydrogens (NH) with solvent hydrogens in globular proteins have provided remarkable insights into the structures of rare high-energy states that populate their folding free-energy surfaces. Lacking, however, has been a unifying theory that rationalizes these high-energy states in terms of the structures and sequences of their resident proteins. The Branched Aliphatic Side Chain (BASiC) hypothesis has been developed to explain the observed patterns of protection in a pair of TIM barrel proteins. This hypothesis supposes that the side chains of isoleucine, leucine, and valine (ILV) residues often form large hydrophobic clusters that very effectively impede the penetration of water to their underlying hydrogen bond networks and, thereby, enhance the protection against solvent exchange. The linkage between the secondary and tertiary structures enables these ILV clusters to serve as cores of stability in high-energy partially folded states. Statistically significant correlations between the locations of large ILV clusters in native conformations and strong protection against exchange for a variety of motifs reported in the literature support the generality of the BASiC hypothesis. The results also illustrate the necessity to elaborate this simple hypothesis to account for the roles of adjacent hydrocarbon moieties in defining stability cores of partially folded states along folding reaction coordinates. © 2015 The Protein Society.

Advances in RNA Structure Determination | Center for Cancer Research

Cancer.gov

The recent years have witnessed a revolution in the field of RNA structure and function. Until recently the main contribution of RNA in cellular and disease functions was considered to be a role defined by the central dogma, namely DNA codes for mRNAs, which in turn encode for proteins, a notion facilitated by non-coding ribosomal RNA and tRNA. It was also assumed at the time

Mechanism of Nucleic Acid Chaperone Function of Retroviral Nuceleocapsid (NC) Proteins

NASA Astrophysics Data System (ADS)

Rouzina, Ioulia; Vo, My-Nuong; Stewart, Kristen; Musier-Forsyth, Karin; Cruceanu, Margareta; Williams, Mark

2006-03-01

Recent studies have highlighted two main activities of HIV-1 NC protein contributing to its function as a universal nucleic acid chaperone. Firstly, it is the ability of NC to weakly destabilize all nucleic acid,(NA), secondary structures, thus resolving the kinetic traps for NA refolding, while leaving the annealed state stable. Secondly, it is the ability of NC to aggregate NA, facilitating the nucleation step of bi-molecular annealing by increasing the local NA concentration. In this work we use single molecule DNA stretching and gel-based annealing assays to characterize these two chaperone activities of NC by using various HIV-1 NC mutants and several other retroviral NC proteins. Our results suggest that two NC functions are associated with its zinc fingers and cationic residues, respectively. NC proteins from other retroviruses have similar activities, although expressed to a different degree. Thus, NA aggregating ability improves, and NA duplex destabilizing activity decreases in the sequence: MLV NC, HIV NC, RSV NC. In contrast, HTLV NC protein works very differently from other NC proteins, and similarly to typical single stranded NA binding proteins. These features of retroviral NCs co-evolved with the structure of their genomes.

Evolution of crystallins for a role in the vertebrate eye lens.

PubMed

Slingsby, Christine; Wistow, Graeme J; Clark, Alice R

2013-04-01

The camera eye lens of vertebrates is a classic example of the re-engineering of existing protein components to fashion a new device. The bulk of the lens is formed from proteins belonging to two superfamilies, the α-crystallins and the βγ-crystallins. Tracing their ancestry may throw light on the origin of the optics of the lens. The α-crystallins belong to the ubiquitous small heat shock proteins family that plays a protective role in cellular homeostasis. They form enormous polydisperse oligomers that challenge modern biophysical methods to uncover the molecular basis of their assembly structure and chaperone-like protein binding function. It is argued that a molecular phenotype of a dynamic assembly suits a chaperone function as well as a structural role in the eye lens where the constraint of preventing protein condensation is paramount. The main cellular partners of α-crystallins, the β- and γ-crystallins, have largely been lost from the animal kingdom but the superfamily is hugely expanded in the vertebrate eye lens. Their structures show how a simple Greek key motif can evolve rapidly to form a complex array of monomers and oligomers. Apart from remaining transparent, a major role of the partnership of α-crystallins with β- and γ-crystallins in the lens is to form a refractive index gradient. Here, we show some of the structural and genetic features of these two protein superfamilies that enable the rapid creation of different assembly states, to match the rapidly changing optical needs among the various vertebrates. Copyright © 2013 The Protein Society.

Influence of metformin on mitochondrial subproteome in the brain of apoE knockout mice.

PubMed

Suski, Maciej; Olszanecki, Rafał; Chmura, Łukasz; Stachowicz, Aneta; Madej, Józef; Okoń, Krzysztof; Adamek, Dariusz; Korbut, Ryszard

2016-02-05

Neurodegenerative diseases are the set of progressive, age-related brain disorders, characterized by an excessive accumulation of mutant proteins in the certain regions of the brain. Such changes, collectively identified as causal factors of neurodegeneration, all impact mitochondria, imminently leading to their dysfunction. These observations predestine mitochondria as an attractive drug target for counteracting degenerative brain damage. The aim of this study was to use a differential proteomic approach to comprehensively assess the changes in mitochondrial protein expression in the brain of apoE-knockout mice (apoE(-/-)) and to investigate the influence of prolonged treatment with metformin - an indirect activator of AMP-activated protein kinase (AMPK) on the brain mitoproteome in apoE(-/-) mice. The quantitative assessment of the brain mitoproteome in apoE(-/-) revealed the changes in 10 proteins expression as compared to healthy C57BL/6J mice and 25 proteins expression in metformin-treated apoE(-/-) mice. Identified proteins mainly included apoptosis regulators, metabolic enzymes and structural proteins. In summary, our study provided proteomic characteristics suggesting the decrease of antioxidant defense and structural disturbances in the brain mitochondria of apoE(-/-) mice as compared to healthy controls. In this setting, the use of metformin changed the expression of several proteins primarily involved in metabolic processes, the regulation of apoptosis and the structural maintenance of mitochondria, what could potentially restore their native functionalities. Copyright © 2015 Elsevier B.V. All rights reserved.

Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation, as a target for anticancer drugs.

PubMed

Jabłońska-Trypuć, Agata; Matejczyk, Marzena; Rosochacki, Stanisław

2016-01-01

The main group of enzymes responsible for the collagen and other protein degradation in extracellular matrix (ECM) are matrix metalloproteinases (MMPs). Collagen is the main structural component of connective tissue and its degradation is a very important process in the development, morphogenesis, tissue remodeling, and repair. Typical structure of MMPs consists of several distinct domains. MMP family can be divided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and other non-classified MMPs. MMPs and their inhibitors have multiple biological functions in all stages of cancer development: from initiation to outgrowth of clinically relevant metastases and likewise in apoptosis and angiogenesis. MMPs and their inhibitors are extensively examined as potential anticancer drugs. MMP inhibitors can be divided into two main groups: synthetic and natural inhibitors. Selected synthetic inhibitors are in clinical trials on humans, e.g. synthetic peptides, non-peptidic molecules, chemically modified tetracyclines, and bisphosphonates. Natural MMP inhibitors are mainly isoflavonoids and shark cartilage.

Structural Disorder Provides Increased Adaptability for Vesicle Trafficking Pathways

PubMed Central

Tompa, Peter

2013-01-01

Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (∼23%) than the other two, COPI (∼9%) and COPII (∼8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and suggest major roles for this structural property in shaping the differences of evolutionary adaptability in the three routes. PMID:23874186

Ribosome-Inactivating and Related Proteins

PubMed Central

Schrot, Joachim; Weng, Alexander; Melzig, Matthias F.

2015-01-01

Ribosome-inactivating proteins (RIPs) are toxins that act as N-glycosidases (EC 3.2.2.22). They are mainly produced by plants and classified as type 1 RIPs and type 2 RIPs. There are also RIPs and RIP related proteins that cannot be grouped into the classical type 1 and type 2 RIPs because of their different sizes, structures or functions. In addition, there is still not a uniform nomenclature or classification existing for RIPs. In this review, we give the current status of all known plant RIPs and we make a suggestion about how to unify those RIPs and RIP related proteins that cannot be classified as type 1 or type 2 RIPs. PMID:26008228

The EBI search engine: EBI search as a service—making biological data accessible for all

PubMed Central

Park, Young M.; Squizzato, Silvano; Buso, Nicola; Gur, Tamer

2017-01-01

Abstract We present an update of the EBI Search engine, an easy-to-use fast text search and indexing system with powerful data navigation and retrieval capabilities. The interconnectivity that exists between data resources at EMBL–EBI provides easy, quick and precise navigation and a better understanding of the relationship between different data types that include nucleotide and protein sequences, genes, gene products, proteins, protein domains, protein families, enzymes and macromolecular structures, as well as the life science literature. EBI Search provides a powerful RESTful API that enables its integration into third-party portals, thus providing ‘Search as a Service’ capabilities, which are the main topic of this article. PMID:28472374

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

Jiang, Lin; Liu, Cong; Leibly, David

Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer’s, Parkinson’s, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer’s disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind tomore » Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers.« less

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.

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

PubMed

Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

2012-09-25

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

Regulation, Signaling, and Physiological Functions of G-Proteins.

PubMed

Syrovatkina, Viktoriya; Alegre, Kamela O; Dey, Raja; Huang, Xin-Yun

2016-09-25

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G-proteins) mainly relay the information from G-protein-coupled receptors (GPCRs) on the plasma membrane to the inside of cells to regulate various biochemical functions. Depending on the targeted cell types, tissues, and organs, these signals modulate diverse physiological functions. The basic schemes of heterotrimeric G-proteins have been outlined. In this review, we briefly summarize what is known about the regulation, signaling, and physiological functions of G-proteins. We then focus on a few less explored areas such as the regulation of G-proteins by non-GPCRs and the physiological functions of G-proteins that cannot be easily explained by the known G-protein signaling pathways. There are new signaling pathways and physiological functions for G-proteins to be discovered and further interrogated. With the advancements in structural and computational biological techniques, we are closer to having a better understanding of how G-proteins are regulated and of the specificity of G-protein interactions with their regulators. Copyright © 2016 Elsevier Ltd. All rights reserved.

Density functional theory study of the conformational space of an infinitely long polypeptide chain

NASA Astrophysics Data System (ADS)

Ireta, Joel; Scheffler, Matthias

2009-08-01

The backbone conformational space of infinitely long polyalanine is investigated with density-functional theory and mapping the potential energy surface in terms of (L, θ) cylindrical coordinates. A comparison of the obtained (L, θ) Ramachandran-like plot with results from an extended set of protein structures shows excellent conformity, with the exception of the polyproline II region. It is demonstrated the usefulness of infinitely long polypeptide models for investigating the influence of hydrogen bonding and its cooperative effect on the backbone conformations. The results imply that hydrogen bonding together with long-range electrostatics is the main actuator for most of the structures assumed by protein residues.

Proteomic plasma profile of psoriatic patients.

PubMed

Gęgotek, Agnieszka; Domingues, Pedro; Wroński, Adam; Wójcik, Piotr; Skrzydlewska, Elżbieta

2018-06-05

Psoriasis is a chronic, immune-mediated inflammatory skin disease with severe consequences for the whole organism. The lack of complete knowledge of the main factors predisposing an individual to the appearance of psoriatic lesions, has recently led to the search for modifications in biochemical pathways participating in the development of this disease. We therefore aimed to investigate changes in the plasma proteomic profile of patients with psoriasis. A proteomics approach was used to analyze the expression of proteins in plasma from psoriatic patients and healthy controls (sex- and age-matched individuals). The analysis was performed using gel electrophoresis, followed by nanoflow LC-MS/MS using a Q-Exactive OrbiTrap mass spectrometer. Proteomic data indicated a significant decrease in the level of proteins involved in lipid metabolism, such as apolipoprotein M, and proteins involved in the management of vitamin D levels in psoriatic patients' plasma. These changes were accompanied by the expression of proteins involved in immune response and signal transduction. This was particularly evident by the level of transcriptional factors, including AT motif binding factor 1, which regulates excessive cellular proliferation and differentiation. It was also suggested that psoriasis development was associated with increased expression of proteins directly involved in signaling molecule secretion [biotinidase and BAI1-associated protein 3]. In addition, the lipid peroxidation product - 4-hydroxynonenal (4-HNE) generates higher level of adducts with proteins in the plasma of psoriatic patients. Moreover, plasma proteins from healthy subjects creating with 4-HNE adducts were mainly characterized as structural, while in the plasma of psoriatic patients, increased levels of 4-HNE-protein adducts with catalytic activity were observed. The results presented herein confirm the current knowledge about the profile of proteins responsible for the immune response and management of vitamin D in the plasma of psoriatic patients. However, several new proteins were also identified, which are involved in signal transduction and lipid metabolism as well as catalytic activity. The expression or structure of these proteins was shown to change through the course of the development of psoriasis. This knowledge may help contribute to the design of more specific pharmacotherapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Redox Thermodynamics of High-Spin and Low-Spin Forms of Chlorite Dismutases with Diverse Subunit and Oligomeric Structures

PubMed Central

2012-01-01

Chlorite dismutases (Clds) are heme b-containing oxidoreductases that convert chlorite to chloride and dioxygen. In this work, the thermodynamics of the one-electron reduction of the ferric high-spin forms and of the six-coordinate low-spin cyanide adducts of the enzymes from Nitrobacter winogradskyi (NwCld) and Candidatus “Nitrospira defluvii” (NdCld) were determined through spectroelectrochemical experiments. These proteins belong to two phylogenetically separated lineages that differ in subunit (21.5 and 26 kDa, respectively) and oligomeric (dimeric and pentameric, respectively) structure but exhibit similar chlorite degradation activity. The E°′ values for free and cyanide-bound proteins were determined to be −119 and −397 mV for NwCld and −113 and −404 mV for NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical experiments revealed that the oxidized state of both proteins is enthalpically stabilized. Molecular dynamics simulations suggest that changes in the protein structure are negligible, whereas solvent reorganization is mainly responsible for the increase in entropy during the redox reaction. Obtained data are discussed with respect to the known structures of the two Clds and the proposed reaction mechanism. PMID:23126649

Structural organization of G-protein-coupled receptors

NASA Astrophysics Data System (ADS)

Lomize, Andrei L.; Pogozheva, Irina D.; Mosberg, Henry I.

1999-07-01

Atomic-resolution structures of the transmembrane 7-α-helical domains of 26 G-protein-coupled receptors (GPCRs) (including opsins, cationic amine, melatonin, purine, chemokine, opioid, and glycoprotein hormone receptors and two related proteins, retinochrome and Duffy erythrocyte antigen) were calculated by distance geometry using interhelical hydrogen bonds formed by various proteins from the family and collectively applied as distance constraints, as described previously [Pogozheva et al., Biophys. J., 70 (1997) 1963]. The main structural features of the calculated GPCR models are described and illustrated by examples. Some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane α-bundle: the formation of extensive networks of interhelical H-bonds and sulfur-aromatic clusters that are spatially organized as 'polarity gradients' the close packing of side-chains throughout the transmembrane domain; and the formation of interhelical disulfide bonds in some receptors and a plausible Zn2+ binding center in retinochrome. Other features of the models are related to biological function and evolution of GPCRs: the formation of a common 'minicore' of 43 evolutionarily conserved residues; a multitude of correlated replacements throughout the transmembrane domain; an Na+-binding site in some receptors, and excellent complementarity of receptor binding pockets to many structurally dissimilar, conformationally constrained ligands, such as retinal, cyclic opioid peptides, and cationic amine ligands. The calculated models are in good agreement with numerous experimental data.

The structure and protein binding of amyloid-specific dye reagents.

PubMed

Stopa, Barbara; Piekarska, Barbara; Konieczny, Leszek; Rybarska, Janina; Spólnik, Paweł; Zemanek, Grzegorz; Roterman, Irena; Król, Marcin

2003-01-01

The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.

Changes in structural and antigenic properties of proteins by radiation

NASA Astrophysics Data System (ADS)

Kume, Tamikazu; Matsuda, Tsukasa

1995-08-01

Radiation effect on structural and antigenic properties of proteins (0.2% in 0.01 M phosphate buffer, pH 7.4) were investigated using ovalbumin (OVA) and bovine serum albumin (BSA). Aggregation of OVA and BSA was induced by radiation and the molecular mass increased significantly in N 2. Significant changes in surface hydrophobicity and [ θ] 222 nm of CD were also observed by radiation showing the destruction of secondary structure of proteins. Antigenicity of irradiated OVA measured by the method of immunodiffusion was decreased by radiation, and the reactivity to anti-OVA antibody was almost diminished at 8 kGy in N 2 and 4 kGy in O 2, respectively. The reactivity of BSA was diminished at 4 kGy both in N 2 and O 2. Changes in hydrophobicity of OVA did not correspond to the decrease in antigenicity, whereas the changes in [ θ] 222 nm relatively well corresponded to the antigenicity. The SDS-PAGE and immunoblotting analysis showed that radiation at higher doses induced the production of protein aggregates and degraded fragments with reactivity to the specific antibodies. These results suggest that the main part of conformation-dependent antigenic structure (conformational epitope) is easily lost by radiation, but some antigenicity, which is mostly due to the amino acid sequence-dependent antigenic structures (sequential epitopes), remains even at higher dose.

Biological and functional relevance of CASP predictions

PubMed Central

Liu, Tianyun; Ish‐Shalom, Shirbi; Torng, Wen; Lafita, Aleix; Bock, Christian; Mort, Matthew; Cooper, David N; Bliven, Spencer; Capitani, Guido; Mooney, Sean D.

2017-01-01

Abstract Our goal is to answer the question: compared with experimental structures, how useful are predicted models for functional annotation? We assessed the functional utility of predicted models by comparing the performances of a suite of methods for functional characterization on the predictions and the experimental structures. We identified 28 sites in 25 protein targets to perform functional assessment. These 28 sites included nine sites with known ligand binding (holo‐sites), nine sites that are expected or suggested by experimental authors for small molecule binding (apo‐sites), and Ten sites containing important motifs, loops, or key residues with important disease‐associated mutations. We evaluated the utility of the predictions by comparing their microenvironments to the experimental structures. Overall structural quality correlates with functional utility. However, the best‐ranked predictions (global) may not have the best functional quality (local). Our assessment provides an ability to discriminate between predictions with high structural quality. When assessing ligand‐binding sites, most prediction methods have higher performance on apo‐sites than holo‐sites. Some servers show consistently high performance for certain types of functional sites. Finally, many functional sites are associated with protein‐protein interaction. We also analyzed biologically relevant features from the protein assemblies of two targets where the active site spanned the protein‐protein interface. For the assembly targets, we find that the features in the models are mainly determined by the choice of template. PMID:28975675

The structural damages of lens crystallins induced by peroxynitrite and methylglyoxal, two causative players in diabetic complications and preventive role of lens antioxidant components.

PubMed

Moghadam, Sogand Sasan; Oryan, Ahmad; Kurganov, Boris I; Tamaddon, Ali-Mohammad; Alavianehr, Mohammad Mehdi; Moosavi-Movahedi, Ali Akbar; Yousefi, Reza

2017-10-01

Peroxynitrite (PON) and methylglyoxal (MGO), two diabetes-associated compounds, are believed to be important causative players in development of diabetic cataracts. In the current study, different spectroscopic methods, gel electrophoresis, lens culture and microscopic assessments were applied to examine the impact of individual, subsequent or simultaneous modification of lens crystallins with MGO and PON on their structure, oligomerization and aggregation. The protein modifications were confirmed with detection of the significantly increased quantity of carbonyl groups and decreased levels of sulfhydryl, tyrosine and tryptophan. Also, lens proteins modification with these chemical agents was accompanied with important structural alteration, oligomerization, disulfide/chromophore mediated protein crosslinking and important proteolytic instability. All these structural damages were more pronounced when the lens proteins were modified in the presence of both mentioned chemical agents, either in sequential or simultaneous manner. Ascorbic acid and glutathione, as the main components of lens antioxidant defense mechanism, were also capable to markedly prevent the damaging effects of PON and MGO on lens crystallins, as indicated by gel electrophoresis. The results of this study may highlight the importance of lens antioxidant defense system in protection of crystallins against the structural insults induced by PON and MGO during chronic hyperglycemia in the diabetic patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Incorporation of organometallic Ru complexes into apo-ferritin cage.

PubMed

Takezawa, Yusuke; Böckmann, Philipp; Sugi, Naoki; Wang, Ziyue; Abe, Satoshi; Murakami, Tatsuya; Hikage, Tatsuo; Erker, Gerhard; Watanabe, Yoshihito; Kitagawa, Susumu; Ueno, Takafumi

2011-03-14

Spherical protein cages such as an iron storage protein, ferritin, have great potential as nanometer-scale capsules to assemble and store metal ions and complexes. We report herein the synthesis of a composite of an apo-ferritin cage and Ru(p-cymene) complexes. Ru complexes were efficiently incorporated into the ferritin cavity without degradation of its cage structure. X-Ray crystallography revealed that the Ru complexes were immobilized on the interior surface of the cage mainly by the coordination of histidine residues.

Impact of gluten-friendly™ technology on wheat kernel endosperm and gluten protein structure in seeds by light and electron microscopy.

PubMed

Landriscina, L; D'Agnello, P; Bevilacqua, A; Corbo, M R; Sinigaglia, M; Lamacchia, C

2017-04-15

The main aim of this paper was to assess the impact of Gluten-Friendly™ (GF) technology (Italian priority patent n° 102015000084813 filed on 17th December 2015) on wheat kernel endosperm morphology and gluten protein structure, using SEM, light and immunofluorescent microscopy. Microscopy was combined with immunodetection with specific antibodies for gliadins, γ-gliadins, LMW subunits and antigenic epitopes to gain a better understanding of the technology at a molecular level. The results showed significant changes to gluten proteins after GF treatment; cross-reactivity towards the antibodies recognizing almost the entire range of gluten proteins as well as the antigenic epitopes through the sequences QQSF, QQSY, PEQPFPQGC and QQPFP was significantly reduced. The present study confirms the results from our previous work and shows, for the first time, the mechanism by which a chemical-physical treatment abolishes the antigenic capacity of gluten. Copyright © 2016 Elsevier Ltd. All rights reserved.

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.

Evidences For Charge Transfer-Induced Conformational Changes In Carbon Nanostructure-Protein Corona

PubMed Central

Podila, R.; Vedantam, P.; Ke, P. C.; Brown, J. M.; Rao, A. M.

2012-01-01

The binding of proteins to a nanostructure often alters protein secondary and tertiary structures. However, the main physical mechanisms that elicit protein conformational changes in the presence of the nanostructure have not yet been fully established. Here we performed a comprehensive spectroscopic study to probe the interactions between bovine serum albumin (BSA) and carbon-based nanostructures of graphene and single-walled carbon nanotubes (SWNTs). Our results showed that the BSA “corona” acted as a weak acceptor to facilitate charge transfer from the carbon nanostructures. Notably, we observed that charge transfer occurred only in the case of SWNTs but not in graphene, resulting from the sharp and discrete electronic density of states of the former. Furthermore, the relaxation of external α–helices in BSA secondary structure increased concomitantly with the charge transfer. These results may help guide controlled nanostructure-biomolecular interactions and prove beneficial for developing novel drug delivery systems, biomedical devices and engineering of safe nanomaterials. PMID:23243478

Extraction of extracellular polymeric substances from aerobic granule with compact interior structure.

PubMed

Adav, Sunil S; Lee, Duu-Jong

2008-06-15

Extracellular polymeric substances (EPS) were extracted from aerobic granules of compact interior structure using seven extraction methods. Ultrasound followed by the chemical reagents formamide and NaOH outperformed other methods in extracting EPS from aerobic granules of compact interior. The collected EPS revealed no contamination by intracellular substances and consisted mainly of proteins, polysaccharides, humic substances and lipids. The quantity of extracted proteins exhibited a weak correlation with quantity of extracted carbohydrates but no correlation with quantity of extracted humic substances. The total polysaccharides/total proteins (PN/PS) ratios for sludge flocs were approximately 0.9 regardless of extraction method. Protein content was significantly enriched in the granules, producing a PN/PS ratio of 3.4-6.2. This experimental result correlated with observations using excitation-emission matrix (EEM) and confocal laser scanning microscope technique. However, detailed study disproved the use of EEM results as a quantitative index of extracted EPS from sludge flocs or from granules.

Therapeutic intervention at cellular quality control systems in Alzheimer's and Parkinson's diseases.

PubMed

Arduino, Daniela M; Esteves, A Raquel; Silva, Diana F F; Martins-Branco, Diogo; Santos, Daniel; Pimentel, Diana F Gomes; Cardoso, Sandra M

2011-01-01

Cellular homeostasis relies on quality control systems so that damaged biologic structures are either repaired or degraded and entirely replaced by newly formed proteins or even organelles. The clearance of dysfunctional cellular structures in long-lived postmitotic cells, like neurons, is essential to eliminate, per example, defective mitochondria, lipofuscin-loaded lysosomes and oxidized proteins. Short-lived proteins are degraded mainly by proteases and proteasomes whether most long-lived proteins and all organelles are digested by autophagy in the lysosomes. Recently, it an interplay was established between the ubiquitin-proteasome system and macroautophagy, so that both degradative mechanisms compensate for each other. In this article we describe each of these clearance systems and their contribution to neuronal quality control. We will highlight some of the findings that provide evidence for the dysfunction of these systems in Alzheimer's and Parkinson's diseases. Ultimately, we provide an outline on potential therapeutic interventions based on the modulation of cellular degradative systems.

Aqueous ionic liquids and their effects on protein structures: an overview on recent theoretical and experimental results

NASA Astrophysics Data System (ADS)

Smiatek, Jens

2017-06-01

Ionic liquids (ILs) are used in a variety of technological and biological applications. Recent experimental and simulation results reveal the influence of aqueous ionic liquids on the stability of protein and enzyme structures. Depending on different parameters like the concentration and the ion composition, one can observe distinct stabilization or denaturation mechanisms for various ILs. In this review, we summarize the main findings and discuss the implications with regard to molecular theories of solutions and specific ion effects. A preferential binding model is introduced in order to discuss protein-IL effects from a statistical mechanics perspective. The value of the preferential binding coefficient determines the strength of the ion influence and indicates a shift of the chemical equilibrium either to the native or the denatured state of the protein. We highlight the role of water in order to explain the self-association behavior of the IL species and discuss recent experimental and simulation results in the light of the observed binding effects.

Large‐scale analysis of intrinsic disorder flavors and associated functions in the protein sequence universe

PubMed Central

Necci, Marco; Piovesan, Damiano

2016-01-01

Abstract Intrinsic disorder (ID) in proteins has been extensively described for the last decade; a large‐scale classification of ID in proteins is mostly missing. Here, we provide an extensive analysis of ID in the protein universe on the UniProt database derived from sequence‐based predictions in MobiDB. Almost half the sequences contain an ID region of at least five residues. About 9% of proteins have a long ID region of over 20 residues which are more abundant in Eukaryotic organisms and most frequently cover less than 20% of the sequence. A small subset of about 67,000 (out of over 80 million) proteins is fully disordered and mostly found in Viruses. Most proteins have only one ID, with short ID evenly distributed along the sequence and long ID overrepresented in the center. The charged residue composition of Das and Pappu was used to classify ID proteins by structural propensities and corresponding functional enrichment. Swollen Coils seem to be used mainly as structural components and in biosynthesis in both Prokaryotes and Eukaryotes. In Bacteria, they are confined in the nucleoid and in Viruses provide DNA binding function. Coils & Hairpins seem to be specialized in ribosome binding and methylation activities. Globules & Tadpoles bind antigens in Eukaryotes but are involved in killing other organisms and cytolysis in Bacteria. The Undefined class is used by Bacteria to bind toxic substances and mediate transport and movement between and within organisms in Viruses. Fully disordered proteins behave similarly, but are enriched for glycine residues and extracellular structures. PMID:27636733

Destabilization of psychrotrophic RNase HI in a localized fashion as revealed by mutational and X-ray crystallographic analyses.

PubMed

Rohman, Muhammad S; Tadokoro, Takashi; Angkawidjaja, Clement; Abe, Yumi; Matsumura, Hiroyoshi; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

2009-01-01

The Arg97 --> Gly and Asp136 --> His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7 degrees C, respectively, in T(m), and 3.5 and 6.1 kJ x mol(-1), respectively, in DeltaG(H2O). These mutations also stabilized the So-RNase HI derivative (4x-RNase HI) with quadruple thermostabilizing mutations in an additive manner. As a result, the resultant sextuple mutant protein (6x-RNase HI) was more stable than the wild-type protein by 28.8 degrees C in T(m) and 27.0 kJ x mol(-1) in DeltaG(H2O). To analyse the effects of the mutations on the protein structure, the crystal structure of the 6x-RNase HI protein was determined at 2.5 A resolution. The main chain fold and interactions of the side-chains of the 6x-RNase HI protein were basically identical to those of the wild-type protein, except for the mutation sites. These results indicate that all six mutations independently affect the protein structure, and are consistent with the fact that the thermostabilizing effects of the mutations are roughly additive. The introduction of favourable interactions and the elimination of unfavourable interactions by the mutations contribute to the stabilization of the 6x-RNase HI protein. We propose that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability.

Insights into the Antimicrobial Mechanism of Action of Human RNase6: Structural Determinants for Bacterial Cell Agglutination and Membrane Permeation

PubMed Central

Pulido, David; Arranz-Trullén, Javier; Prats-Ejarque, Guillem; Velázquez, Diego; Torrent, Marc; Moussaoui, Mohammed; Boix, Ester

2016-01-01

Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1–45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance. PMID:27089320

EsxB, a secreted protein from Bacillus anthracis forms two distinct helical bundles

DOE PAGES

Fan, Yao; Tan, Kemin; Chhor, Gekleng; ...

2015-07-03

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for twomore » alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.« less

Interaction of bovine serum albumin with N-acyl amino acid based anionic surfactants: Effect of head-group hydrophobicity.

PubMed

Ghosh, Subhajit; Dey, Joykrishna

2015-11-15

The function of a protein depends upon its structure and surfactant molecules are known to alter protein structure. For this reason protein-surfactant interaction is important in biological, pharmaceutical, and cosmetic industries. In the present work, interactions of a series of anionic surfactants having the same hydrocarbon chain length, but different amino acid head group, such as l-alanine, l-valine, l-leucine, and l-phenylalanine with the transport protein, bovine serum albumin (BSA), were studied at low surfactant concentrations using fluorescence and circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The results of fluorescence measurements suggest that the surfactant molecules bind simultaneously to the drug binding site I and II of the protein subdomain IIA and IIIA, respectively. The fluorescence as well as CD spectra suggest that the conformation of BSA goes to a more structured state upon surfactant binding at low concentrations. The binding constants of the surfactants were determined by the use of fluorescence as well as ITC measurements and were compared with that of the corresponding glycine-derived surfactant. The binding constant values clearly indicate a significant head-group effect on the BSA-surfactant interaction and the interaction is mainly hydrophobic in nature. Copyright © 2015 Elsevier Inc. All rights reserved.

STRUM: structure-based prediction of protein stability changes upon single-point mutation.

PubMed

Quan, Lijun; Lv, Qiang; Zhang, Yang

2016-10-01

Mutations in human genome are mainly through single nucleotide polymorphism, some of which can affect stability and function of proteins, causing human diseases. Several methods have been proposed to predict the effect of mutations on protein stability; but most require features from experimental structure. Given the fast progress in protein structure prediction, this work explores the possibility to improve the mutation-induced stability change prediction using low-resolution structure modeling. We developed a new method (STRUM) for predicting stability change caused by single-point mutations. Starting from wild-type sequences, 3D models are constructed by the iterative threading assembly refinement (I-TASSER) simulations, where physics- and knowledge-based energy functions are derived on the I-TASSER models and used to train STRUM models through gradient boosting regression. STRUM was assessed by 5-fold cross validation on 3421 experimentally determined mutations from 150 proteins. The Pearson correlation coefficient (PCC) between predicted and measured changes of Gibbs free-energy gap, ΔΔG, upon mutation reaches 0.79 with a root-mean-square error 1.2 kcal/mol in the mutation-based cross-validations. The PCC reduces if separating training and test mutations from non-homologous proteins, which reflects inherent correlations in the current mutation sample. Nevertheless, the results significantly outperform other state-of-the-art methods, including those built on experimental protein structures. Detailed analyses show that the most sensitive features in STRUM are the physics-based energy terms on I-TASSER models and the conservation scores from multiple-threading template alignments. However, the ΔΔG prediction accuracy has only a marginal dependence on the accuracy of protein structure models as long as the global fold is correct. These data demonstrate the feasibility to use low-resolution structure modeling for high-accuracy stability change prediction upon point mutations. http://zhanglab.ccmb.med.umich.edu/STRUM/ CONTACT: qiang@suda.edu.cn and zhng@umich.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

STRUM: structure-based prediction of protein stability changes upon single-point mutation

PubMed Central

Quan, Lijun; Lv, Qiang; Zhang, Yang

2016-01-01

Motivation: Mutations in human genome are mainly through single nucleotide polymorphism, some of which can affect stability and function of proteins, causing human diseases. Several methods have been proposed to predict the effect of mutations on protein stability; but most require features from experimental structure. Given the fast progress in protein structure prediction, this work explores the possibility to improve the mutation-induced stability change prediction using low-resolution structure modeling. Results: We developed a new method (STRUM) for predicting stability change caused by single-point mutations. Starting from wild-type sequences, 3D models are constructed by the iterative threading assembly refinement (I-TASSER) simulations, where physics- and knowledge-based energy functions are derived on the I-TASSER models and used to train STRUM models through gradient boosting regression. STRUM was assessed by 5-fold cross validation on 3421 experimentally determined mutations from 150 proteins. The Pearson correlation coefficient (PCC) between predicted and measured changes of Gibbs free-energy gap, ΔΔG, upon mutation reaches 0.79 with a root-mean-square error 1.2 kcal/mol in the mutation-based cross-validations. The PCC reduces if separating training and test mutations from non-homologous proteins, which reflects inherent correlations in the current mutation sample. Nevertheless, the results significantly outperform other state-of-the-art methods, including those built on experimental protein structures. Detailed analyses show that the most sensitive features in STRUM are the physics-based energy terms on I-TASSER models and the conservation scores from multiple-threading template alignments. However, the ΔΔG prediction accuracy has only a marginal dependence on the accuracy of protein structure models as long as the global fold is correct. These data demonstrate the feasibility to use low-resolution structure modeling for high-accuracy stability change prediction upon point mutations. Availability and Implementation: http://zhanglab.ccmb.med.umich.edu/STRUM/ Contact: qiang@suda.edu.cn and zhng@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27318206

A comparative analysis on the physicochemical properties of tick-borne encephalitis virus envelope protein residues that affect its antigenic properties.

PubMed

Bukin, Yu S; Dzhioev, Yu P; Tkachev, S E; Kozlova, I V; Paramonov, A I; Ruzek, D; Qu, Z; Zlobin, V I

2017-06-15

This work is dedicated to the study of the variability of the main antigenic envelope protein E among different strains of tick-borne encephalitis virus at the level of physical and chemical properties of the amino acid residues. E protein variants were extracted from then NCBI database. Four amino acid residues properties in the polypeptide sequences were investigated: the average volume of the amino acid residue in the protein tertiary structure, the number of amino acid residue hydrogen bond donors, the charge of amino acid residue lateral radical and the dipole moment of the amino acid residue. These physico-chemical properties are involved in antigen-antibody interactions. As a result, 103 different variants of the antigenic determinants of the tick-borne encephalitis virus E protein were found, significantly different by physical and chemical properties of the amino acid residues in their structure. This means that some strains among the natural variants of tick-borne encephalitis virus can potentially escape the immune response induced by the standard vaccine. Copyright © 2017 Elsevier B.V. All rights reserved.

Autogrid-based clustering of kinases: selection of representative conformations for docking purposes.

PubMed

Marzaro, Giovanni; Ferrarese, Alessandro; Chilin, Adriana

2014-08-01

The selection of the most appropriate protein conformation is a crucial aspect in molecular docking experiments. In order to reduce the errors arising from the use of a single protein conformation, several authors suggest the use of several tridimensional structures for the target. However, the selection of the most appropriate protein conformations still remains a challenging goal. The protein 3D-structures selection is mainly performed based on pairwise root-mean-square-deviation (RMSD) values computation, followed by hierarchical clustering. Herein we report an alternative strategy, based on the computation of only two atom affinity map for each protein conformation, followed by multivariate analysis and hierarchical clustering. This methodology was applied on seven different kinases of pharmaceutical interest. The comparison with the classical RMSD-based strategy was based on cross-docking of co-crystallized ligands. In the case of epidermal growth factor receptor kinase, also the docking performance on 220 known ligands were evaluated, followed by 3D-QSAR studies. In all the cases, the herein proposed methodology outperformed the RMSD-based one.

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells.

PubMed

Afroze, Syeda H; Uddin, M Nasir; Cao, Xiaobo; Asea, Alexzander; Gizachew, Dawit

2011-08-01

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.

HotSpot Wizard 3.0: web server for automated design of mutations and smart libraries based on sequence input information.

PubMed

Sumbalova, Lenka; Stourac, Jan; Martinek, Tomas; Bednar, David; Damborsky, Jiri

2018-05-23

HotSpot Wizard is a web server used for the automated identification of hotspots in semi-rational protein design to give improved protein stability, catalytic activity, substrate specificity and enantioselectivity. Since there are three orders of magnitude fewer protein structures than sequences in bioinformatic databases, the major limitation to the usability of previous versions was the requirement for the protein structure to be a compulsory input for the calculation. HotSpot Wizard 3.0 now accepts the protein sequence as input data. The protein structure for the query sequence is obtained either from eight repositories of homology models or is modeled using Modeller and I-Tasser. The quality of the models is then evaluated using three quality assessment tools-WHAT_CHECK, PROCHECK and MolProbity. During follow-up analyses, the system automatically warns the users whenever they attempt to redesign poorly predicted parts of their homology models. The second main limitation of HotSpot Wizard's predictions is that it identifies suitable positions for mutagenesis, but does not provide any reliable advice on particular substitutions. A new module for the estimation of thermodynamic stabilities using the Rosetta and FoldX suites has been introduced which prevents destabilizing mutations among pre-selected variants entering experimental testing. HotSpot Wizard is freely available at http://loschmidt.chemi.muni.cz/hotspotwizard.

The reticulons: Guardians of the structure and function of the endoplasmic reticulum

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

Di Sano, Federica; Bernardoni, Paolo; Piacentini, Mauro, E-mail: mauro.piacentini@uniroma2.it

2012-07-01

The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by a specific class of curvature stabilizing proteins, the reticulons and DP1; however it is still unclear how the sheets are assembled. The ER is the cellular compartment responsible for secretory and membrane protein synthesis. The reducing conditions of ER lead to the intra/inter-chain formation of new disulphide bonds into polypeptides during protein folding assessed by enzymatic or spontaneous reactions. Moreover, ER represents the main intracellular calcium storage site and it plays an important role in calcium signalingmore » that impacts many cellular processes. Accordingly, the maintenance of ER function represents an essential condition for the cell, and ER morphology constitutes an important prerogative of it. Furthermore, it is well known that ER undergoes prominent shape transitions during events such as cell division and differentiation. Thus, maintaining the correct ER structure is an essential feature for cellular physiology. Now, it is known that proper ER-associated proteins play a fundamental role in ER tubules formation. Among these ER-shaping proteins are the reticulons (RTN), which are acquiring a relevant position. In fact, beyond the structural role of reticulons, in very recent years new and deeper functional implications of these proteins are emerging in relation to their involvement in several cellular processes.« less

Floral nectary, nectar production dynamics and chemical composition in five nocturnal Oenothera species (Onagraceae) in relation to floral visitors.

PubMed

Antoń, Sebastian; Komoń-Janczara, Elwira; Denisow, Bożena

2017-12-01

Main conclusion The floral nectars were sucrose-dominant; however, nectar protein and amino acid contents differed, indicating that composition of nitrogenous compounds may vary considerably even between closely related plant species, irrespectively of nectary structure. Numerous zoophilous plants attract their pollinators by offering floral nectar; an aqueous solution produced by specialized secretory tissues, known as floral nectaries. Although many papers on nectaries and nectar already exist, there has been a little research into the structure of nectaries and/or nectar production and composition in species belonging to the same genus. To redress this imbalance, we sought, in the present paper, to describe the floral nectary, nectar production, and nectar composition in five nocturnal Oenothera species with respect to their floral visitors. The structure of nectaries was similar for all the species investigated, and comprised the epidermis (with nectarostomata), numerous layers of nectary parenchyma, and subsecretory parenchyma. Anthesis for a single flower was short (ca. 10-12 h), and flowers lasted only one night. The release of floral nectar commenced at the bud stage (approx. 4 h before anthesis) and nectar was available to pollinators until petal closure. Nectar concentration was relatively low (ca. 27%) and the nectar was sucrose-dominant, and composed mainly of sucrose, glucose and fructose. The protein content of the nectar was also relatively low (on average, 0.31 µg ml -1 ). Nevertheless, a great variety of amino acids, including both protein and non-protein types, was detected in the nectar profile of the investigated taxa. We noted both diurnal and nocturnal generalist, opportunistic floral insect visitors.

Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein*

PubMed Central

Rahman, M. Azizur; Oomori, Tamotsu; Wörheide, Gert

2011-01-01

Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

[FOXP2 and the molecular biology of language: new evidence. II. Molecular aspects and implications for the ontogenesis and phylogeny of language].

PubMed

Benítez-Burraco, A

FOXP2 is the first gene linked to a hereditary variant of specific language impairment and seems to code for a transcriptional repressor that intervenes in the regulation of the development and the functioning of certain thalamic-cortical-striatal circuits. In the last three years, significant progress has been made in the determination of the structural and functional properties of the gene. These advances essentially have to do with the precise analysis of the most important structural motifs of the protein that it codes for and the main parameters that determine its interaction with DNA. They also concern the determination of the functional and behavioural properties in vivo of the main isoforms of the FOXP2 protein, the exact determination of the pattern of expression of new orthologues of the gene, and the identification of the different target genes for factor FOXP2. This new evidence suggests that protein FOXP2 protein has a high degree of versatility in vivo when it comes to binding to DNA; that its different isoforms are biologically functional; and that the FOXP2 gene is functional during embryonic development and during the adult phase. It also suggests that it is involved in the development and/or functioning of the thalamic-cortical-striatal circuits associated to motor planning, sequential behaviour and procedural learning (a significant saving in developmental terms of the regulatory mechanism in which the gene is involved), as well as the accuracy of the models of linguistic processing that consider language to be, to a large extent, the result of an interaction between certain cortical and subcortical structures.

Recombinant protein blends: silk beyond natural design.

PubMed

Dinjaski, Nina; Kaplan, David L

2016-06-01

Recombinant DNA technology and new material concepts are shaping future directions in biomaterial science for the design and production of the next-generation biomaterial platforms. Aside from conventionally used synthetic polymers, numerous natural biopolymers (e.g., silk, elastin, collagen, gelatin, alginate, cellulose, keratin, chitin, polyhydroxyalkanoates) have been investigated for properties and manipulation via bioengineering. Genetic engineering provides a path to increase structural and functional complexity of these biopolymers, and thereby expand the catalog of available biomaterials beyond that which exists in nature. In addition, the integration of experimental approaches with computational modeling to analyze sequence-structure-function relationships is starting to have an impact in the field by establishing predictive frameworks for determining material properties. Herein, we review advances in recombinant DNA-mediated protein production and functionalization approaches, with a focus on hybrids or combinations of proteins; recombinant protein blends or 'recombinamers'. We highlight the potential biomedical applications of fibrous protein recombinamers, such as Silk-Elastin Like Polypeptides (SELPs) and Silk-Bacterial Collagens (SBCs). We also discuss the possibility for the rationale design of fibrous proteins to build smart, stimuli-responsive biomaterials for diverse applications. We underline current limitations with production systems for these proteins and discuss the main trends in systems/synthetic biology that may improve recombinant fibrous protein design and production. Copyright © 2016. Published by Elsevier Ltd.

2S Albumin Storage Proteins: What Makes them Food Allergens?

PubMed

Moreno, F Javier; Clemente, Alfonso

2008-01-01

2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response.

2S Albumin Storage Proteins: What Makes them Food Allergens?

PubMed Central

Moreno, F. Javier; Clemente, Alfonso

2008-01-01

2S albumin storage proteins are becoming of increasing interest in nutritional and clinical studies as they have been reported as major food allergens in seeds of many mono- and di-cotyledonous plants. This review describes the main biochemical, structural and functional properties of these proteins thought to play a role in determining their potential allergenicity. 2S albumins are considered to sensitize directly via the gastrointestinal tract (GIT). The high stability of their intrinsic protein structure, dominated by a well-conserved skeleton of cysteine residues, to the harsh conditions present in the GIT suggests that these proteins are able to cross the gut mucosal barrier to sensitize the mucosal immune system and/or elicit an allergic response. The flexible and solvent-exposed hypervariable region of these proteins is immunodominant and has the ability to bind IgE from allergic patients´ sera. Several linear IgE-binding epitopes of 2S albumins spanning this region have been described to play a major role in allergenicity; the role of conformational epitopes of these proteins in food allergy is far from being understood and need to be investigated. Finally, the interaction of these proteins with other components of the food matrix might influence the absorption rates of immunologically reactive 2S albumins but also in their immune response. PMID:18949071

Properties and structure of a low-potential, penta-heme cytochrome c 552 from a thermophilic purple sulfur photosynthetic bacterium Thermochromatium tepidum.

PubMed

Chen, Jing-Hua; Yu, Long-Jiang; Boussac, Alain; Wang-Otomo, Zheng-Yu; Kuang, Tingyun; Shen, Jian-Ren

2018-04-24

The thermophilic purple sulfur bacterium Thermochromatium tepidum possesses four main water-soluble redox proteins involved in the electron transfer behavior. Crystal structures have been reported for three of them: a high potential iron-sulfur protein, cytochrome c', and one of two low-potential cytochrome c 552 (which is a flavocytochrome c) have been determined. In this study, we purified another low-potential cytochrome c 552 (LPC), determined its N-terminal amino acid sequence and the whole gene sequence, characterized it with absorption and electron paramagnetic spectroscopy, and solved its high-resolution crystal structure. This novel cytochrome was found to contain five c-type hemes. The overall fold of LPC consists of two distinct domains, one is the five heme-containing domain and the other one is an Ig-like domain. This provides a representative example for the structures of multiheme cytochromes containing an odd number of hemes, although the structures of multiheme cytochromes with an even number of hemes are frequently seen in the PDB database. Comparison of the sequence and structure of LPC with other proteins in the databases revealed several characteristic features which may be important for its functioning. Based on the results obtained, we discuss the possible intracellular function of this LPC in Tch. tepidum.

Uriniferous tubule: structural and functional organization.

PubMed

Christensen, Erik Ilsø; Wagner, Carsten A; Kaissling, Brigitte

2012-04-01

The uriniferous tubule is divided into the proximal tubule, the intermediate (thin) tubule, the distal tubule and the collecting duct. The present chapter is based on the chapters by Maunsbach and Christensen on the proximal tubule, and by Kaissling and Kriz on the distal tubule and collecting duct in the 1992 edition of the Handbook of Physiology, Renal Physiology. It describes the fine structure (light and electron microscopy) of the entire mammalian uriniferous tubule, mainly in rats, mice, and rabbits. The structural data are complemented by recent data on the location of the major transport- and transport-regulating proteins, revealed by morphological means(immunohistochemistry, immunofluorescence, and/or mRNA in situ hybridization). The structural differences along the uriniferous tubule strictly coincide with the distribution of the major luminal and basolateral transport proteins and receptors and both together provide the basis for the subdivision of the uriniferous tubule into functional subunits. Data on structural adaptation to defined functional changes in vivo and to genetical alterations of specified proteins involved in transepithelial transport importantly deepen our comprehension of the correlation of structure and function in the kidney, of the role of each segment or cell type in the overall renal function,and our understanding of renal pathophysiology. © 2012 American Physiological Society. Compr Physiol 2:933-996, 2012.

Structure-based Markov random field model for representing evolutionary constraints on functional sites.

PubMed

Jeong, Chan-Seok; Kim, Dongsup

2016-02-24

Elucidating the cooperative mechanism of interconnected residues is an important component toward understanding the biological function of a protein. Coevolution analysis has been developed to model the coevolutionary information reflecting structural and functional constraints. Recently, several methods have been developed based on a probabilistic graphical model called the Markov random field (MRF), which have led to significant improvements for coevolution analysis; however, thus far, the performance of these models has mainly been assessed by focusing on the aspect of protein structure. In this study, we built an MRF model whose graphical topology is determined by the residue proximity in the protein structure, and derived a novel positional coevolution estimate utilizing the node weight of the MRF model. This structure-based MRF method was evaluated for three data sets, each of which annotates catalytic site, allosteric site, and comprehensively determined functional site information. We demonstrate that the structure-based MRF architecture can encode the evolutionary information associated with biological function. Furthermore, we show that the node weight can more accurately represent positional coevolution information compared to the edge weight. Lastly, we demonstrate that the structure-based MRF model can be reliably built with only a few aligned sequences in linear time. The results show that adoption of a structure-based architecture could be an acceptable approximation for coevolution modeling with efficient computation complexity.

Sequence Identification, Recombinant Production, and Analysis of the Self-Assembly of Egg Stalk Silk Proteins from Lacewing Chrysoperla carnea.

PubMed

Neuenfeldt, Martin; Scheibel, Thomas

2017-06-13

Egg stalk silks of the common green lacewing Chrysoperla carnea likely comprise at least three different silk proteins. Based on the natural spinning process, it was hypothesized that these proteins self-assemble without shear stress, as adult lacewings do not use a spinneret. To examine this, the first sequence identification and determination of the gene expression profile of several silk proteins and various transcript variants thereof was conducted, and then the three major proteins were recombinantly produced in Escherichia coli encoded by their native complementary DNA (cDNA) sequences. Circular dichroism measurements indicated that the silk proteins in aqueous solutions had a mainly intrinsically disordered structure. The largest silk protein, which we named ChryC1, exhibited a lower critical solution temperature (LCST) behavior and self-assembled into fibers or film morphologies, depending on the conditions used. The second silk protein, ChryC2, self-assembled into nanofibrils and subsequently formed hydrogels. Circular dichroism and Fourier transform infrared spectroscopy confirmed conformational changes of both proteins into beta sheet rich structures upon assembly. ChryC3 did not self-assemble into any morphology under the tested conditions. Thereby, through this work, it could be shown that recombinant lacewing silk proteins can be produced and further used for studying the fiber formation of lacewing egg stalks.

Systematic comparison of the response properties of protein and RNA mediated gene regulatory motifs.

PubMed

Iyengar, Bharat Ravi; Pillai, Beena; Venkatesh, K V; Gadgil, Chetan J

2017-05-30

We present a framework enabling the dissection of the effects of motif structure (feedback or feedforward), the nature of the controller (RNA or protein), and the regulation mode (transcriptional, post-transcriptional or translational) on the response to a step change in the input. We have used a common model framework for gene expression where both motif structures have an activating input and repressing regulator, with the same set of parameters, to enable a comparison of the responses. We studied the global sensitivity of the system properties, such as steady-state gain, overshoot, peak time, and peak duration, to parameters. We find that, in all motifs, overshoot correlated negatively whereas peak duration varied concavely with peak time. Differences in the other system properties were found to be mainly dependent on the nature of the controller rather than the motif structure. Protein mediated motifs showed a higher degree of adaptation i.e. a tendency to return to baseline levels; in particular, feedforward motifs exhibited perfect adaptation. RNA mediated motifs had a mild regulatory effect; they also exhibited a lower peaking tendency and mean overshoot. Protein mediated feedforward motifs showed higher overshoot and lower peak time compared to the corresponding feedback motifs.

Preformed cell structure and cell heredity

PubMed Central

2008-01-01

This review will first recall the phenomena of “cortical inheritance” observed and genetically demonstrated in Paramecium 40 years ago, and later in other ciliates (Tetrahymena, Oxytricha, Paraurostyla), and will analyze the deduced concept of “cytotaxis” or “structural memory.” The significance of these phenomena, all related (but not strictly restricted) to the properties of ciliary basal bodies and their mode of duplication, will be interpreted in the light of present knowledge on the mechanism and control of basal body/centriole duplication. Then other phenomena described in a variety of organisms will be analyzed or mentioned which show the relevance of the concept of cytotaxis to other cellular processes, mainly (1) cytoskeleton assembly and organization with examples on ciliates, trypanosome, mammalian cells and plants, and (2) transmission of polarities with examples on yeast, trypanosome and metazoa. Finally, I will discuss some aspects of this particular type of non-DNA inheritance: (1) why so few documented examples if structural memory is a basic parameter in cell heredity, and (2) how are these phenomena (which all rely on protein/protein interactions, and imply a formatting role of preexisting proteinic complexes on neo-formed proteins and their assembly) related to prions? PMID:19164887

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

Fan, Yao; Tan, Kemin; Chhor, Gekleng

The EsxB protein from Bacillus anthracis belongs to the WXG100 family, a group of proteins secreted by a specialized secretion system. We have determined the crystal structures of recombinant EsxB and discovered that the small protein (~10 kDa), comprised of a helix-loop-helix (HLH) hairpin, is capable of associating into two different helical bundles. The two basic quaternary assemblies of EsxB are an antiparallel (AP) dimer and a rarely observed bisecting U (BU) dimer. This structural duality of EsxB is believed to originate from the heptad repeat sequence diversity of the first helix of its HLH hairpin, which allows for twomore » alternative helix packing. The flexibility of EsxB and the ability to form alternative helical bundles underscore the possibility that this protein can serve as an adaptor in secretion and can form hetero-oligomeric helix bundle(s) with other secreted members of the WXG100 family, such as EsxW. The highly conserved WXG motif is located within the loop of the HLH hairpin and is mostly buried within the helix bundle suggesting that its role is mainly structural. The exact functions of the motif, including a proposed role as a secretion signal, remain unknown.« less

Ready to use bioinformatics analysis as a tool to predict immobilisation strategies for protein direct electron transfer (DET).

PubMed

Cazelles, R; Lalaoui, N; Hartmann, T; Leimkühler, S; Wollenberger, U; Antonietti, M; Cosnier, S

2016-11-15

Direct electron transfer (DET) to proteins is of considerable interest for the development of biosensors and bioelectrocatalysts. While protein structure is mainly used as a method of attaching the protein to the electrode surface, we employed bioinformatics analysis to predict the suitable orientation of the enzymes to promote DET. Structure similarity and secondary structure prediction were combined underlying localized amino-acids able to direct one of the enzyme's electron relays toward the electrode surface by creating a suitable bioelectrocatalytic nanostructure. The electro-polymerization of pyrene pyrrole onto a fluorine-doped tin oxide (FTO) electrode allowed the targeted orientation of the formate dehydrogenase enzyme from Rhodobacter capsulatus (RcFDH) by means of hydrophobic interactions. Its electron relays were directed to the FTO surface, thus promoting DET. The reduction of nicotinamide adenine dinucleotide (NAD(+)) generating a maximum current density of 1μAcm(-2) with 10mM NAD(+) leads to a turnover number of 0.09electron/s/molRcFDH. This work represents a practical approach to evaluate electrode surface modification strategies in order to create valuable bioelectrocatalysts. Copyright © 2016 Elsevier B.V. All rights reserved.

Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

PubMed

Kriechbaumer, Verena; Botchway, Stanley W; Slade, Susan E; Knox, Kirsten; Frigerio, Lorenzo; Oparka, Karl; Hawes, Chris

2015-11-01

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane. © 2015 American Society of Plant Biologists. All Rights Reserved.

The crystal structure of the Split End protein SHARP adds a new layer of complexity to proteins containing RNA recognition motifs.

PubMed

Arieti, Fabiana; Gabus, Caroline; Tambalo, Margherita; Huet, Tiphaine; Round, Adam; Thore, Stéphane

2014-06-01

The Split Ends (SPEN) protein was originally discovered in Drosophila in the late 1990s. Since then, homologous proteins have been identified in eukaryotic species ranging from plants to humans. Every family member contains three predicted RNA recognition motifs (RRMs) in the N-terminal region of the protein. We have determined the crystal structure of the region of the human SPEN homolog that contains these RRMs-the SMRT/HDAC1 Associated Repressor Protein (SHARP), at 2.0 Å resolution. SHARP is a co-regulator of the nuclear receptors. We demonstrate that two of the three RRMs, namely RRM3 and RRM4, interact via a highly conserved interface. Furthermore, we show that the RRM3-RRM4 block is the main platform mediating the stable association with the H12-H13 substructure found in the steroid receptor RNA activator (SRA), a long, non-coding RNA previously shown to play a crucial role in nuclear receptor transcriptional regulation. We determine that SHARP association with SRA relies on both single- and double-stranded RNA sequences. The crystal structure of the SHARP-RRM fragment, together with the associated RNA-binding studies, extend the repertoire of nucleic acid binding properties of RRM domains suggesting a new hypothesis for a better understanding of SPEN protein functions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta

DOE PAGES

Jiang, Lin; Liu, Cong; Leibly, David; ...

2013-07-16

Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer’s, Parkinson’s, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer’s disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind tomore » Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers.« less

[NUCLEAR STRUCTURE IN THE SECRETORY CELLS OF MAMMARY GLANDS IN LACTATING AND NON-LACTATING RATS].

PubMed

Tyutina, K V; Skopichev, V G; Bogolyubov, D S; Bogolyubova, I O

2016-01-01

The features of structural and functional organization of the main nuclear compartments and distribution of their key molecular components (chromatin-remodeling protein ATRX, RNA polymerase I and II, and the splicing factor SC35) has been studied in the nuclei of mammary gland cells at different functional states. No significant differences between the nuclei of the cells in the lactating and non-lactating mammary glands have been revealed at the ultrastructural level. At the same time, photometric analysis has revealed higher intensity of nucleoplasmic immunofluorescent staining of mammary glands in the lactating animals when antibodies against the proteins ATRX and SC35 were used. Apparently, this observation reflects the changes of the structural and functional status of chromatin as well as the redistribution of splicing factors between the sites of their deposition and transcription.

Cryo-EM Structure of the TOM Core Complex from Neurospora crassa.

PubMed

Bausewein, Thomas; Mills, Deryck J; Langer, Julian D; Nitschke, Beate; Nussberger, Stephan; Kühlbrandt, Werner

2017-08-10

The TOM complex is the main entry gate for protein precursors from the cytosol into mitochondria. We have determined the structure of the TOM core complex by cryoelectron microscopy (cryo-EM). The complex is a 148 kDa symmetrical dimer of ten membrane protein subunits that create a shallow funnel on the cytoplasmic membrane surface. In the core of the dimer, the β-barrels of the Tom40 pore form two identical preprotein conduits. Each Tom40 pore is surrounded by the transmembrane segments of the α-helical subunits Tom5, Tom6, and Tom7. Tom22, the central preprotein receptor, connects the two Tom40 pores at the dimer interface. Our structure offers detailed insights into the molecular architecture of the mitochondrial preprotein import machinery. Copyright © 2017 Elsevier Inc. All rights reserved.

The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

NASA Astrophysics Data System (ADS)

Beedle, Amy E. M.; Lezamiz, Ainhoa; Stirnemann, Guillaume; Garcia-Manyes, Sergi

2015-08-01

Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.

Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA

PubMed Central

Ciganda, Martin; Williams, Noreen

2012-01-01

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D) for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC. PMID:22253864

A designed repeat protein as an affinity capture reagent

PubMed Central

Speltz, Elizabeth B.; Brown, Rebecca S.H.; Hajare, Holly S.; Schlieker, Christian; Regan, Lynne

2017-01-01

Repeat proteins are an attractive target for protein engineering and design. We have focused our attention on the design and engineering of one particular class - tetratricopeptide repeat (TPR) proteins. In previous work we have shown that the structure and stability of TPR proteins can be manipulated in a rational fashion [Cortajarena 2011; Main 2003]. Building on those studies, we have designed and characterized a number of different peptide-binding TPR modules and we have also assembled these modules into supramolecular arrays [Cortajarena 2009; Cortajarena 2008; Jackrel 2009; Kajander 2007]. Here we focus on the development of one such TPR-peptide interaction for a practical application – affinity purification. We illustrate the general utility of our designed protein interaction. Furthermore, this example highlights how basic research on protein-peptide interactions can lead to the development of novel reagents with important practical applications. PMID:26517897

Protein contact prediction using patterns of correlation.

PubMed

Hamilton, Nicholas; Burrage, Kevin; Ragan, Mark A; Huber, Thomas

2004-09-01

We describe a new method for using neural networks to predict residue contact pairs in a protein. The main inputs to the neural network are a set of 25 measures of correlated mutation between all pairs of residues in two "windows" of size 5 centered on the residues of interest. While the individual pair-wise correlations are a relatively weak predictor of contact, by training the network on windows of correlation the accuracy of prediction is significantly improved. The neural network is trained on a set of 100 proteins and then tested on a disjoint set of 1033 proteins of known structure. An average predictive accuracy of 21.7% is obtained taking the best L/2 predictions for each protein, where L is the sequence length. Taking the best L/10 predictions gives an average accuracy of 30.7%. The predictor is also tested on a set of 59 proteins from the CASP5 experiment. The accuracy is found to be relatively consistent across different sequence lengths, but to vary widely according to the secondary structure. Predictive accuracy is also found to improve by using multiple sequence alignments containing many sequences to calculate the correlations. Copyright 2004 Wiley-Liss, Inc.

Water binding of proteins in the processing frankfurter-type sausages. Part. 1. Water-binding ability of freeze-dried meat fractions containing myofibrillar and stromal proteins.

PubMed

Heinevetter, L; Gassmann, B; Kroll, J

1987-01-01

As soon as possible and 48 h after slaughter respectively, from both blade-bone muscle groups of cattle and pig carcasses the "thick pieces" were excised, extracted, and fractionated. Residues and precipitates from water and salt extracts resulted were freeze-dried, and an improved Baumann capillary suction apparatus was used to measure their water binding capacity (WBC) with and without addition of 2% sodium chloride and/or heating to 80 degrees C. With one exception the WBC results followed a relative pattern demonstrating the final residues (stromal proteins and leavings of myofibrillar proteins) binding the highest amount of added water, precipitates of dialysis (mainly containing myofibrillar proteins) a remarkable amount and powdered meats the least. As scanning electron micrographs confirmed, there were no fibrous structures in the precipitates resulted from dialysis of salt solutions (1.0 mol/1). Heating decreased the spontaneous water uptake of all fractions. Addition of sodium chloride had only a noticeable capillary-suction and swelling effect on unheated samples. Hence swelling of undissolved protein structures (extraction of myosin and possibly of actomyosin) is therefore not the only way for water binding in frankfurter-type sausages.

Recombinant Production, Reconstruction in Lipid-Protein Nanodiscs, and Electron Microscopy of Full-Length α-Subunit of Human Potassium Channel Kv7.1.

PubMed

Shenkarev, Z O; Karlova, M G; Kulbatskii, D S; Kirpichnikov, M P; Lyukmanova, E N; Sokolova, O S

2018-05-01

Voltage-gated potassium channel Kv7.1 plays an important role in the excitability of cardiac muscle. The α-subunit of Kv7.1 (KCNQ1) is the main structural element of this channel. Tetramerization of KCNQ1 in the membrane results in formation of an ion channel, which comprises a pore and four voltage-sensing domains. Mutations in the human KCNQ1 gene are one of the major causes of inherited arrhythmias, long QT syndrome in particular. The construct encoding full-length human KCNQ1 protein was synthesized in this work, and an expression system in the Pichia pastoris yeast cells was developed. The membrane fraction of the yeast cells containing the recombinant protein (rKCNQ1) was solubilized with CHAPS detergent. To better mimic the lipid environment of the channel, lipid-protein nanodiscs were formed using solubilized membrane fraction and MSP2N2 protein. The rKCNQ1/nanodisc and rKCNQ1/CHAPS samples were purified using the Rho1D4 tag introduced at the C-terminus of the protein. Protein samples were examined using transmission electron microscopy with negative staining. In both cases, homogeneous rKCNQ1 samples were observed based on image analysis. Statistical analysis of the images of individual protein particles solubilized in the detergent revealed the presence of a tetrameric structure confirming intact subunit assembly. A three-dimensional channel structure reconstructed at 2.5-nm resolution represents a compact density with diameter of the membrane part of ~9 nm and height ~11 nm. Analysis of the images of rKCNQ1 in nanodiscs revealed additional electron density corresponding to the lipid bilayer fragment and the MSP2N2 protein. These results indicate that the nanodiscs facilitate protein isolation, purification, and stabilization in solution and can be used for further structural studies of human Kv7.1.

Storage Stability of Food Protein Hydrolysates-A Review.

PubMed

Rao, Qinchun; Klaassen Kamdar, Andre; Labuza, Theodore P

2016-05-18

In recent years, mainly due to the specific health benefits associated with (1) the discovery of bioactive peptides in protein hydrolysates, (2) the reduction of protein allergenicity by protein hydrolysis, and (3) the improved protein digestibility and absorption of protein hydrolysates, the utilization of protein hydrolysates in functional foods and beverages has significantly increased. Although the specific health benefits from different hydrolysates are somewhat proven, the delivery and/or stability of these benefits is debatable during distribution, storage, and consumption. In this review, we discuss (1) the quality changes in different food protein hydrolysates during storage; (2) the resulting changes in the structure and texture of three food matrices, i.e., low moisture foods (LMF, aw < 0.6), intermediate moisture foods (IMF, 0.6 ≤ aw < 0.85), and high moisture foods (HMF, aw ≥ 0.85); and (3) the potential solutions to improve storage stability of food protein hydrolysates. In addition, we note there is a great need for evaluation of biofunction availability of bioactive peptides in food protein hydrolysates during storage.

ω-Turn: a novel β-turn mimic in globular proteins stabilized by main-chain to side-chain C−H···O interaction.

PubMed

Dhar, Jesmita; Chakrabarti, Pinak; Saini, Harpreet; Raghava, Gajendra Pal Singh; Kishore, Raghuvansh

2015-02-01

Mimicry of structural motifs is a common feature in proteins. The 10-membered hydrogen-bonded ring involving the main-chain C − O in a β-turn can be formed using a side-chain carbonyl group leading to Asx-turn. We show that the N − H component of hydrogen bond can be replaced by a C(γ) -H group in the side chain, culminating in a nonconventional C − H···O interaction. Because of its shape this β-turn mimic is designated as ω-turn, which is found to occur ∼ three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C − H···O interaction occurring between the terminal residues, constraining the torsion angles ϕi + 1, ψi + 1, ϕi + 2 and χ'1(i + 2) (using the interacting C(γ) atom). Based on these angles there are two types of ω-turns, each of which can be further divided into two groups. C(β) -branched side-chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal-binding sites. N-linked glycosylation occurs at the consensus pattern Asn-Xaa-Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω-turn, which may be the recognition site for protein modification. Location between two β-strands is the most common occurrence in protein tertiary structure, and being generally exposed ω-turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design. © 2014 Wiley Periodicals, Inc.

Insights on Structure and Function of a Late Embryogenesis Abundant Protein from Amaranthus cruentus: An Intrinsically Disordered Protein Involved in Protection against Desiccation, Oxidant Conditions, and Osmotic Stress

PubMed Central

Saucedo, Alma L.; Hernández-Domínguez, Eric E.; de Luna-Valdez, Luis A.; Guevara-García, Angel A.; Escobedo-Moratilla, Abraham; Bojorquéz-Velázquez, Esaú; del Río-Portilla, Federico; Fernández-Velasco, Daniel A.; Barba de la Rosa, Ana P.

2017-01-01

Late embryogenesis abundant (LEA) proteins are part of a large protein family that protect other proteins from aggregation due to desiccation or osmotic stresses. Recently, the Amaranthus cruentus seed proteome was characterized by 2D-PAGE and one highly accumulated protein spot was identified as a LEA protein and was named AcLEA. In this work, AcLEA cDNA was cloned into an expression vector and the recombinant protein was purified and characterized. AcLEA encodes a 172 amino acid polypeptide with a predicted molecular mass of 18.34 kDa and estimated pI of 8.58. Phylogenetic analysis revealed that AcLEA is evolutionarily close to the LEA3 group. Structural characteristics were revealed by nuclear magnetic resonance and circular dichroism methods. We have shown that recombinant AcLEA is an intrinsically disordered protein in solution even at high salinity and osmotic pressures, but it has a strong tendency to take a secondary structure, mainly folded as α-helix, when an inductive additive is present. Recombinant AcLEA function was evaluated using Escherichia coli as in vivo model showing the important protection role against desiccation, oxidant conditions, and osmotic stress. AcLEA recombinant protein was localized in cytoplasm of Nicotiana benthamiana protoplasts and orthologs were detected in seeds of wild and domesticated amaranth species. Interestingly AcLEA was detected in leaves, stems, and roots but only in plants subjected to salt stress. This fact could indicate the important role of AcLEA protection during plant stress in all amaranth species studied. PMID:28439280

Structural consequences of cutting a binding loop: two circularly permuted variants of streptavidin

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

Le Trong, Isolde; University of Washington, Box 357742, Seattle, WA 98195-7742; Chu, Vano

2013-06-01

The crystal structures of two circularly permuted streptavidins probe the role of a flexible loop in the tight binding of biotin. Molecular-dynamics calculations for one of the mutants suggests that increased fluctuations in a hydrogen bond between the protein and biotin are associated with cleavage of the binding loop. Circular permutation of streptavidin was carried out in order to investigate the role of a main-chain amide in stabilizing the high-affinity complex of the protein and biotin. Mutant proteins CP49/48 and CP50/49 were constructed to place new N-termini at residues 49 and 50 in a flexible loop involved in stabilizing themore » biotin complex. Crystal structures of the two mutants show that half of each loop closes over the binding site, as observed in wild-type streptavidin, while the other half adopts the open conformation found in the unliganded state. The structures are consistent with kinetic and thermodynamic data and indicate that the loop plays a role in enthalpic stabilization of the bound state via the Asn49 amide–biotin hydrogen bond. In wild-type streptavidin, the entropic penalties of immobilizing a flexible portion of the protein to enhance binding are kept to a manageable level by using a contiguous loop of medium length (six residues) which is already constrained by its anchorage to strands of the β-barrel protein. A molecular-dynamics simulation for CP50/49 shows that cleavage of the binding loop results in increased structural fluctuations for Ser45 and that these fluctuations destabilize the streptavidin–biotin complex.« less

Evolution of crystallins for a role in the vertebrate eye lens

PubMed Central

Slingsby, Christine; Wistow, Graeme J; Clark, Alice R

2013-01-01

The camera eye lens of vertebrates is a classic example of the re-engineering of existing protein components to fashion a new device. The bulk of the lens is formed from proteins belonging to two superfamilies, the α-crystallins and the βγ-crystallins. Tracing their ancestry may throw light on the origin of the optics of the lens. The α-crystallins belong to the ubiquitous small heat shock proteins family that plays a protective role in cellular homeostasis. They form enormous polydisperse oligomers that challenge modern biophysical methods to uncover the molecular basis of their assembly structure and chaperone-like protein binding function. It is argued that a molecular phenotype of a dynamic assembly suits a chaperone function as well as a structural role in the eye lens where the constraint of preventing protein condensation is paramount. The main cellular partners of α-crystallins, the β- and γ-crystallins, have largely been lost from the animal kingdom but the superfamily is hugely expanded in the vertebrate eye lens. Their structures show how a simple Greek key motif can evolve rapidly to form a complex array of monomers and oligomers. Apart from remaining transparent, a major role of the partnership of α-crystallins with β- and γ-crystallins in the lens is to form a refractive index gradient. Here, we show some of the structural and genetic features of these two protein superfamilies that enable the rapid creation of different assembly states, to match the rapidly changing optical needs among the various vertebrates. PMID:23389822

sc-PDB: an annotated database of druggable binding sites from the Protein Data Bank.

PubMed

Kellenberger, Esther; Muller, Pascal; Schalon, Claire; Bret, Guillaume; Foata, Nicolas; Rognan, Didier

2006-01-01

The sc-PDB is a collection of 6 415 three-dimensional structures of binding sites found in the Protein Data Bank (PDB). Binding sites were extracted from all high-resolution crystal structures in which a complex between a protein cavity and a small-molecular-weight ligand could be identified. Importantly, ligands are considered from a pharmacological and not a structural point of view. Therefore, solvents, detergents, and most metal ions are not stored in the sc-PDB. Ligands are classified into four main categories: nucleotides (< 4-mer), peptides (< 9-mer), cofactors, and organic compounds. The corresponding binding site is formed by all protein residues (including amino acids, cofactors, and important metal ions) with at least one atom within 6.5 angstroms of any ligand atom. The database was carefully annotated by browsing several protein databases (PDB, UniProt, and GO) and storing, for every sc-PDB entry, the following features: protein name, function, source, domain and mutations, ligand name, and structure. The repository of ligands has also been archived by diversity analysis of molecular scaffolds, and several chemoinformatics descriptors were computed to better understand the chemical space covered by stored ligands. The sc-PDB may be used for several purposes: (i) screening a collection of binding sites for predicting the most likely target(s) of any ligand, (ii) analyzing the molecular similarity between different cavities, and (iii) deriving rules that describe the relationship between ligand pharmacophoric points and active-site properties. The database is periodically updated and accessible on the web at http://bioinfo-pharma.u-strasbg.fr/scPDB/.

Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity.

PubMed

Korkuć, Paula; Walther, Dirk

2015-01-01

To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous system) suggesting specific molecular and physiological roles of promiscuous metabolites.

Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity

PubMed Central

Korkuć, Paula; Walther, Dirk

2015-01-01

To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous system) suggesting specific molecular and physiological roles of promiscuous metabolites. PMID:26442281

Mechanism of ubiquinol oxidation by the bc(1) complex: role of the iron sulfur protein and its mobility.

PubMed

Crofts, A R; Guergova-Kuras, M; Huang, L; Kuras, R; Zhang, Z; Berry, E A

1999-11-30

Native structures of ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and structures with inhibitors in place, show a 16-22 A displacement of the [2Fe-2S] cluster and the position of the C-terminal extrinsic domain of the iron sulfur protein. None of the structures shows a static configuration that would allow catalysis of all partial reactions of quinol oxidation. We have suggested that the different conformations reflect a movement of the subunit necessary for catalysis. The displacement from an interface with cytochrome c(1) in native crystals to an interface with cytochrome b is induced by stigmatellin or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and involves ligand formation between His-161 of the [2Fe-2S] binding cluster and the inhibitor. The movement is a rotational displacement, so that the same conserved docking surface on the iron sulfur protein interacts with cytochrome c(1) and with cytochrome b. The mobile extrinsic domain retains essentially the same tertiary structure, and the anchoring N-terminal tail remains in the same position. The movement occurs through an extension of a helical segment in the short linking span. We report details of the protein structure for the two main configurations in the chicken heart mitochondrial complex and discuss insights into mechanism provided by the structures and by mutant strains in which the docking at the cytochrome b interface is impaired. The movement of the iron sulfur protein represents a novel mechanism of electron transfer, in which a tethered mobile head allows electron transfer through a distance without the entropic loss from free diffusion.

Analysis of zinc binding sites in protein crystal structures.

PubMed

Alberts, I L; Nadassy, K; Wodak, S J

1998-08-01

The geometrical properties of zinc binding sites in a dataset of high quality protein crystal structures deposited in the Protein Data Bank have been examined to identify important differences between zinc sites that are directly involved in catalysis and those that play a structural role. Coordination angles in the zinc primary coordination sphere are compared with ideal values for each coordination geometry, and zinc coordination distances are compared with those in small zinc complexes from the Cambridge Structural Database as a guide of expected trends. We find that distances and angles in the primary coordination sphere are in general close to the expected (or ideal) values. Deviations occur primarily for oxygen coordinating atoms and are found to be mainly due to H-bonding of the oxygen coordinating ligand to protein residues, bidentate binding arrangements, and multi-zinc sites. We find that H-bonding of oxygen containing residues (or water) to zinc bound histidines is almost universal in our dataset and defines the elec-His-Zn motif. Analysis of the stereochemistry shows that carboxyl elec-His-Zn motifs are geometrically rigid, while water elec-His-Zn motifs show the most geometrical variation. As catalytic motifs have a higher proportion of carboxyl elec atoms than structural motifs, they provide a more rigid framework for zinc binding. This is understood biologically, as a small distortion in the zinc position in an enzyme can have serious consequences on the enzymatic reaction. We also analyze the sequence pattern of the zinc ligands and residues that provide elecs, and identify conserved hydrophobic residues in the endopeptidases that also appear to contribute to stabilizing the catalytic zinc site. A zinc binding template in protein crystal structures is derived from these observations.

A structural portrait of the PDZ domain family.

PubMed

Ernst, Andreas; Appleton, Brent A; Ivarsson, Ylva; Zhang, Yingnan; Gfeller, David; Wiesmann, Christian; Sidhu, Sachdev S

2014-10-23

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular and functional analyses of a maize autoactive NB-LRR protein identify precise structural requirements for activity.

PubMed

Wang, Guan-Feng; Ji, Jiabing; El-Kasmi, Farid; Dangl, Jeffery L; Johal, Guri; Balint-Kurti, Peter J

2015-02-01

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.

Emerging Synaptic Molecules as Candidates in the Etiology of Neurological Disorders

PubMed Central

Torres, Viviana I.; Vallejo, Daniela

2017-01-01

Synapses are complex structures that allow communication between neurons in the central nervous system. Studies conducted in vertebrate and invertebrate models have contributed to the knowledge of the function of synaptic proteins. The functional synapse requires numerous protein complexes with specialized functions that are regulated in space and time to allow synaptic plasticity. However, their interplay during neuronal development, learning, and memory is poorly understood. Accumulating evidence links synapse proteins to neurodevelopmental, neuropsychiatric, and neurodegenerative diseases. In this review, we describe the way in which several proteins that participate in cell adhesion, scaffolding, exocytosis, and neurotransmitter reception from presynaptic and postsynaptic compartments, mainly from excitatory synapses, have been associated with several synaptopathies, and we relate their functions to the disease phenotype. PMID:28331639

From Never Born Proteins to Minimal Living Cells: two projects in synthetic biology.

PubMed

Luisi, Pier Luigi; Chiarabelli, Cristiano; Stano, Pasquale

2006-12-01

The Never Born Proteins (NBPs) and the Minimal Cell projects are two currently developed research lines belonging to the field of synthetic biology. The first deals with the investigation of structural and functional properties of de novo proteins with random sequences, selected and isolated using phage display methods. The minimal cell is the simplest cellular construct which displays living properties, such as self-maintenance, self-reproduction and evolvability. The semi-synthetic approach to minimal cells involves the use of extant genes and proteins in order to build a supramolecular construct based on lipid vesicles. Results and outlooks on these two research lines are shortly discussed, mainly focusing on their relevance to the origin of life studies.

Comparative protein profiles of Butea superba tubers under seasonal changes.

PubMed

Leelahawong, Chonchanok; Srisomsap, Chantragan; Cherdshewasart, Wichai; Chokchaichamnankit, Daranee; Vinayavekhin, Nawaporn; Sangvanich, Polkit

2016-07-01

Seasonal changes are major factors affecting environmental conditions which induce multiple stresses in plants, leading to changes in protein relative abundance in the complex cellular plant metabolic pathways. Proteomics was applied to study variations in proteome composition of Butea. superba tubers during winter, summer and rainy season throughout the year using two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight tandem mass spectrometry. A total of 191 protein spots were identified and also classified into 12 functional groups. The majority of these were mainly involved in carbohydrate and energy metabolism (30.37 %) and defense and stress (18.32 %). The results exhibited the highest numbers of identified proteins in winter-harvested samples. Forty-five differential proteins were found in different seasons, involving important metabolic pathways. Further analysis indicated that changes in the protein levels were due mainly to temperature stress during summer and to water stress during winter, which affected cellular structure, photosynthesis, signal transduction and homeostasis, amino-acid biosynthesis, protein destination and storage, protein biosynthesis and stimulated defense and stress mechanisms involving glycolytic enzymes and relative oxygen species catabolizing enzymes. The proteins with differential relative abundances might induce an altered physiological status within plant tubers for survival. The work provided new insights into the better understanding of the molecular basis of plant proteomes and stress tolerance mechanisms, especially during seasonal changes. The finding suggested proteins that might potentially be used as protein markers in differing seasons in other plants and aid in selecting B. superba tubers with the most suitable medicinal properties in the future.

Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

PubMed

Deryusheva, Evgeniia I; Machulin, Andrey V; Selivanova, Olga M; Galzitskaya, Oxana V

2017-04-01

Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Extracellular protein analysis of activated sludge and their functions in wastewater treatment plant by shotgun proteomics.

PubMed

Zhang, Peng; Shen, Yu; Guo, Jin-Song; Li, Chun; Wang, Han; Chen, You-Peng; Yan, Peng; Yang, Ji-Xiang; Fang, Fang

2015-07-10

In this work, proteins in extracellular polymeric substances extracted from anaerobic, anoxic and aerobic sludges of wastewater treatment plant (WWTP) were analyzed to probe their origins and functions. Extracellular proteins in WWTP sludges were identified using shotgun proteomics, and 130, 108 and 114 proteins in anaerobic, anoxic and aerobic samples were classified, respectively. Most proteins originated from cell and cell part, and their most major molecular functions were catalytic activity and binding activity. The results exhibited that the main roles of extracellular proteins in activated sludges were multivalence cations and organic molecules binding, as well as in catalysis and degradation. The catalytic activity proteins were more widespread in anaerobic sludge compared with those in anoxic and aerobic sludges. The structure difference between anaerobic and aerobic sludges could be associated with their catalytic activities proteins. The results also put forward a relation between the macro characteristics of activated sludges and micro functions of extracellular proteins in biological wastewater treatment process.

Structural characterization and mutational assessment of podocin - a novel drug target to nephrotic syndrome - an in silico approach.

PubMed

Tabassum, Asra; Rajeshwari, Tadigadapa; Soni, Nidhi; Raju, D S B; Yadav, Mukesh; Nayarisseri, Anuraj; Jahan, Parveen

2014-03-01

Non-synonymous single nucleotide changes (nSNC) are coding variants that introduce amino acid changes in their corresponding proteins. They can affect protein function; they are believed to have the largest impact on human health compared with SNCs in other regions of the genome. Such a sequence alteration directly affects their structural stability through conformational changes. Presence of these conformational changes near catalytic site or active site may alter protein function and as a consequence receptor-ligand complex interactions. The present investigation includes assessment of human podocin mutations (G92C, P118L, R138Q, and D160G) on its structure. Podocin is an important glomerular integral membrane protein thought to play a key role in steroid resistant nephrotic syndrome. Podocin has a hairpin like structure with 383 amino acids, it is an integral protein homologous to stomatin, and acts as a molecular link in a stretch-sensitive system. We modeled 3D structure of podocin by means of Modeller and validated via PROCHECK to get a Ramachandran plot (88.5% in most favored region), main chain, side chain, bad contacts, gauche and pooled standard deviation. Further, a protein engineering tool Triton was used to induce mutagenesis corresponding to four variants G92C, P118L, R138Q and D160G in the wild type. Perusal of energies of wild and mutated type of podocin structures confirmed that mutated structures were thermodynamically more stable than wild type and therefore biological events favored synthesis of mutated forms of podocin than wild type. As a conclusive part, two mutations G92C (-8179.272 kJ/mol) and P118L (-8136.685 kJ/mol) are more stable and probable to take place in podocin structure over wild podocin structure (-8105.622 kJ/mol). Though there is lesser difference in mutated and wild type (approximately, 74 and 35 kJ/mol), it may play a crucial role in deciding why mutations are favored and occur at the genetic level.

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.

Computational prediction of atomic structures of helical membrane proteins aided by EM maps.

PubMed

Kovacs, Julio A; Yeager, Mark; Abagyan, Ruben

2007-09-15

Integral membrane proteins pose a major challenge for protein-structure prediction because only approximately 100 high-resolution structures are available currently, thereby impeding the development of rules or empirical potentials to predict the packing of transmembrane alpha-helices. However, when an intermediate-resolution electron microscopy (EM) map is available, it can be used to provide restraints which, in combination with a suitable computational protocol, make structure prediction feasible. In this work we present such a protocol, which proceeds in three stages: 1), generation of an ensemble of alpha-helices by flexible fitting into each of the density rods in the low-resolution EM map, spanning a range of rotational angles around the main helical axes and translational shifts along the density rods; 2), fast optimization of side chains and scoring of the resulting conformations; and 3), refinement of the lowest-scoring conformations with internal coordinate mechanics, by optimizing the van der Waals, electrostatics, hydrogen bonding, torsional, and solvation energy contributions. In addition, our method implements a penalty term through a so-called tethering map, derived from the EM map, which restrains the positions of the alpha-helices. The protocol was validated on three test cases: GpA, KcsA, and MscL.

The Generalized Born solvation model: What is it?

NASA Astrophysics Data System (ADS)

Onufriev, Alexey

2004-03-01

Implicit solvation models provide, for many applications, an effective way of describing the electrostatic effects of aqueous solvation. Here we outline the main approximations behind the popular Generalized Born solvation model. We show how its accuracy, relative to the Poisson-Boltzmann treatment, can be significantly improved in a computationally inexpensive manner to make the model useful in the studies of large-scale conformational transitions at the atomic level. The improved model is tested in a molecular dynamics simulation of folding of a 46-residue (three helix bundle) protein. Starting from an extended structure at 450K, the protein folds to the lowest energy conformation within 6 ns of simulation time, and the predicted structure differs from the native one by 2.4 A (backbone RMSD).

Fractionation, partial characterization and bioactivity of water-soluble polysaccharides and polysaccharide-protein complexes from Pleurotus geesteranus.

PubMed

Zhang, Mei; Zhu, Lin; Cui, Steve W; Wang, Qi; Zhou, Ting; Shen, Hengsheng

2011-01-01

Fractionation and purification of mushroom polysaccharides is a critical process for mushroom clinical application. After a hot-water treatment, the crude Pleurotus geesteranus (PG) was further fractionated into four fractions (PG-1, -2, -3, -4) using gradient precipitation with water and ammonia sulphate. By controlling the initial polymer concentration and ratio of solvents, this process produced PG fractions with high chemical uniformity and narrow Mw distribution without free proteins. Structurally, PG-1 and PG-2 are pure homopolysaccharide mainly composed of glucose; and PG-3 and PG-4 are heteropolysaccharide-protein complexes. PG-2, a high M(w) fraction mainly composed of glucose presented significant cytotoxicity at the concentration of 200 and 100 μg/ml to human breast cancer cells. Here, we report a new mushroom polysaccharides extraction and fractionation method, with which we produced four fractions of PG with PG-2 appearing effective anti-tumour activity. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Mechanics of Lipid Bilayer Membranes

NASA Astrophysics Data System (ADS)

Powers, Thomas R.

All cells have membranes. The plasma membrane encapsulates the cell's interior, acting as a barrier against the outside world. In cells with nuclei (eukaryotic cells), membranes also form internal compartments (organelles) which carry out specialized tasks, such as protein modification and sorting in the case of the Golgi apparatus, and ATP production in the case of mitochondria. The main components of membranes are lipids and proteins. The proteins can be channels, carriers, receptors, catalysts, signaling molecules, or structural elements, and typically contribute a substantial fraction of the total membrane dry weight. The equilibrium properties of pure lipid membranes are relatively well-understood, and will be the main focus of this article. The framework of elasticity theory and statistical mechanics that we will develop will serve as the foundation for understanding biological phenomena such as the nonequilibrium behavior of membranes laden with ion pumps, the role of membrane elasticity in ion channel gating, and the dynamics of vesicle fission and fusion. Understanding the mechanics of lipid membranes is also important for drug encapsulation and delivery.

Seaweed as a source of novel nutraceuticals: sulfated polysaccharides and peptides.

PubMed

Jiménez-Escrig, A; Gómez-Ordóñez, E; Rupérez, P

2011-01-01

Seaweeds and seaweed-derived products are underexploited marine bioresources and a source of natural ingredients for functional foods. Nutritional studies on seaweeds indicate that brown and red seaweeds possess a good nutritional quality and could be used as an alternative source of dietary fiber, protein, and minerals. Moreover, bioactive sulfated polysaccharides are the main components of soluble fiber in seaweeds and also bioactive peptides can be prepared from seaweed protein. This chapter gives an overview of the main biological properties of sulfated polysaccharides and peptides from brown and red seaweeds. Recent studies have provided evidence that sulfated polysaccharides from seaweeds can play a vital role in human health and nutrition. Besides, peptides derived from algal protein are most promising as antihypertensive agents. Further research work, especially in vivo studies, are needed in order to gain a better knowledge of the relation structure-function by which bioactive compounds from seaweeds exert their bioactivity. Copyright © 2011 Elsevier Inc. All rights reserved.

Automated main-chain model building by template matching and iterative fragment extension.

PubMed

Terwilliger, Thomas C

2003-01-01

An algorithm for the automated macromolecular model building of polypeptide backbones is described. The procedure is hierarchical. In the initial stages, many overlapping polypeptide fragments are built. In subsequent stages, the fragments are extended and then connected. Identification of the locations of helical and beta-strand regions is carried out by FFT-based template matching. Fragment libraries of helices and beta-strands from refined protein structures are then positioned at the potential locations of helices and strands and the longest segments that fit the electron-density map are chosen. The helices and strands are then extended using fragment libraries consisting of sequences three amino acids long derived from refined protein structures. The resulting segments of polypeptide chain are then connected by choosing those which overlap at two or more C(alpha) positions. The fully automated procedure has been implemented in RESOLVE and is capable of model building at resolutions as low as 3.5 A. The algorithm is useful for building a preliminary main-chain model that can serve as a basis for refinement and side-chain addition.

Functional diversity of potassium channel voltage-sensing domains.

PubMed

Islas, León D

2016-01-01

Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.

Functional diversity of potassium channel voltage-sensing domains

PubMed Central

Islas, León D.

2016-01-01

Abstract Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology. PMID:26794852

The basic helix-loop-helix region of the transcriptional repressor hairy and enhancer of split 1 is preorganized to bind DNA.

PubMed

Popovic, Matija; Wienk, Hans; Coglievina, Maristella; Boelens, Rolf; Pongor, Sándor; Pintar, Alessandro

2014-04-01

Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions. Copyright © 2014 Wiley Periodicals, Inc.

Urea-mediated protein denaturation: a consensus view.

PubMed

Das, Atanu; Mukhopadhyay, Chaitali

2009-09-24

We have performed all-atom molecular dynamics simulations of three structurally similar small globular proteins in 8 M urea and compared the results with pure aqueous simulations. Protein denaturation is preceded by an initial loss of water from the first solvation shell and consequent in-flow of urea toward the protein. Urea reaches the first solvation shell of the protein mainly due to electrostatic interaction with a considerable contribution coming from the dispersion interaction. Urea shifts the equilibrium from the native to denatured ensemble by making the protein-protein contact less stable than protein-urea contact, which is just the reverse of the condition in pure water, where protein-protein contact is more stable than protein-water contact. We have also seen that water follows urea and reaches the protein interior at later stages of denaturation, while urea preferentially and efficiently solvates different parts of the protein. Solvation of the protein backbone via hydrogen bonding, favorable electrostatic interaction with hydrophilic residues, and dispersion interaction with hydrophobic residues are the key steps through which urea intrudes the core of the protein and denatures it. Why urea is preferred over water for binding to the protein backbone and how urea orients itself toward the protein backbone have been identified comprehensively. All the key components of intermolecular forces are found to play a significant part in urea-induced protein denaturation and also toward the stability of the denatured state ensemble. Changes in water network/structure and dynamical properties and higher degree of solvation of the hydrophobic residues validate the presence of "indirect mechanism" along with the "direct mechanism" and reinforce the effect of urea on protein.

A Complete Structural Inventory of the Mycobacterial Microcompartment Shell Proteins Constrains Models of Global Architecture and Transport*

PubMed Central

Mallette, Evan

2017-01-01

Bacterial microcompartments are bacterial analogs of eukaryotic organelles in that they spatially segregate aspects of cellular metabolism, but they do so by building not a lipid membrane but a thin polyhedral protein shell. Although multiple shell protein structures are known for several microcompartment types, additional uncharacterized components complicate systematic investigations of shell architecture. We report here the structures of all four proteins proposed to form the shell of an uncharacterized microcompartment designated the Rhodococcus and Mycobacterium microcompartment (RMM), which, along with crystal interactions and docking studies, suggests possible models for the particle's vertex and edge organization. MSM0272 is a typical hexameric β-sandwich shell protein thought to form the bulk of the facet. MSM0273 is a pentameric β-barrel shell protein that likely plugs the vertex of the particle. MSM0271 is an unusual double-ringed bacterial microcompartment shell protein whose rings are organized in an offset position relative to all known related proteins. MSM0275 is related to MSM0271 but self-organizes as linear strips that may line the facet edge; here, the presence of a novel extendable loop may help ameliorate poor packing geometry of the rigid main particle at the angled edges. In contrast to previously characterized homologs, both of these proteins show closed pores at both ends. This suggests a model where key interactions at the vertex and edges are mediated at the inner layer of the shell by MSM0271 (encircling MSM0273) and MSM0275, and the facet is built from MSM0272 hexamers tiling in the outer layer of the shell. PMID:27927988

A Complete Structural Inventory of the Mycobacterial Microcompartment Shell Proteins Constrains Models of Global Architecture and Transport.

PubMed

Mallette, Evan; Kimber, Matthew S

2017-01-27

Bacterial microcompartments are bacterial analogs of eukaryotic organelles in that they spatially segregate aspects of cellular metabolism, but they do so by building not a lipid membrane but a thin polyhedral protein shell. Although multiple shell protein structures are known for several microcompartment types, additional uncharacterized components complicate systematic investigations of shell architecture. We report here the structures of all four proteins proposed to form the shell of an uncharacterized microcompartment designated the Rhodococcus and Mycobacterium microcompartment (RMM), which, along with crystal interactions and docking studies, suggests possible models for the particle's vertex and edge organization. MSM0272 is a typical hexameric β-sandwich shell protein thought to form the bulk of the facet. MSM0273 is a pentameric β-barrel shell protein that likely plugs the vertex of the particle. MSM0271 is an unusual double-ringed bacterial microcompartment shell protein whose rings are organized in an offset position relative to all known related proteins. MSM0275 is related to MSM0271 but self-organizes as linear strips that may line the facet edge; here, the presence of a novel extendable loop may help ameliorate poor packing geometry of the rigid main particle at the angled edges. In contrast to previously characterized homologs, both of these proteins show closed pores at both ends. This suggests a model where key interactions at the vertex and edges are mediated at the inner layer of the shell by MSM0271 (encircling MSM0273) and MSM0275, and the facet is built from MSM0272 hexamers tiling in the outer layer of the shell. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of high pressure homogenization on the structure and the interfacial and emulsifying properties of β-lactoglobulin.

PubMed

Ali, Ali; Le Potier, Isabelle; Huang, Nicolas; Rosilio, Véronique; Cheron, Monique; Faivre, Vincent; Turbica, Isabelle; Agnely, Florence; Mekhloufi, Ghozlene

2018-02-15

The effect of high pressure homogenization (HPH) on the structure of β-lactoglobulin (β-lg) was studied by combining spectroscopic, chromatographic, and electrophoretic methods. The consequences of the resulting structure modifications on oil/water (O/W) interfacial properties were also assessed. Moderated HPH treatment (100 MPa/4 cycles) showed no significant modification of protein structure and interfacial properties. However, a harsher HPH treatment (300 MPa/5 cycles) induced structural transformation, mainly from β-sheets to random coils, wide loss in lipocalin core, and protein aggregation via intermolecular disulfide bridges. HPH-modified β-lg displayed higher surface hydrophobicity leading to a faster adsorption rate at the interface and an earlier formation of an elastic interfacial film at C β-lg  = 0.1 wt%. However, no modification of the interfacial properties was observed at C β-lg  = 1 wt%. At this protein concentration, the prior denaturation of β-lg by HPH did not modify the droplet size of nanoemulsions prepared with these β-lg solutions as the aqueous phases. A slightly increased creaming rate was however observed. The effects of HPH and heat denaturations appeared qualitatively similar, but with differences in their extent. Copyright © 2017 Elsevier B.V. All rights reserved.

GPCR-ModSim: A comprehensive web based solution for modeling G-protein coupled receptors

PubMed Central

Esguerra, Mauricio; Siretskiy, Alexey; Bello, Xabier; Sallander, Jessica; Gutiérrez-de-Terán, Hugo

2016-01-01

GPCR-ModSim (http://open.gpcr-modsim.org) is a centralized and easy to use service dedicated to the structural modeling of G-protein Coupled Receptors (GPCRs). 3D molecular models can be generated from amino acid sequence by homology-modeling techniques, considering different receptor conformations. GPCR-ModSim includes a membrane insertion and molecular dynamics (MD) equilibration protocol, which can be used to refine the generated model or any GPCR structure uploaded to the server, including if desired non-protein elements such as orthosteric or allosteric ligands, structural waters or ions. We herein revise the main characteristics of GPCR-ModSim and present new functionalities. The templates used for homology modeling have been updated considering the latest structural data, with separate profile structural alignments built for inactive, partially-active and active groups of templates. We have also added the possibility to perform multiple-template homology modeling in a unique and flexible way. Finally, our new MD protocol considers a series of distance restraints derived from a recently identified conserved network of helical contacts, allowing for a smoother refinement of the generated models which is particularly advised when there is low homology to the available templates. GPCR- ModSim has been tested on the GPCR Dock 2013 competition with satisfactory results. PMID:27166369

FTIR spectroscopy structural analysis of the interaction between Lactobacillus kefir S-layers and metal ions

NASA Astrophysics Data System (ADS)

Gerbino, E.; Mobili, P.; Tymczyszyn, E.; Fausto, R.; Gómez-Zavaglia, A.

2011-02-01

FTIR spectroscopy was used to structurally characterize the interaction of S-layer proteins extracted from two strains of Lactobacillus kefir (the aggregating CIDCA 8348 and the non-aggregating JCM 5818) with metal ions (Cd +2, Zn +2, Pb +2 and Ni +2). The infrared spectra indicate that the metal/protein interaction occurs mainly through the carboxylate groups of the side chains of Asp and Glut residues, with some contribution of the NH groups belonging to the peptide backbone. The frequency separation between the νCOO - anti-symmetric and symmetric stretching vibrations in the spectra of the S-layers in presence of the metal ions was found to be ca. 190 cm -1 for S-layer CIDCA 8348 and ca. 170 cm -1 for JCM 5818, denoting an unidentate coordination in both cases. Changes in the secondary structures of the S-layers induced by the interaction with the metal ions were also noticed: a general trend to increase the amount of β-sheet structures and to reduce the amount of α-helices was observed. These changes allow the proteins to adjust their structure to the presence of the metal ions at minimum energy expense, and accordingly, these adjustments were found to be more important for the bigger ions.

ATR-IR study of skin components: Lipids, proteins and water. Part I: Temperature effect

NASA Astrophysics Data System (ADS)

Olsztyńska-Janus, S.; Pietruszka, A.; Kiełbowicz, Z.; Czarnecki, M. A.

2018-01-01

In this work we report the studies of the effect of temperature on skin components, such as lipids, proteins and water. Modifications of lipids structure induced by increasing temperature (from 20 to 90 °C) have been studied using ATR-IR (Attenuated Total Reflectance Infrared) spectroscopy, which is a powerful tool for characterization of the molecular structure and properties of tissues, such as skin. Due to the small depth of penetration (0.6-5.6 μm), ATR-IR spectroscopy probes only the outermost layer of the skin, i.e. the stratum corneum (SC). The assignment of main spectral features of skin components allows for the determination of phase transitions from the temperature dependencies of band intensities [e.g. νas(CH2) and νs(CH2)]. The phase transitions were determined by using two methods: the first one was based on the first derivative of the Boltzmann function and the second one employed tangent lines of sigmoidal, aforementioned dependencies. The phase transitions in lipids were correlated with modifications of the structure of water and proteins.

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms.

PubMed

Simões, Manuel; Pereira, Maria O; Sillankorva, Sanna; Azeredo, Joana; Vieira, Maria J

2007-01-01

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm(2), and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.

Improved method for predicting protein fold patterns with ensemble classifiers.

PubMed

Chen, W; Liu, X; Huang, Y; Jiang, Y; Zou, Q; Lin, C

2012-01-27

Protein folding is recognized as a critical problem in the field of biophysics in the 21st century. Predicting protein-folding patterns is challenging due to the complex structure of proteins. In an attempt to solve this problem, we employed ensemble classifiers to improve prediction accuracy. In our experiments, 188-dimensional features were extracted based on the composition and physical-chemical property of proteins and 20-dimensional features were selected using a coupled position-specific scoring matrix. Compared with traditional prediction methods, these methods were superior in terms of prediction accuracy. The 188-dimensional feature-based method achieved 71.2% accuracy in five cross-validations. The accuracy rose to 77% when we used a 20-dimensional feature vector. These methods were used on recent data, with 54.2% accuracy. Source codes and dataset, together with web server and software tools for prediction, are available at: http://datamining.xmu.edu.cn/main/~cwc/ProteinPredict.html.

Automatic rebuilding and optimization of crystallographic structures in the Protein Data Bank

PubMed Central

Joosten, Robbie P.; Joosten, Krista; Cohen, Serge X.; Vriend, Gert; Perrakis, Anastassis

2011-01-01

Motivation: Macromolecular crystal structures in the Protein Data Bank (PDB) are a key source of structural insight into biological processes. These structures, some >30 years old, were constructed with methods of their era. With PDB_REDO, we aim to automatically optimize these structures to better fit their corresponding experimental data, passing the benefits of new methods in crystallography on to a wide base of non-crystallographer structure users. Results: We developed new algorithms to allow automatic rebuilding and remodeling of main chain peptide bonds and side chains in crystallographic electron density maps, and incorporated these and further enhancements in the PDB_REDO procedure. Applying the updated PDB_REDO to the oldest, but also to some of the newest models in the PDB, corrects existing modeling errors and brings these models to a higher quality, as judged by standard validation methods. Availability and Implementation: The PDB_REDO database and links to all software are available at http://www.cmbi.ru.nl/pdb_redo. Contact: r.joosten@nki.nl; a.perrakis@nki.nl Supplementary Information: Supplementary data are available at Bioinformatics online. PMID:22034521

Recognition of the murine coronavirus genomic RNA packaging signal depends on the second RNA-binding domain of the nucleocapsid protein.

PubMed

Kuo, Lili; Koetzner, Cheri A; Hurst, Kelley R; Masters, Paul S

2014-04-01

The coronavirus nucleocapsid (N) protein forms a helical ribonucleoprotein with the viral positive-strand RNA genome and binds to the principal constituent of the virion envelope, the membrane (M) protein, to facilitate assembly and budding. Besides these structural roles, N protein associates with a component of the replicase-transcriptase complex, nonstructural protein 3, at a critical early stage of infection. N protein has also been proposed to participate in the replication and selective packaging of genomic RNA and the transcription and translation of subgenomic mRNA. Coronavirus N proteins contain two structurally distinct RNA-binding domains, an unusual characteristic among RNA viruses. To probe the functions of these domains in the N protein of the model coronavirus mouse hepatitis virus (MHV), we constructed mutants in which each RNA-binding domain was replaced by its counterpart from the N protein of severe acute respiratory syndrome coronavirus (SARS-CoV). Mapping of revertants of the resulting chimeric viruses provided evidence for extensive intramolecular interactions between the two RNA-binding domains. Through analysis of viral RNA that was packaged into virions we identified the second of the two RNA-binding domains as a principal determinant of MHV packaging signal recognition. As expected, the interaction of N protein with M protein was not affected in either of the chimeric viruses. Moreover, the SARS-CoV N substitutions did not alter the fidelity of leader-body junction formation during subgenomic mRNA synthesis. These results more clearly delineate the functions of N protein and establish a basis for further exploration of the mechanism of genomic RNA packaging. This work describes the interactions of the two RNA-binding domains of the nucleocapsid protein of a model coronavirus, mouse hepatitis virus. The main finding is that the second of the two domains plays an essential role in recognizing the RNA structure that allows the selective packaging of genomic RNA into assembled virions.

Trichomonas vaginalis vast BspA-like gene family: evidence for functional diversity from structural organisation and transcriptomics

PubMed Central

2010-01-01

Background Trichomonas vaginalis is the most common non-viral human sexually transmitted pathogen and importantly, contributes to facilitating the spread of HIV. Yet very little is known about its surface and secreted proteins mediating interactions with, and permitting the invasion and colonisation of, the host mucosa. Initial annotations of T. vaginalis genome identified a plethora of candidate extracellular proteins. Results Data mining of the T. vaginalis genome identified 911 BspA-like entries (TvBspA) sharing TpLRR-like leucine-rich repeats, which represent the largest gene family encoding potential extracellular proteins for the pathogen. A broad range of microorganisms encoding BspA-like proteins was identified and these are mainly known to live on mucosal surfaces, among these T. vaginalis is endowed with the largest gene family. Over 190 TvBspA proteins with inferred transmembrane domains were characterised by a considerable structural diversity between their TpLRR and other types of repetitive sequences and two subfamilies possessed distinct classic sorting signal motifs for endocytosis. One TvBspA subfamily also shared a glycine-rich protein domain with proteins from Clostridium difficile pathogenic strains and C. difficile phages. Consistent with the hypothesis that TvBspA protein structural diversity implies diverse roles, we demonstrated for several TvBspA genes differential expression at the transcript level in different growth conditions. Identified variants of repetitive segments between several TvBspA paralogues and orthologues from two clinical isolates were also consistent with TpLRR and other repetitive sequences to be functionally important. For one TvBspA protein cell surface expression and antibody responses by both female and male T. vaginalis infected patients were also demonstrated. Conclusions The biased mucosal habitat for microbial species encoding BspA-like proteins, the characterisation of a vast structural diversity for the TvBspA proteins, differential expression of a subset of TvBspA genes and the cellular localisation and immunological data for one TvBspA; all point to the importance of the TvBspA proteins to various aspects of T. vaginalis pathobiology at the host-pathogen interface. PMID:20144183

Thermodynamic Effects of Noncoded and Coded Methionine Substitutions in Calmodulin

PubMed Central

Yamniuk, Aaron P.; Ishida, Hiroaki; Lippert, Dustin; Vogel, Hans J.

2009-01-01

The methionine residues in the calcium (Ca2+) regulatory protein calmodulin (CaM) are structurally and functionally important. They are buried within the N- and C-domains of apo-CaM but become solvent-exposed in Ca2+-CaM, where they interact with numerous target proteins. Previous structural studies have shown that methionine substitutions to the noncoded amino acids selenomethionine, ethionine, or norleucine, or mutation to leucine do not impact the main chain structure of CaM. Here we used differential scanning calorimetry to show that these substitutions enhance the stability of both domains, with the largest increase in melting temperature (19–26°C) achieved with leucine or norleucine in the apo-C-domain. Nuclear magnetic resonance spectroscopy experiments also revealed the loss of a slow conformational exchange process in the Leu-substituted apo-C-domain. In addition, isothermal titration calorimetry experiments revealed considerable changes in the enthalpy and entropy of target binding to apo-CaM and Ca2+-CaM, but the free energy of binding was largely unaffected due to enthalpy-entropy compensation. Collectively, these results demonstrate that noncoded and coded methionine substitutions can be accommodated in CaM because of the structural plasticity of the protein. However, adjustments in side-chain packing and dynamics lead to significant differences in protein stability and the thermodynamics of target binding. PMID:19217866

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

Characterization of a novel glycine-rich protein from the cell wall of maize silk tissues.

PubMed

Tao, T Y; Ouellet, T; Dadej, K; Miller, S S; Johnson, D A; Singh, J

2006-08-01

The isolation, characterization and regulation of expression of a maize silk-specific gene is described. zmgrp5 (Zea mays glycine-rich protein 5) encodes a 187 amino acid glycine-rich protein that displays developmentally regulated silk-specific expression. Northern, Western, in situ mRNA hybridization and transient gene expression analyses indicate that zmgrp5 is expressed in silk hair and in cells of the vascular bundle and pollen tube transmitting tissue elements. The protein is secreted into the extracellular matrix and is localized in the cell wall fraction mainly through interactions mediated by covalent disulphide bridges. Taken together, these results suggest that the protein may play a role in maintaining silk structure during development. This is the first documented isolation of a stigma-specific gene from maize, an important agronomic member of the Poaceae family.

[The identification investigation of the material evidence of biological origin after multifactorial exposure].

PubMed

Iakovlev, D Iu; Solodun, Iu V; Proskurin, V N

1999-01-01

The possibility of investigating pieces of material evidence of biological origin after exposure to various factors is evaluated. The possibility of detecting proteins of liquid media of human organism by electrophoresis in polyacrylamide denatured gel is investigated. The method is intended for identification of biological material in a state of grave destruction. Methodology of such studies is proposed. The data indicate that the structural integrity and qualitative composition of the spectrum of main serum proteins are retained after combined exposure to damaging factors and complete destruction of blood cells.

Biophysical characterization of α-synuclein and its controversial structure

PubMed Central

Alderson, T Reid; Markley, John L

2013-01-01

α-synuclein, a presynaptic protein of poorly defined function, constitutes the main component of Parkinson disease-associated Lewy bodies. Extensive biophysical investigations have provided evidence that isolated α-synuclein is an intrinsically disordered protein (IDP) in vitro. Subsequently serving as a model IDP in numerous studies, α-synuclein has aided in the development of many technologies used to characterize IDPs and arguably represents the most thoroughly analyzed IDP to date. Recent reports, however, have challenged the disordered nature of α-synuclein inside cells and have instead proposed a physiologically relevant helical tetramer. Despite α-synuclein’s rich biophysical history, a single coherent picture has not yet emerged concerning its in vivo structure, dynamics, and physiological role(s). We present herein a review of the biophysical discoveries, developments, and models pertinent to the characterization of α-synuclein’s structure and analysis of the native tetramer controversy. PMID:24634806

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool.

PubMed

Nagy, Gergely N; Leveles, Ibolya; Vértessy, Beáta G

2014-09-01

The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chemical reactions or radiation effects on bases within the DNA structure. Several enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a number of representatives in clear structural and kinetic detail. In this review, we focus in detail on those examples where clear evidence has been produced using high-resolution structural studies. Comparing the protein fold and architecture of the enzyme active sites, two main classes of sanitizing deoxyribonucleoside triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes associated with attack at the α-phosphorus, it is shown that coordination of the γ-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the β-phosphorus atom mainly coordinate the α- and the β-phosphate only. Characteristic differences are also observed with respect to the role of the metal ion cofactor (Mg(2+) ) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution. © 2014 FEBS.

Atomic resolution model of the antibody Fc interaction with the complement C1q component.

PubMed

Schneider, Sebastian; Zacharias, Martin

2012-05-01

The globular C1q heterotrimer is a subunit of the C1 complement factor. Binding of the C1q subunit to the constant (Fc) part of antibody molecules is a first step and key event of complement activation. Although three-dimensional structures of C1q and antibody Fc subunits have been determined experimentally no atomic resolution structure of the C1q-Fc complex is known so far. Based on systematic protein-protein docking searches and Molecular Dynamics simulations a structural model of the C1q-IgG1-Fc-binding geometry has been obtained. The structural model is compatible with available experimental data on the interaction between the two partner proteins. It predicts a binding geometry that involves mainly the B-subunit of the C1q-trimer and both subunits of the IgG1-Fc-dimer with small conformational adjustments with respect to the unbound partners to achieve high surface complementarity. In addition to several charge-charge and polar contacts in the rim region of the interface it also involves nonpolar contacts between the two proteins and is compatible with the carbohydrate moiety of the Fc subunit. The model for the complex structure provides a working model for rationalizing available biochemical data on this important interaction and can form the basis for the design of Fc variants with a greater capacity to activate the complement system for example on binding to cancer cells or other target structures. Copyright © 2012 Elsevier Ltd. All rights reserved.

Abundant RNA editing sites of chloroplast protein-coding genes in Ginkgo biloba and an evolutionary pattern analysis.

PubMed

He, Peng; Huang, Sheng; Xiao, Guanghui; Zhang, Yuzhou; Yu, Jianing

2016-12-01

RNA editing is a posttranscriptional modification process that alters the RNA sequence so that it deviates from the genomic DNA sequence. RNA editing mainly occurs in chloroplasts and mitochondrial genomes, and the number of editing sites varies in terrestrial plants. Why and how RNA editing systems evolved remains a mystery. Ginkgo biloba is one of the oldest seed plants and has an important evolutionary position. Determining the patterns and distribution of RNA editing in the ancient plant provides insights into the evolutionary trend of RNA editing, and helping us to further understand their biological significance. In this paper, we investigated 82 protein-coding genes in the chloroplast genome of G. biloba and identified 255 editing sites, which is the highest number of RNA editing events reported in a gymnosperm. All of the editing sites were C-to-U conversions, which mainly occurred in the second codon position, biased towards to the U_A context, and caused an increase in hydrophobic amino acids. RNA editing could change the secondary structures of 82 proteins, and create or eliminate a transmembrane region in five proteins as determined in silico. Finally, the evolutionary tendencies of RNA editing in different gene groups were estimated using the nonsynonymous-synonymous substitution rate selection mode. The G. biloba chloroplast genome possesses the highest number of RNA editing events reported so far in a seed plant. Most of the RNA editing sites can restore amino acid conservation, increase hydrophobicity, and even influence protein structures. Similar purifying selections constitute the dominant evolutionary force at the editing sites of essential genes, such as the psa, some psb and pet groups, and a positive selection occurred in the editing sites of nonessential genes, such as most ndh and a few psb genes.

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity

PubMed Central

Lai, Julian; Koh, Chuan Hock; Tjota, Monika; Pieuchot, Laurent; Raman, Vignesh; Chandrababu, Karthik Balakrishna; Yang, Daiwen; Wong, Limsoon; Jedd, Gregory

2012-01-01

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity. PMID:22955885

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

NASA Astrophysics Data System (ADS)

Fitter, Jörg

2003-08-01

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

Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

PubMed Central

Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

2017-01-01

Many bacterial pathogens employ the type III secretion system (T3SS) to translocate effector proteins into eukaryotic cells to overcome host defenses. To date, most of our knowledge about the T3SS molecular architecture comes from the studies on animal pathogens. In plant pathogens, nine Hrc proteins are believed to be structural components of the T3SS, of which HrcC and HrcJ form the outer and inner rings of the T3SS, respectively. Here, we demonstrated that a novel outer membrane-bound protein (HpaM) of Xanthomonas campestris pv. campestris is critical for the type III secretion and is structurally and functionally conserved in phytopathogenic Xanthomonas spp. We showed that the C-terminus of HpaM extends into the periplasm to interact physically with HrcJ and the middle part of HpaM interacts physically with HrcC. It is clear that the outer and inner rings compose the main basal body of the T3SS apparatus in animal pathogens. Therefore, we presume that HpaM may act as a T3SS structural component, or play a role in assisting assembling or affecting the stability of the T3SS apparatus. HpaM is a highly prevalent and specific protein in Xanthomonas spp., suggesting that the T3SS of Xanthomonas is distinctive in some aspects from other pathogens. PMID:28198457

Investigation of arc repressor DNA-binding specificity by comparative molecular dynamics simulations.

PubMed

Song, Wei; Guo, Jun-Tao

2015-01-01

Transcription factors regulate gene expression through binding to specific DNA sequences. How transcription factors achieve high binding specificity is still not well understood. In this paper, we investigated the role of protein flexibility in protein-DNA-binding specificity by comparative molecular dynamics (MD) simulations. Protein flexibility has been considered as a key factor in molecular recognition, which is intrinsically a dynamic process involving fine structural fitting between binding components. In this study, we performed comparative MD simulations on wild-type and F10V mutant P22 Arc repressor in both free and complex conformations. The F10V mutant has lower DNA-binding specificity though both the bound and unbound main-chain structures between the wild-type and F10V mutant Arc are highly similar. We found that the DNA-binding motif of wild-type Arc is structurally more flexible than the F10V mutant in the unbound state, especially for the six DNA base-contacting residues in each dimer. We demonstrated that the flexible side chains of wild-type Arc lead to a higher DNA-binding specificity through forming more hydrogen bonds with DNA bases upon binding. Our simulations also showed a possible conformational selection mechanism for Arc-DNA binding. These results indicate the important roles of protein flexibility and dynamic properties in protein-DNA-binding specificity.

The Protein-DNA Interface database

PubMed Central

2010-01-01

The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 Å or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface. We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes. PMID:20482798

The Protein-DNA Interface database.

PubMed

Norambuena, Tomás; Melo, Francisco

2010-05-18

The Protein-DNA Interface database (PDIdb) is a repository containing relevant structural information of Protein-DNA complexes solved by X-ray crystallography and available at the Protein Data Bank. The database includes a simple functional classification of the protein-DNA complexes that consists of three hierarchical levels: Class, Type and Subtype. This classification has been defined and manually curated by humans based on the information gathered from several sources that include PDB, PubMed, CATH, SCOP and COPS. The current version of the database contains only structures with resolution of 2.5 A or higher, accounting for a total of 922 entries. The major aim of this database is to contribute to the understanding of the main rules that underlie the molecular recognition process between DNA and proteins. To this end, the database is focused on each specific atomic interface rather than on the separated binding partners. Therefore, each entry in this database consists of a single and independent protein-DNA interface.We hope that PDIdb will be useful to many researchers working in fields such as the prediction of transcription factor binding sites in DNA, the study of specificity determinants that mediate enzyme recognition events, engineering and design of new DNA binding proteins with distinct binding specificity and affinity, among others. Finally, due to its friendly and easy-to-use web interface, we hope that PDIdb will also serve educational and teaching purposes.

Caveolae, caveolin-1 and cavin-1: Emerging roles in pulmonary hypertension.

PubMed

Chettimada, Sukrutha; Yang, Jincheng; Moon, Hyung-Geun; Jin, Yang

2015-07-28

Caveolae are flask-shaped invaginations of cell membrane that play a significant structural and functional role. Caveolae harbor a variety of signaling molecules and serve to receive, concentrate and transmit extracellular signals across the membrane. Caveolins are the main structural proteins residing in the caveolae. Caveolins and another category of newly identified caveolae regulatory proteins, named cavins, are not only responsible for caveolae formation, but also interact with signaling complexes in the caveolae and regulate transmission of signals across the membrane. In the lung, two of the three caveolin isoforms, i.e ., cav-1 and -2, are expressed ubiquitously. Cavin protein family is composed of four proteins, named cavin-1 (or PTRF for polymerase Ⅰ and transcript release factor), cavin-2 (or SDPR for serum deprivation protein response), cavin-3 (or SRBC for sdr-related gene product that binds to-c-kinase) and cavin-4 (or MURC for muscle restricted coiled-coiled protein or cavin-4). All the caveolin and cavin proteins are essential regulators for caveolae dynamics. Recently, emerging evidence suggest that caveolae and its associated proteins play crucial roles in development and progression of pulmonary hypertension. The focus of this review is to outline and discuss the contrast in alteration of cav-1 (cav-1),-2 and cavin-1 (PTRF) expression and downstream signaling mechanisms between human and experimental models of pulmonary hypertension.

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

PubMed Central

Wolfram, Stefanie; Wielsch, Natalie; Hupfer, Yvonne; Mönch, Bettina; Lu-Walther, Hui-Wen; Heintzmann, Rainer; Werz, Oliver; Svatoš, Aleš; Pohnert, Georg

2015-01-01

Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways. PMID:26496085

Proteomic analysis of the crayfish gastrolith chitinous extracellular matrix reveals putative protein complexes and a central role for GAP 65.

PubMed

Glazer, Lilah; Roth, Ziv; Weil, Simy; Aflalo, Eliahu D; Khalaila, Isam; Sagi, Amir

2015-10-14

Chitin is a major component of arthropod cuticles, where it forms a three-dimensional network that constitutes the scaffold upon which cuticles form. The chitin fibers that form this network are closely associated with specific structural proteins, while the cuticular matrix contains many additional structural, enzymatic and other proteins. We study the crayfish gastrolith as a simple model for the assembly of calcified cuticular structures, with particular focus on the proteins involved in this process. The present study integrates a gastrolith-forming epithelium transcriptomic library with data from mass spectrometry analysis of proteins extracted from the gastrolith matrix to obtain a near-complete picture of gastrolith protein content. Using native protein separation we identified 24 matrix proteins, of which 14 are novel. Further analysis led to discovery of three putative protein complexes, all containing GAP 65 the most abundant gastrolith structural protein. Using immunological methods we further studied the role of GAP 65 in the gastrolith matrix and forming epithelium, as well as in the newly identified protein complexes. We propose that gastrolith matrix construction is a sequential process in which protein complexes are dynamically assembled and disassembled around GAP 65, thus changing their functional properties to perform each step in the construction process. The scientific interest on which this study is based arises from three main features of gastroliths: (1) Gastroliths possess partial analogy to cuticles both in structural and molecular properties, and may be regarded, with the appropriate reservations (see Introduction), as simple models for cuticle assembly. At the same time, gastroliths are terminally assembled during a well-defined period, which can be controlled in the laboratory, making them significantly easier to study than cuticles. (2) Gastroliths, like the crayfish exoskeleton, contain stable amorphous calcium carbonate (ACC) rather than crystalline calcite. The biological mechanism for the stabilization of a naturally unstable, but at the same time biologically highly available, calcium carbonate polymorph is of great interest from the pharmaceutical point of view. (3) The gastrolith organic matrix is based on a highly structured chitin network that interacts with a variety of substances. This biologically manipulated, biodegradable structure is in itself of biotechnological and pharmaceutical potential. A growing body of evidence indicates that proteins play central roles in all above aspects of gastrolith construction. This study offers the first comprehensive screening of gastrolith proteins, and we believe that the analysis presented in this work can not only help reveal basic biological questions regarding assembly of mineralized and non-mineralized cuticular structures, but may also serve as basis for applied research in the fields of agriculture (e.g. cuticle-based pest management), health (e.g. bioavailable calcium supplements and biodegradable drug carriers) and materials science (e.g. non-toxic scaffolds for water purification). Copyright © 2015. Published by Elsevier B.V.

Modeling and experimental assessment of a buried Leu–Ile mutation in dengue envelope domain III

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

Kulkarni, Manjiri R.; Numoto, Nobutaka; Ito, Nobutoshi

Envelope protein domain III (ED3) of the dengue virus is important for both antibody binding and host cell interaction. Here, we focused on how a L387I mutation in the protein core could take place in DEN4 ED3, but cannot be accommodated in DEN3 ED3 without destabilizing its structure. To this end, we modeled a DEN4-L387I structure using the Penultimate Rotamer Library and taking the DEN4 ED3 main-chain as a fixed template. We found that three out of seven Ile{sup 387} conformers fit in DEN4 ED3 without introducing the severe atomic clashes that are observed when DEN3 serotype’s ED3 is usedmore » as a template. A more extensive search using 273 side-chain rotamers of the residues surrounding Ile{sup 387} confirmed this prediction. In order to assess the prediction, we determined the crystal structure of DEN4-L387I at 2 Å resolution. Ile{sup 387} indeed adopted one of the three predicted rotamers. Altogether, this study demonstrates that the effects of single mutations are to a large extent successfully predicted by systematically modeling the side-chain structures of the mutated as well as those of its surrounding residues using fixed main-chain structures and assessing inter-atomic steric clashes. More accurate and reliable predictions require considering sub-angstrom main-chain deformation, which remains a challenging task. - Highlights: • We mutated L387I of DEN4 ED3 and examined its effects on structure and stability. • We modeled the side-chain of Ile{sup 387} using DEN4 ED3's structure as a template. • We determined the crystal structure of DEN4-L387I and confirmed the modeling. • Side-chain repacking occurring around Ile{sup 387} involved >3 inter-connected residues. • These results explained why L387I mutation in DEN4 ED3 conserves thermostability.« less

FTIR studies of gluten matrix dehydration after fibre polysaccharide addition.

PubMed

Nawrocka, Agnieszka; Krekora, Magdalena; Niewiadomski, Zbigniew; Miś, Antoni

2018-06-30

FTIR spectroscopy was used to determine changes in secondary structure, as well as water state, in gluten and model doughs supplemented by four fibre polysaccharides (microcrystalline cellulose, inulin, apple pectin and citrus pectin). The gluten and model doughs were obtained from commercially available wheat gluten and model flour, respectively. The polysaccharides were used in five concentrations: 3%, 6%, 9%, 12% and 18%. Analysis of the FTIR spectra indicated that polysaccharides could be divided into two groups: first - microcrystalline cellulose and inulin, second - apple and citrus pectins that induced opposite structural changes. Changes in secondary structure concern mainly β-sheets and β-turns that form aggregated β-structures, suggesting dehydration of the gluten matrix as a result of competition for water between gluten proteins and polysaccharides. Moreover, the positive band at ca. 1226 cm -1 in the spectra of pectin-modified samples indicates formation of 'ether' type hydrogen bonds between gluten proteins and pectins. Copyright © 2018 Elsevier Ltd. All rights reserved.

The RCSB Protein Data Bank: new resources for research and education

PubMed Central

Rose, Peter W.; Bi, Chunxiao; Bluhm, Wolfgang F.; Christie, Cole H.; Dimitropoulos, Dimitris; Dutta, Shuchismita; Green, Rachel K.; Goodsell, David S.; Prlić, Andreas; Quesada, Martha; Quinn, Gregory B.; Ramos, Alexander G.; Westbrook, John D.; Young, Jasmine; Zardecki, Christine; Berman, Helen M.; Bourne, Philip E.

2013-01-01

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) develops tools and resources that provide a structural view of biology for research and education. The RCSB PDB web site (http://www.rcsb.org) uses the curated 3D macromolecular data contained in the PDB archive to offer unique methods to access, report and visualize data. Recent activities have focused on improving methods for simple and complex searches of PDB data, creating specialized access to chemical component data and providing domain-based structural alignments. New educational resources are offered at the PDB-101 educational view of the main web site such as Author Profiles that display a researcher’s PDB entries in a timeline. To promote different kinds of access to the RCSB PDB, Web Services have been expanded, and an RCSB PDB Mobile application for the iPhone/iPad has been released. These improvements enable new opportunities for analyzing and understanding structure data. PMID:23193259

Thermal transitions in serum amyloid A in solution and on the lipid: implications for structure and stability of acute-phase HDL[S

PubMed Central

Jayaraman, Shobini; Haupt, Christian; Gursky, Olga

2015-01-01

Serum amyloid A (SAA) is an acute-phase protein that circulates mainly on plasma HDL. SAA interactions with its functional ligands and its pathogenic deposition in reactive amyloidosis depend, in part, on the structural disorder of this protein and its propensity to oligomerize. In vivo, SAA can displace a substantial fraction of the major HDL protein, apoA-I, and thereby influence the structural remodeling and functions of acute-phase HDL in ways that are incompletely understood. We use murine SAA1.1 to report the first structural stability study of human plasma HDL that has been enriched with SAA. Calorimetric and spectroscopic analyses of these and other SAA-lipid systems reveal two surprising findings. First, progressive displacement of the exchangeable fraction of apoA-I by SAA has little effect on the structural stability of HDL and its fusion and release of core lipids. Consequently, the major determinant for HDL stability is the nonexchangeable apoA-I. A structural model explaining this observation is proposed, which is consistent with functional studies in acute-phase HDL. Second, we report an α-helix folding/unfolding transition in SAA in the presence of lipid at near-physiological temperatures. This new transition may have potentially important implications for normal functions of SAA and its pathogenic misfolding. PMID:26022803

The Origin and Early Evolution of Membrane Proteins

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

2006-01-01

The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

ZrO2 nanoparticles labeled via a native protein corona: detection by fluorescence microscopy and Raman microspectroscopy in rat lungs.

PubMed

Silge, Anja; Bräutigam, Katharina; Bocklitz, Thomas; Rösch, Petra; Vennemann, Antje; Schmitz, Inge; Popp, Jürgen; Wiemann, Martin

2015-08-07

ZrO2 nanoparticles are frequently used in composite materials such as dental fillers from where they may be released and inhaled upon polishing and grinding. Since the overall distribution of ZrO2 NP inside the lung parenchyma can hardly be observed by routine histology, here a labeling with a fluorphore was used secondary to the adsorption of serum proteins. Particles were then intratracheally instilled into rat lungs. After 3 h fluorescent structures consisted of agglomerates scattered throughout the lung parenchyma, which were mainly concentrated in alveolar macrophages after 3 d. A detection method based on Raman microspectroscopy was established to investigate the chemical composition of those fluorescent structures in detail. Raman measurements were arranged such that no spectral interference with the protein-bound fluorescence label was evident. Applying chemometrical methods, Raman signals of the ZrO2 nanomaterial were co-localized with the fluorescence label, indicating the stability of the nanomaterial-protein-dye complex inside the rat lung. The combination of Raman microspectroscopy and adsorptive fluorescence labeling may, therefore, become a useful tool for studying the localization of protein-coated nanomaterials in cells and tissues.

Structure and function of matrix proteins and peptides in the biomineral formation in crustaceans.

PubMed

Nagasawa, Hiromichi

2011-01-01

Crustaceans have hard cuticle with layered structure, which is composed mainly of chitin, proteins, and calcium carbonate. Crustaceans grow by shedding the old cuticle and replacing it with a new one. Decalcification in the cuticle during the pre-molt stage and concomitant calcification in the stomach to form gastroliths observed in some crustacean species are triggered by the molting hormone. Various proteins and peptides have been identified from calcified cuticle and gastroliths, and their functions have been examined in terms of calcification and interaction with chitin. Acidic nature of matrix proteins is important for recruitment of calcium ions and interaction with calcium carbonate. Examination of the relationship between amino acid sequence containing acidic amino acid residues and calcification inhibitory activity revealed that the potency did not depend on the sequence but on the number of acidic amino acid residues. Calcium carbonate in the calcified tissues of crustaceans is amorphous in many cases. Crustaceans take a strategy to induce and maintain amorphous calcium carbonate by using low-molecular-weight phosphorus compounds.

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.

The Cell Wall-Associated Proteins in the Dimorphic Pathogenic Species of Paracoccidioides.

PubMed

Puccia, Rosana; Vallejo, Milene C; Longo, Larissa V G

2017-01-01

Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis (PCM). They are dimorphic ascomycetes that grow as filaments at mild temperatures up to 28oC and as multibudding pathogenic yeast cells at 37oC. Components of the fungal cell wall have an important role in the interaction with the host because they compose the cell outermost layer. The Paracoccidioides cell wall is composed mainly of polysaccharides, but it also contains proportionally smaller rates of proteins, lipids, and melanin. The polysaccharide cell wall composition and structure of Paracoccidioides yeast cells, filamentous and transition phases were studied in detail in the past. Other cell wall components have been better analyzed in the last decades. The present work gives to the readers a detailed updated view of cell wall-associated proteins. Proteins that have been localized at the cell wall compartment using antibodies are individually addressed. We also make an overview about PCM, the Paracoccidioides cell wall structure, secretion mechanisms, and fungal extracellular vesicles. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Identification of a conserved neutralizing linear B-cell epitope in the VP1 proteins of duck hepatitis A virus type 1 and 3.

PubMed

Zhang, Ruihua; Zhou, Guomei; Xin, Yinghao; Chen, Junhao; Lin, Shaoli; Tian, Ye; Xie, Zhijing; Jiang, Shijin

2015-11-18

Duck virus hepatitis (DVH), mainly caused by duck hepatitis A virus (DHAV), is a severe disease threaten to duck industry and has worldwide distribution. As the major structural protein, the VP1 protein of DHAV is able to induce neutralizing antibody in ducks. In this study, a monoclonal antibody (mAb) 4F8 against the intact DHAV-1 particles was used to identify the possible epitope in the three serotypes of DHAV. The mAb 4F8 had weak neutralizing activities to both DHAV-1 and DHAV-3, and reacted with the conserved linear B-cell epitopes of (75)GEIILT(80) in DHAV-1 VP1 and (75)GEVILT(80) in DHAV-3 VP1 protein, respectively, while not with DHAV-2 VP1. This was the first report about identification of the common conserved neutralizing linear B-cell epitope of DHAV-1 and DHAV-3, which will facilitate understanding of the antigenic structure of VP1 and the serologic diagnosis of DHAV infection. Copyright © 2015 Elsevier B.V. All rights reserved.

Visualisation of Collagen in fixed skeletal muscle tissue using fluorescently tagged Collagen binding protein CNA35.

PubMed

Mohammadkhah, Melika; Simms, Ciaran K; Murphy, Paula

2017-02-01

Detection and visualisation of Collagen structure are important to understand the relationship between mechanical behaviour and microstructure in skeletal muscle since Collagen is the main structural protein in animal connective tissues, and is primarily responsible for their passive load-bearing properties. In the current study, the direct detection and visualization of Collagen using fluorescently tagged CNA35 binding protein (fused to EGFP or tdTomato) is reported for the first time on fixed skeletal muscle tissue. This Technical Note also establishes a working protocol by examining tissue preparation, dilution factor, exposure time etc. for sensitivity and specificity. Penetration of the binding protein into intact mature skeletal muscle was found to be very limited, but detection works well on tissue sections with higher sensitivity on wax embedded sections compared to frozen sections. CNA35 fused to tdTomato has a higher sensitivity than CNA35 fused to EGFP but both show specific detection. Best results were obtained with 15μm wax embedded sections, with blocking of non-specific binding in 1% BSA and antigen retrieval in Sodium Citrate. There was a play-off between dilution of the binding protein and time of incubation but both CNA35-tdTomato and CNA35-EGFP worked well with approximately 100μg/ml of purified protein with overnight incubation, while CNA35-tdTomato could be utilized at 5 fold less concentration. This approach can be applied to study the relationship between skeletal muscle micro-structure and to observe mechanical response to applied deformation. It can be used more broadly to detect Collagen in a variety of fixed tissues, useful for structure-functions studies, constitutive modelling, tissue engineering and assessment of muscle tissue pathologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Correlation of cell surface proteins of distinct Beauveria bassiana cell types and adaption to varied environment and interaction with the host insect.

PubMed

Yang, Zhi; Jiang, Hongyan; Zhao, Xin; Lu, Zhuoyue; Luo, Zhibing; Li, Xuebing; Zhao, Jing; Zhang, Yongjun

2017-02-01

The insect fungal pathogen Beauveria bassiana produces a number of distinct cell types that include aerial conidia, blastospores and haemolymph-derived cells, termed hyphal bodies, to adapt varied environment niches and within the host insect. These cells display distinct biochemical properties and surface structures, and a highly ordered outermost brush-like structure uniquely present on hyphal bodies, but not on any in vitro cells. Here, we found that the outermost structure on the hyphal bodies mainly consisted of proteins associated to structural wall components in that most of it could be removed by dithiothreitol (DTT) or proteinase K. DTT-treatment also caused delayed germination, decreased tolerance to ultraviolet irradiation and virulence of conidia or blastospores, with decreased adherence and alternated carbohydrate epitopes, suggesting involvement in fungal development, stress responses and virulence. To characterize these cell surface molecules, proteins were released from the living cells using DTT, and identified and quantitated using label-free quantitative mass spectrometry. Thereafter, a series of bioinformatics programs were used to predict cell surface-associated proteins (CSAPs), and 96, 166 and 54 CSAPs were predicted from the identified protein pools of conidia, blastospores and hyphal bodies, respectively, which were involved in utilization of carbohydrate, nitrogen, and lipid, detoxification, pathogen-host interaction, and likely other cellular processes. Thirteen, sixty-nine and six CSAPs were exclusive in conidia, blastospores and hyphal bodies, respectively, which were verified by eGFP-tagged proteins at their N-terminus. Our data provide a crucial cue to understand mechanism of B. bassiana to adapt to varied environment and interaction with insect host. Copyright © 2016 Elsevier Inc. All rights reserved.

Anchoring protein crystals to mounting loops with hydrogel using inkjet technology.

PubMed

Shinoda, Akira; Tanaka, Yoshikazu; Yao, Min; Tanaka, Isao

2014-11-01

X-ray crystallography is an important technique for structure-based drug discovery, mainly because it is the only technique that can reveal whether a ligand binds to the target protein as well as where and how it binds. However, ligand screening by X-ray crystallography involves a crystal-soaking experiment, which is usually performed manually. Thus, the throughput is not satisfactory for screening large numbers of candidate ligands. In this study, a technique to anchor protein crystals to mounting loops by using gel and inkjet technology has been developed; the method allows soaking of the mounted crystals in ligand-containing solution. This new technique may assist in the design of a fully automated drug-screening pipeline.

Structural basis of cargo recognitions for class V myosins

PubMed Central

Wei, Zhiyi; Liu, Xiaotian; Yu, Cong; Zhang, Mingjie

2013-01-01

Class V myosins (MyoV), the most studied unconventional myosins, recognize numerous cargos mainly via the motor’s globular tail domain (GTD). Little is known regarding how MyoV-GTD recognizes such a diverse array of cargos specifically. Here, we solved the crystal structures of MyoVa-GTD in its apo-form and in complex with two distinct cargos, melanophilin and Rab interacting lysosomal protein-like 2. The apo-MyoVa-GTD structure indicates that most mutations found in patients with Griscelli syndrome, microvillus inclusion disease, or cancers or in “dilute” rodents likely impair the folding of GTD. The MyoVa-GTD/cargo complex structure reveals two distinct cargo-binding surfaces, one primarily via charge–charge interaction and the other mainly via hydrophobic interactions. Structural and biochemical analysis reveal the specific cargo-binding specificities of various isoforms of mammalian MyoV as well as very different cargo recognition mechanisms of MyoV between yeast and higher eukaryotes. The MyoVa-GTD structures resolved here provide a framework for future functional studies of vertebrate class V myosins. PMID:23798443

Identification of DNA-Binding Proteins Using Mixed Feature Representation Methods.

PubMed

Qu, Kaiyang; Han, Ke; Wu, Song; Wang, Guohua; Wei, Leyi

2017-09-22

DNA-binding proteins play vital roles in cellular processes, such as DNA packaging, replication, transcription, regulation, and other DNA-associated activities. The current main prediction method is based on machine learning, and its accuracy mainly depends on the features extraction method. Therefore, using an efficient feature representation method is important to enhance the classification accuracy. However, existing feature representation methods cannot efficiently distinguish DNA-binding proteins from non-DNA-binding proteins. In this paper, a multi-feature representation method, which combines three feature representation methods, namely, K-Skip-N-Grams, Information theory, and Sequential and structural features (SSF), is used to represent the protein sequences and improve feature representation ability. In addition, the classifier is a support vector machine. The mixed-feature representation method is evaluated using 10-fold cross-validation and a test set. Feature vectors, which are obtained from a combination of three feature extractions, show the best performance in 10-fold cross-validation both under non-dimensional reduction and dimensional reduction by max-relevance-max-distance. Moreover, the reduced mixed feature method performs better than the non-reduced mixed feature technique. The feature vectors, which are a combination of SSF and K-Skip-N-Grams, show the best performance in the test set. Among these methods, mixed features exhibit superiority over the single features.

The EBI search engine: EBI search as a service-making biological data accessible for all.

PubMed

Park, Young M; Squizzato, Silvano; Buso, Nicola; Gur, Tamer; Lopez, Rodrigo

2017-07-03

We present an update of the EBI Search engine, an easy-to-use fast text search and indexing system with powerful data navigation and retrieval capabilities. The interconnectivity that exists between data resources at EMBL-EBI provides easy, quick and precise navigation and a better understanding of the relationship between different data types that include nucleotide and protein sequences, genes, gene products, proteins, protein domains, protein families, enzymes and macromolecular structures, as well as the life science literature. EBI Search provides a powerful RESTful API that enables its integration into third-party portals, thus providing 'Search as a Service' capabilities, which are the main topic of this article. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

PubMed

Delaforge, Elise; Kragelj, Jaka; Tengo, Laura; Palencia, Andrés; Milles, Sigrid; Bouvignies, Guillaume; Salvi, Nicola; Blackledge, Martin; Jensen, Malene Ringkjøbing

2018-01-24

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R 1ρ , Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

Competitive folding of anti-terminator/terminator hairpins monitored by single molecule FRET.

PubMed

Clerte, Caroline; Declerck, Nathalie; Margeat, Emmanuel

2013-02-01

The control of transcription termination by RNA-binding proteins that modulate RNA-structures is an important regulatory mechanism in bacteria. LicT and SacY from Bacillus subtilis prevent the premature arrest of transcription by binding to an anti-terminator RNA hairpin that overlaps an intrinsic terminator located in the 5'-mRNA leader region of the gene to be regulated. In order to investigate the molecular determinants of this anti-termination/termination balance, we have developed a fluorescence-based nucleic acids system that mimics the competition between the LicT or SacY anti-terminator targets and the overlapping terminators. Using Förster Resonance Energy Transfer on single diffusing RNA hairpins, we could monitor directly their opening or closing state, and thus investigate the effects on this equilibrium of the binding of anti-termination proteins or terminator-mimicking oligonucleotides. We show that the anti-terminator hairpins adopt spontaneously a closed structure and that their structural dynamics is mainly governed by the length of their basal stem. The induced stability of the anti-terminator hairpins determines both the affinity and specificity of the anti-termination protein binding. Finally, we show that stabilization of the anti-terminator hairpin, by an extended basal stem or anti-termination protein binding can efficiently counteract the competing effect of the terminator-mimic.

In Situ FTIR Microspectroscopy of Brain Tissue from a Transgenic Mouse Model of Alzheimer Disease

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

Rak,M.; Del Bigio, M.; Mai, S.

2007-01-01

Plaques composed of the A{beta} peptide are the main pathological feature of Alzheimer's disease. Dense-core plaques are fibrillar deposits of A{beta}, showing all the classical properties of amyloid including {beta}-sheet secondary structure, while diffuse plaques are amorphous deposits. We studied both plaque types, using synchrotron infrared (IR) microspectroscopy, a technique that allows the chemical composition and average protein secondary structure to be investigated in situ. We examined plaques in hippocampal, cortical and caudal tissue from 5- to 21-month-old TgCRND8 mice, a transgenic model expressing doubly mutant amyloid precursor protein, and displaying impaired hippocampal function and robust pathology from an earlymore » age. Spectral analysis confirmed that the congophilic plaque cores were composed of protein in a {beta}-sheet conformation. The amide I maximum of plaque cores was at 1623 cm-1, and unlike for in vitro A{beta} fibrils, the high-frequency (1680-1690 cm-1) component attributed to antiparallel {beta}-sheet was not observed. A significant elevation in phospholipids was found around dense-core plaques in TgCRND8 mice ranging in age from 5 to 21 months. In contrast, diffuse plaques were not associated with IR detectable changes in protein secondary structure or relative concentrations of any other tissue components.« less

Microcystin-LR and Cylindrospermopsin Induced Alterations in Chromatin Organization of Plant Cells

PubMed Central

Máthé, Csaba; M-Hamvas, Márta; Vasas, Gábor

2013-01-01

Cyanobacteria produce metabolites with diverse bioactivities, structures and pharmacological properties. The effects of microcystins (MCYs), a family of peptide type protein-phosphatase inhibitors and cylindrospermopsin (CYN), an alkaloid type of protein synthesis blocker will be discussed in this review. We are focusing mainly on cyanotoxin-induced changes of chromatin organization and their possible cellular mechanisms. The particularities of plant cells explain the importance of such studies. Preprophase bands (PPBs) are premitotic cytoskeletal structures important in the determination of plant cell division plane. Phragmoplasts are cytoskeletal structures involved in plant cytokinesis. Both cyanotoxins induce the formation of multipolar spindles and disrupted phragmoplasts, leading to abnormal sister chromatid segregation during mitosis. Thus, MCY and CYN are probably inducing alterations of chromosome number. MCY induces programmed cell death: chromatin condensation, nucleus fragmentation, necrosis, alterations of nuclease and protease enzyme activities and patterns. The above effects may be related to elevated reactive oxygen species (ROS) and/or disfunctioning of microtubule associated proteins. Specific effects: MCY-LR induces histone H3 hyperphosphorylation leading to incomplete chromatid segregation and the formation of micronuclei. CYN induces the formation of split or double PPB directly related to protein synthesis inhibition. Cyanotoxins are powerful tools in the study of plant cell organization. PMID:24084787

Functional inclusion bodies produced in the yeast Pichia pastoris.

PubMed

Rueda, Fabián; Gasser, Brigitte; Sánchez-Chardi, Alejandro; Roldán, Mònica; Villegas, Sandra; Puxbaum, Verena; Ferrer-Miralles, Neus; Unzueta, Ugutz; Vázquez, Esther; Garcia-Fruitós, Elena; Mattanovich, Diethard; Villaverde, Antonio

2016-10-01

Bacterial inclusion bodies (IBs) are non-toxic protein aggregates commonly produced in recombinant bacteria. They are formed by a mixture of highly stable amyloid-like fibrils and releasable protein species with a significant extent of secondary structure, and are often functional. As nano structured materials, they are gaining biomedical interest because of the combination of submicron size, mechanical stability and biological activity, together with their ability to interact with mammalian cell membranes for subsequent cell penetration in absence of toxicity. Since essentially any protein species can be obtained as IBs, these entities, as well as related protein clusters (e.g., aggresomes), are being explored in biocatalysis and in biomedicine as mechanically stable sources of functional protein. One of the major bottlenecks for uses of IBs in biological interfaces is their potential contamination with endotoxins from producing bacteria. To overcome this hurdle, we have explored here the controlled production of functional IBs in the yeast Pichia pastoris (Komagataella spp.), an endotoxin-free host system for recombinant protein production, and determined the main physicochemical and biological traits of these materials. Quantitative and qualitative approaches clearly indicate the formation of IBs inside yeast, similar in morphology, size and biological activity to those produced in E. coli, that once purified, interact with mammalian cell membranes and penetrate cultured mammalian cells in absence of toxicity. Structurally and functionally similar from those produced in E. coli, the controlled production of IBs in P. pastoris demonstrates that yeasts can be used as convenient platforms for the biological fabrication of self-organizing protein materials in absence of potential endotoxin contamination and with additional advantages regarding, among others, post-translational modifications often required for protein functionality.

Sequentially distant but structurally similar proteins exhibit fold specific patterns based on their biophysical properties.

PubMed

Rajendran, Senthilnathan; Jothi, Arunachalam

2018-05-16

The Three-dimensional structure of a protein depends on the interaction between their amino acid residues. These interactions are in turn influenced by various biophysical properties of the amino acids. There are several examples of proteins that share the same fold but are very dissimilar at the sequence level. For proteins to share a common fold some crucial interactions should be maintained despite insignificant sequence similarity. Since the interactions are because of the biophysical properties of the amino acids, we should be able to detect descriptive patterns for folds at such a property level. In this line, the main focus of our research is to analyze such proteins and to characterize them in terms of their biophysical properties. Protein structures with sequence similarity lesser than 40% were selected for ten different subfolds from three different mainfolds (according to CATH classification) and were used for this analysis. We used the normalized values of the 49 physio-chemical, energetic and conformational properties of amino acids. We characterize the folds based on the average biophysical property values. We also observed a fold specific correlational behavior of biophysical properties despite a very low sequence similarity in our data. We further trained three different binary classification models (Naive Bayes-NB, Support Vector Machines-SVM and Bayesian Generalized Linear Model-BGLM) which could discriminate mainfold based on the biophysical properties. We also show that among the three generated models, the BGLM classifier model was able to discriminate protein sequences coming under all beta category with 81.43% accuracy and all alpha, alpha-beta proteins with 83.37% accuracy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis

PubMed Central

Bel, Yolanda; Banyuls, Núria; Chakroun, Maissa; Escriche, Baltasar; Ferré, Juan

2017-01-01

Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices. PMID:28387713

On the molecular interaction between lactoferrin and the dye Red HE-3B. A novel approach for docking a charged and highly flexible molecule to protein surfaces

NASA Astrophysics Data System (ADS)

Grasselli, Mariano; Cascone, Osvaldo; Anspach, F. Birger; Delfino, Jose M.

2002-12-01

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.

On the molecular interaction between lactoferrin and the dye Red HE-3b. A novel approach for docking a charged and highly flexible molecule to protein surfaces.

PubMed

Grasselli, Mariano; Cascone, Osvaldo; Birger Anspach, F; Delfino, Jose M

2002-12-01

Lactoferrin (Lf) is a non-heme, iron binding protein present in many physiological fluids of vertebrates where its main role is the microbicidal activity. It has been isolated by different methods, including dye-affinity chromatography. Red HE-3B is one of the most common triazinic dyes applied in protein purification, but scant knowledge is available on structural details and on the energetics of its interaction with proteins. In this work we present a computational approach useful for identifying possible binding sites for Red HE-3B in apo and holo forms of Lfs from human and bovine source. A new geometrical description of Red HE-3B is introduced which greatly simplifies the conformational analysis. This approach proved to be of particular advantage for addressing conformational ensembles of highly flexible molecules. Predictions from this analysis were correlated with experimentally observed dye-binding sites, as mapped by protection from proteolysis in Red HE-3B/Lf complexes. This method could bear relevance for the screening of possible dye-binding sites in proteins whose structure is known and as a potential tool for the design of engineered protein variants which could be purified by dye-affinity chromatography.

Interactions between avidin and graphene for development of a biosensing platform.

PubMed

Macwan, Isaac; Khan, Md Daud Hossain; Aphale, Ashish; Singh, Shrishti; Liu, Juan; Hingorani, Manju; Patra, Prabir

2017-03-15

Fundamental understanding of interactions at the interface of biological molecules, such as proteins, and nanomaterials is crucial for developing various biocompatible hybrid materials and biosensing platforms. Biosensors comprising of graphene-based conductive nanomaterials offer the advantage of higher sensitivity and reliable diagnosis mainly due to their superior specific surface area and ballistic conductivity. Furthermore, conductive nanocomposite structures that immobilize proteins can synergize the properties of both transducers and molecular recognition elements improving the performance of the biosensing device. Here we report for the first time, using a combined molecular dynamics simulations and experimental approach, the interactions between avidin and graphene for the development of a sensing platform that can be used for the detection of biological macromolecules such as mismatch repair proteins through biotinylated DNA substrates. We find that the interactive forces between avidin and graphene are mainly hydrophobic, along with some van der Waals, electrostatic and hydrogen bonding interactions. Notably, the structure and function of the avidin molecule are largely preserved after its adsorption on the graphene surface. The MD results agree well with scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) analysis of avidin immobilized on a graphenated polypyrrole (G-PPy) conductive nanocomposite confirming the adsorption of avidin on graphene nanoplatelets as observed from the Fourier-transform infrared spectroscopy (FTIR). Copyright © 2016 Elsevier B.V. All rights reserved.

The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein

PubMed Central

Nepravishta, Ridvan; Mandaliti, Walter; Melino, Sonia; Margon, Alja; Scaini, Denis; Mazzei, Pierluigi; Piccolo, Alessandro; Legname, Giuseppe; Paci, Maurizio; Leita, Liviana

2017-01-01

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment. PMID:29161325

Seasonal carcass composition and energy balance of female black ducks in Maine

USGS Publications Warehouse

Reinecke, K.J.; Stone, T.L.; Owen, R.B.

1982-01-01

Female Black Ducks (Anas rubripes) collected in Maine during the summer, fall, and winter of 1974-1976 showed significant seasonal variation in body weight, nonfat dry weight, gizzard and pectoral muscle weight, and fat, moisture, and protein content. Variation of body weight within and among seasons was correlated more strongly with carcass protein content, and with fat content during seasons of heavy lipid deposition, than with three structural size variables (culmen, tarsus, and sternum). Regression equations including fat and protein as independent variables accounted for 80-90% of the annual and seasonal variation in body weight; structural size variables alone accounted for less than 30%. Immature females averaged 54 and 99 g lighter, and carried 54 and 59 g less fat than adults during the fall and winter. Ducks of both age classes lost weight in December and January. Adult and immature females metabolized 59 and 64 g of fat and 17 and 25 g of protein in winter compared with 46 g of fat during the nesting season. Nutrient reserves are thus equally as important for the winter survival of these birds as for successfurl eproduction. Seasonal changes in carcass composition suggest that (1) fat deposited in late fall provides an energy reserve during winter, (2) a reduction in lean weight during winter may lower daily energy requirements and increase the effective amount of energy reserves, and (3) declining body weights during late winter may be an endogenous rhythm that reflects a shift in the expected benefits of an energy reserve compared to the costs of carrying additional weight,

Protein tyrosine nitration in plants: Present knowledge, computational prediction and future perspectives.

PubMed

Kolbert, Zsuzsanna; Feigl, Gábor; Bordé, Ádám; Molnár, Árpád; Erdei, László

2017-04-01

Nitric oxide (NO) and related molecules (reactive nitrogen species) regulate diverse physiological processes mainly through posttranslational modifications such as protein tyrosine nitration (PTN). PTN is a covalent and specific modification of tyrosine (Tyr) residues resulting in altered protein structure and function. In the last decade, great efforts have been made to reveal candidate proteins, target Tyr residues and functional consequences of nitration in plants. This review intends to evaluate the accumulated knowledge about the biochemical mechanism, the structural and functional consequences and the selectivity of plants' protein nitration and also about the decomposition or conversion of nitrated proteins. At the same time, this review emphasizes yet unanswered or uncertain questions such as the reversibility/irreversibility of tyrosine nitration, the involvement of proteasomes in the removal of nitrated proteins or the effect of nitration on Tyr phosphorylation. The different NO producing systems of algae and higher plants raise the possibility of diversely regulated protein nitration. Therefore studying PTN from an evolutionary point of view would enrich our present understanding with novel aspects. Plant proteomic research can be promoted by the application of computational prediction tools such as GPS-YNO 2 and iNitro-Tyr software. Using the reference Arabidopsis proteome, Authors performed in silico analysis of tyrosine nitration in order to characterize plant tyrosine nitroproteome. Nevertheless, based on the common results of the present prediction and previous experiments the most likely nitrated proteins were selected thus recommending candidates for detailed future research. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Fabrication of Protein Microparticles and Microcapsules with Biomolecular Tools

NASA Astrophysics Data System (ADS)

Cheung, Kwan Yee; Lai, Kwok Kei; Mak, Wing Cheung

2018-05-01

Microparticles have attracted much attention for medical, analytical and biological applications. Calcium carbonate (CaCO3) templating method with the advantages of having narrow size distribution, controlled morphology and good biocompatibility that has been widely used for the synthesis of various protein-based microparticles. Despite CaCO3 template is biocompatible, most of the conventional methods to create stable protein microparticles are mainly driven by chemical crosslink reagents which may induce potential harmful effect and remains undesirable especially for biomedical or clinical applications. In this article, we demonstrate the fabrication of protein microparticles and microcapsules with an innovative method using biomolecular tools such as enzymes and affinity molecules to trigger the assembling of protein molecules within a porous CaCO3 template followed by a template removal step. We demonstrated the enzyme-assisted fabrication of collagen microparticles triggered by transglutaminase, as well as the affinity-assisted fabrication of BSA-biotin avidin microcapsules triggered by biotin-avidin affinity interaction, respectively. Based on the different protein assemble mechanisms, the collagen microparticles appeared as a solid-structured particles, while the BSA-biotin avidin microcapsules appeared as hollow-structured morphology. The fabrication procedures are simple and robust that allows producing protein microparticles or microcapsules under mild conditions at physiological pH and temperature. In addition, the microparticle morphologies, protein compositions and the assemble mechanisms were studied. Our technology provides a facile approach to design and fabricate protein microparticles and microcapsules that are useful in the area of biomaterials, pharmaceuticals and analytical chemistry.

Relationship between β-relaxation and structural stability of lysozyme: Microscopic insight on thermostabilization mechanism by trehalose from Raman spectroscopy experiments

NASA Astrophysics Data System (ADS)

Hédoux, Alain; Paccou, Laurent; Guinet, Yannick

2014-06-01

Raman investigations were carried out in the low-frequency and amide I regions on lysozyme aqueous solutions in absence and presence of trehalose. Raman spectroscopy gives the unique opportunity to analyze the protein and solvent dynamics in the low-frequency range while monitoring the unfolding process by capturing the spectrum of the amide I band. From the analysis of the quasielastic intensity, a dynamic change is firstly observed in a highly hydrated protein, around 70 °C, and interpreted in relation with the denaturation mechanism of the protein. The use of heavy water and partly deuterated trehalose gives clear information on protein-trehalose interactions in the native state of lysozyme (at room temperature) and during the thermal denaturation process of lysozyme. At room temperature, it was found that trehalose is preferentially excluded from the protein surface, and has a main effect on the tetrahedral local order of water molecules corresponding to a stiffening of the H-bond network in the solvent. The consequence is a significant reduction of the amplitude of fast relaxational motions, inducing a less marked dynamic transition shifted toward the high temperatures. Upon heating, interaction between trehalose and lysozyme is detected during the solvent penetration within the protein, i.e., while the native globular state softens into a molten globule (MG) state. Addition of trehalose reduces the protein flexibility in the MG state, improving the structural stability of the protein, and inhibiting the protein aggregation.

Correlative nanoscale imaging of actin filaments and their complexes

NASA Astrophysics Data System (ADS)

Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil; Gimzewski, James K.

2013-06-01

Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

Molecular modelling indicates that the pathological conformations of prion proteins might be beta-helical.

PubMed Central

Downing, D T; Lazo, N D

1999-01-01

Creutzfeldt-Jakob disease, kuru, scrapie and bovine spongiform encephalopathy are diseases of the mammalian central nervous system that involve the conversion of a cellular protein into an insoluble extracellular isoform. Spectroscopic studies have shown that the precursor protein contains mainly alpha-helical and random-coil conformations, whereas the prion isoform is largely in the beta conformation. The pathogenic prion is resistant to denaturation and protease digestion and can promote the conversion of the precursor protein to the pathogenic form. These properties have yet to be explained in terms of the structural conformations of the proteins. In the present study, molecular modelling showed that prion proteins could adopt the beta-helical conformation, which has been established for a number of fibrous proteins and has been suggested previously as the basis of amyloid fibrils. The beta-helical conformation provides explanations for the biophysical and biochemical stability of prions, their ability to form templates for the transmission of pathological conformation, and the existence of phenotypical strains of the prion diseases. PMID:10510313

How to Choose the Suitable Template for Homology Modelling of GPCRs: 5-HT7 Receptor as a Test Case.

PubMed

Shahaf, Nir; Pappalardo, Matteo; Basile, Livia; Guccione, Salvatore; Rayan, Anwar

2016-09-01

G protein-coupled receptors (GPCRs) are a super-family of membrane proteins that attract great pharmaceutical interest due to their involvement in almost every physiological activity, including extracellular stimuli, neurotransmission, and hormone regulation. Currently, structural information on many GPCRs is mainly obtained by the techniques of computer modelling in general and by homology modelling in particular. Based on a quantitative analysis of eighteen antagonist-bound, resolved structures of rhodopsin family "A" receptors - also used as templates to build 153 homology models - it was concluded that a higher sequence identity between two receptors does not guarantee a lower RMSD between their structures, especially when their pair-wise sequence identity (within trans-membrane domain and/or in binding pocket) lies between 25 % and 40 %. This study suggests that we should consider all template receptors having a sequence identity ≤50 % with the query receptor. In fact, most of the GPCRs, compared to the currently available resolved structures of GPCRs, fall within this range and lack a correlation between structure and sequence. When testing suitability for structure-based drug design, it was found that choosing as a template the most similar resolved protein, based on sequence resemblance only, led to unsound results in many cases. Molecular docking analyses were carried out, and enrichment factors as well as attrition rates were utilized as criteria for assessing suitability for structure-based drug design. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic analysis of intestinal tissues from mice fed with Lentinula edodes-derived polysaccharides.

PubMed

Xu, Xiaofei; Yang, Jiguo; Ning, Zhengxiang; Zhang, Xuewu

2016-01-01

Lentinula edodes-derived polysaccharides are well known for their immunomodulation and antitumor activities. However, the mechanisms of action have not been fully elucidated. This study presents proteomic analysis of the colon and small intestine from mice fed with an immunostimulating heteropolysaccharide L2 from the fruit body of L. edodes. Two-dimensional gel electrophoresis (2-DE) and MALDI-TOF-TOF MS/MS were employed to characterize the protein profiles. Twenty nine gel spots representing 20 proteins in colon tissues and 38 gel spots in small intestine tissues representing 23 proteins were identified as showing significant changes in abundance. These differential proteins in abundance are mainly involved in metabolism, binding, structural components, and response to stimulus. Protein-protein interaction network analysis demonstrated mapping of the 20 colon proteins to a 7-protein and a 3-protein sub-network, and mapping of the 23 small intestine proteins to a 9-protein and a 5-protein sub-network. All the 40 altered proteins were integrated into a unified network containing 25 proteins, suggesting the existence of a concerted mechanism, although acting on the colon and small intestine separately. These findings facilitate the understanding of the regulatory mechanism in response to L2 treatment.

In silico identification of functional regions in proteins.

PubMed

Nimrod, Guy; Glaser, Fabian; Steinberg, David; Ben-Tal, Nir; Pupko, Tal

2005-06-01

In silico prediction of functional regions on protein surfaces, i.e. sites of interaction with DNA, ligands, substrates and other proteins, is of utmost importance in various applications in the emerging fields of proteomics and structural genomics. When a sufficient number of homologs is found, powerful prediction schemes can be based on the observation that evolutionarily conserved regions are often functionally important, typically, only the principal functionally important region of the protein is detected, while secondary functional regions with weaker conservation signals are overlooked. Moreover, it is challenging to unambiguously identify the boundaries of the functional regions. We present a new methodology, called PatchFinder, that automatically identifies patches of conserved residues that are located in close proximity to each other on the protein surface. PatchFinder is based on the following steps: (1) Assignment of conservation scores to each amino acid position on the protein surface. (2) Assignment of a score to each putative patch, based on its likelihood to be functionally important. The patch of maximum likelihood is considered to be the main functionally important region, and the search is continued for non-overlapping patches of secondary importance. We examined the accuracy of the method using the IGPS enzyme, the SH2 domain and a benchmark set of 112 proteins. These examples demonstrated that PatchFinder is capable of identifying both the main and secondary functional patches. The PatchFinder program is available at: http://ashtoret.tau.ac.il/~nimrodg/

Molecular Effects of Concentrated Solutes on Protein Hydration, Dynamics, and Electrostatics.

PubMed

Abriata, Luciano A; Spiga, Enrico; Peraro, Matteo Dal

2016-08-23

Most studies of protein structure and function are performed in dilute conditions, but proteins typically experience high solute concentrations in their physiological scenarios and biotechnological applications. High solute concentrations have well-known effects on coarse protein traits like stability, diffusion, and shape, but likely also perturb other traits through finer effects pertinent at the residue and atomic levels. Here, NMR and molecular dynamics investigations on ubiquitin disclose variable interactions with concentrated solutes that lead to localized perturbations of the protein's surface, hydration, electrostatics, and dynamics, all dependent on solute size and chemical properties. Most strikingly, small polar uncharged molecules are sticky on the protein surface, whereas charged small molecules are not, but the latter still perturb the internal protein electrostatics as they diffuse nearby. Meanwhile, interactions with macromolecular crowders are favored mainly through hydrophobic, but not through polar, surface patches. All the tested small solutes strongly slow down water exchange at the protein surface, whereas macromolecular crowders do not exert such strong perturbation. Finally, molecular dynamics simulations predict that unspecific interactions slow down microsecond- to millisecond-timescale protein dynamics despite having only mild effects on pico- to nanosecond fluctuations as corroborated by NMR. We discuss our results in the light of recent advances in understanding proteins inside living cells, focusing on the physical chemistry of quinary structure and cellular organization, and we reinforce the idea that proteins should be studied in native-like media to achieve a faithful description of their function. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC).

PubMed

De Pinto, Vito; Messina, Angela; Accardi, Rosita; Aiello, Rita; Guarino, Francesca; Tomasello, Marianna Flora; Tommasino, Massimo; Tasco, Gianluca; Casadio, Rita; Benz, Roland; De Giorgi, Francesca; Ichas, François; Baker, Mark; Lawen, Alfons

2003-03-01

Mitochondrial porin or VDAC (Voltage Dependent Anion selective Channels) was identified for the first time in 1976, on the basis of the evolutionary similarity between the gram negative and mitochondrial outer membranes. Since this achievement VDAC has been extensively investigated: its functional features have been sharply defined upon reconstitution in artificial membranes and its sequence has been determined in many genomes. Unfortunately the tertiary structure has not yet been solved, mainly because it proved to be very difficult to get suitable crystals. Despite this established knowledge, in the last few years this protein has attracted renewed interest. There are two main reasons for this interest: the discovery, in most eukaryotes, of a family of genes encoding VDAC isoforms and the claims of VDAC involvement in the intrinsic pathway of apoptosis and in particular in the mechanism of cytochrome c release from mitochondria. We can affirm that nowadays the eukaryotic porin (or VDAC) is studied in a more general cellular contest, looking at the interactions and integration with other molecules, since VDAC is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. In this minireview we will briefly focus our attention onto the following topics: 1) recent advances about the structure of VDAC; 2) the VDAC-related multigene families; 3) the presence, targeting and function of VDAC in various cell membranes.

Investigation of Natural Bombyx mori Silk Fibroin Proteins Using INS

NASA Astrophysics Data System (ADS)

Crain, Christopher; Strange, Nicholas; Larese, J. Z.

The mechanical properties of many protein comprised biomaterials are a direct reflection of non-covalent (i.e. weak) interacting ions such as F-actin in muscles, tubulin in the cytoskeleton of cells, viral capsids, and silk. Porter and Vollrath underscored the two main factors that are critical for understanding the high mechanical strength of silks: the nanoscale semi-crystalline folding structure, which gives it exceptional toughness and strength, and the degree of hydration of the disordered fraction, which acts to modify these properties. Understanding and controlling these two principal factors are the key to the functionality of protein elastomers, and render silk an ideal model protein for (bio)material design. We will describe our investigation of electrospun silk of the Bombyx mori (silk worm), using Inelastic Neutron Scattering (INS). These techniques were used to investigate the microscopic dynamics of the dry and hydrated protein.

Study of the nitric oxide system in the rat cerebellum during aging.

PubMed

Blanco, Santos; Molina, Francisco J; Castro, Lourdes; Del Moral, Maria L; Hernandez, Raquel; Jimenez, Ana; Rus, Alma; Martinez-Lara, Esther; Siles, Eva; Peinado, Maria A

2010-06-24

The cerebellum is the neural structure with the highest levels of nitric oxide, a neurotransmitter that has been proposed to play a key role in the brain aging, although knowledge concerning its contribution to cerebellar senescence is still unclear, due mainly to absence of integrative studies that jointly evaluate the main factors involved in its cell production and function. Consequently, in the present study, we investigate the expression, location, and activity of nitric oxide synthase isoenzymes; the protein nitration; and the production of nitric oxide in the cerebellum of adult and old rats. Our results show no variation in the expression of nitric oxide synthase isoforms with aging, although, we have detected some changes in the cellular distribution pattern of the inducible isoform particularly in the cerebellar nuclei. There is also an increase in nitric oxide synthase activity, as well as greater protein-nitration levels, and maintenance of nitrogen oxides (NOx) levels in the senescent cerebellum. The nitric oxide/nitric oxide synthases system suffers from a number of changes, mainly in the inducible nitric oxide synthase distribution and in overall nitric oxide synthases activity in the senescent cerebellum, which result in an increase of the protein nitration. These changes might be related to the oxidative damage detected with aging in the cerebellum.

Dynamic Conformational Changes in MUNC18 Prevent Syntaxin Binding

PubMed Central

Bar-On, Dana; Nachliel, Esther; Gutman, Menachem; Ashery, Uri

2011-01-01

The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphoryalation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins. PMID:21390273

Differential protein folding and chemical changes in lung tissues exposed to asbestos or particulates

PubMed Central

Pascolo, Lorella; Borelli, Violetta; Canzonieri, Vincenzo; Gianoncelli, Alessandra; Birarda, Giovanni; Bedolla, Diana E.; Salomé, Murielle; Vaccari, Lisa; Calligaro, Carla; Cotte, Marine; Hesse, Bernhard; Luisi, Fernando; Zabucchi, Giuliano; Melato, Mauro; Rizzardi, Clara

2015-01-01

Environmental and occupational inhalants may induce a large number of pulmonary diseases, with asbestos exposure being the most risky. The mechanisms are clearly related to chemical composition and physical and surface properties of materials. A combination of X-ray fluorescence (μXRF) and Fourier Transform InfraRed (μFTIR) microscopy was used to chemically characterize and compare asbestos bodies versus environmental particulates (anthracosis) in lung tissues from asbestos exposed and control patients. μXRF analyses revealed heterogeneously aggregated particles in the anthracotic structures, containing mainly Si, K, Al and Fe. Both asbestos and particulates alter lung iron homeostasis, with a more marked effect in asbestos exposure. μFTIR analyses revealed abundant proteins on asbestos bodies but not on anthracotic particles. Most importantly, the analyses demonstrated that the asbestos coating proteins contain high levels of β-sheet structures. The occurrence of conformational changes in the proteic component of the asbestos coating provides new insights into long-term asbestos effects. PMID:26159651

Interaction of lysozyme with a tear film lipid layer model: A molecular dynamics simulation study.

PubMed

Wizert, Alicja; Iskander, D Robert; Cwiklik, Lukasz

2017-12-01

The tear film is a thin multilayered structure covering the cornea. Its outermost layer is a lipid film underneath of which resides on an aqueous layer. This tear film lipid layer (TFLL) is itself a complex structure, formed by both polar and nonpolar lipids. It was recently suggested that due to tear film dynamics, TFLL contains inhomogeneities in the form of polar lipid aggregates. The aqueous phase of tear film contains lachrymal-origin proteins, whereby lysozyme is the most abundant. These proteins can alter TFLL properties, mainly by reducing its surface tension. However, a detailed nature of protein-lipid interactions in tear film is not known. We investigate the interactions of lysozyme with TFLL in molecular details by employing coarse-grained molecular dynamics simulations. We demonstrate that lysozyme, due to lateral restructuring of TFLL, is able to penetrate the tear lipid film embedded in inverse micellar aggregates. Copyright © 2017 Elsevier B.V. All rights reserved.

The multi-copy simultaneous search methodology: a fundamental tool for structure-based drug design.

PubMed

Schubert, Christian R; Stultz, Collin M

2009-08-01

Fragment-based ligand design approaches, such as the multi-copy simultaneous search (MCSS) methodology, have proven to be useful tools in the search for novel therapeutic compounds that bind pre-specified targets of known structure. MCSS offers a variety of advantages over more traditional high-throughput screening methods, and has been applied successfully to challenging targets. The methodology is quite general and can be used to construct functionality maps for proteins, DNA, and RNA. In this review, we describe the main aspects of the MCSS method and outline the general use of the methodology as a fundamental tool to guide the design of de novo lead compounds. We focus our discussion on the evaluation of MCSS results and the incorporation of protein flexibility into the methodology. In addition, we demonstrate on several specific examples how the information arising from the MCSS functionality maps has been successfully used to predict ligand binding to protein targets and RNA.

Ten Good Reasons for the Use of the Tellurium-Centered Anderson-Evans Polyoxotungstate in Protein Crystallography.

PubMed

Bijelic, Aleksandar; Rompel, Annette

2017-06-20

Protein crystallography represents at present the most productive and most widely used method to obtain structural information on target proteins and protein-ligand complexes within the atomic resolution range. The knowledge obtained in this way is essential for understanding the biology, chemistry, and biochemistry of proteins and their functions but also for the development of compounds of high pharmacological and medicinal interest. Here, we address the very central problem in protein crystallography: the unpredictability of the crystallization process. Obtaining protein crystals that diffract to high resolutions represents the essential step to perform any structural study by X-ray crystallography; however, this method still depends basically on trial and error making it a very time- and resource-consuming process. The use of additives is an established process to enable or improve the crystallization of proteins in order to obtain high quality crystals. Therefore, a more universal additive addressing a wider range of proteins is desirable as it would represent a huge advance in protein crystallography and at the same time drastically impact multiple research fields. This in turn could add an overall benefit for the entire society as it profits from the faster development of novel or improved drugs and from a deeper understanding of biological, biochemical, and pharmacological phenomena. With this aim in view, we have tested several compounds belonging to the emerging class of polyoxometalates (POMs) for their suitability as crystallization additives and revealed that the tellurium-centered Anderson-Evans polyoxotungstate [TeW 6 O 24 ] 6- (TEW) was the most suitable POM-archetype. After its first successful application as a crystallization additive, we repeatedly reported on TEW's positive effects on the crystallization behavior of proteins with a particular focus on the protein-TEW interactions. As electrostatic interactions are the main force for TEW binding to proteins, TEW with its highly negative charge addresses in principle all proteins possessing positively charged patches. Furthermore, due to its high structural and chemical diversity, TEW exhibits major advantages over some commonly used crystallization additives. Therefore, we summarized all features of TEW, which are beneficial for protein crystallization, and present ten good reasons to promote the use of TEW in protein crystallography as a powerful additive. Our results demonstrate that TEW is a compound that is, in many respects, predestined as a crystallization additive. We assume that many crystallographers and especially researchers, who are not experts in this field but willing to crystallize their structurally unknown target protein, could benefit from the use of TEW as it is able to promote both the crystallization process itself and the subsequent structure elucidation by providing valuable anomalous signals, which are helpful for the phasing step.

Structural characterisation of parotid and whole mouth salivary pellicles adsorbed onto DPI and QCMD hydroxyapatite sensors.

PubMed

Ash, Anthony; Burnett, Gary R; Parker, Roger; Ridout, Mike J; Rigby, Neil M; Wilde, Peter J

2014-04-01

In this study we investigated the differences in the properties of pellicles formed from stimulated parotid saliva (PS), which contains little or no mucin; and stimulated whole mouth saliva (WMS), which contains mainly two types of mucin: MUC5B and MUC7. By contacting WMS and PS with quartz-crystal microbalance with dissipation monitoring (QCM-D) and dual polarisation interferometer (DPI) hydroxyapatite (the main component of enamel) coated sensors, we observed the formation and structure of the respective salivary pellicles. As this was the first time that DPI hydroxyapatite sensors have been used to measure salivary pellicle adsorption; the techniques combined allowed us to measure the hydrated mass, dry mass, thickness and viscoelastic properties of the pellicle; but also to record the density of the PS and WMS formed pellicles. Subsequently, the PS pellicle was shown to form a denser layer than WMS pellicle; which would suggest that the proteins present in PS are also responsible for forming the dense basal layer of the acquired enamel pellicle. Whereas proteins present in the WMS are more likely to help form the softer outer layer of the pellicle. The data presented help to further define the mechanisms leading to the multi-layered structure of the salivary pellicle and demonstrate that salivary composition has an important effect on the structural properties of the adsorbed pellicle. Copyright © 2013 Elsevier B.V. All rights reserved.

Exploring the structural insights on human laforin mutation K87A in Lafora disease--a molecular dynamics study.

PubMed

Srikumar, P S; Rohini, K

2013-10-01

Lafora disease (LD) is an autosomal recessive, progressive form of myoclonus epilepsy which affects worldwide. LD occurs mainly in countries like southern Europe, northern Africa, South India, and in the Middle East. LD occurs with its onset mainly in teenagers and leads to decline and death within 2 to 10 years. The genes EPM2A and EPM2B are commonly involved in 90 % of LD cases. EPM2A codes for protein laforin which contains an amino terminal carbohydrate binding module (CBM) belonging to the CBM20 family and a carboxy terminal dual specificity phosphatase domain. Mutations in laforin are found to abolish glycogen binding and have been reported in wet lab methods. In order to investigate on structural insights on laforin mutation K81A, we performed molecular dynamics (MD) simulation studies for native and mutant protein. MD simulation results showed loss of stability due to mutation K87A which confirmed the structural reason for conformational changes observed in laforin. The conformational change of mutant laforin was confirmed by analysis using root mean square deviation, root mean square fluctuation, solvent accessibility surface area, radius of gyration, hydrogen bond, and principle component analysis. Our results identified that the flexibility of K87A mutated laforin structure, with replacement of acidic amino acid to aliphatic amino acid in functional CBM domain, have more impact in abolishing glycogen binding that favors LD.

Enhanced plastic deformations of nanofibrillated cellulose film by adsorbed moisture and protein-mediated interactions.

PubMed

Malho, Jani-Markus; Ouellet-Plamondon, Claudiane; Rüggeberg, Markus; Laaksonen, Päivi; Ikkala, Olli; Burgert, Ingo; Linder, Markus B

2015-01-12

Biological composites are typically based on an adhesive matrix that interlocks rigid reinforcing elements in fiber composite or brick-and-mortar assemblies. In nature, the adhesive matrix is often made up of proteins, which are also interesting model systems, as they are unique among polymers in that we know how to engineer their structures with atomic detail and to select protein elements for specific interactions with other components. Here we studied how fusion proteins that consist of cellulose binding proteins linked to proteins that show a natural tendency to form multimer complexes act as an adhesive matrix in combination with nanofibrillated cellulose. We found that the fusion proteins are retained with the cellulose and that the proteins mainly affect the plastic yield behavior of the cellulose material as a function of water content. Interestingly, the proteins increased the moisture absorption of the composite, but the well-known plastifying effect of water was clearly decreased. The work helps to understand the functional basis of nanocellulose composites as materials and aims toward building model systems for molecular biomimetic materials.

Dynamic Fluctuations of Protein-Carbohydrate Interactions Promote Protein Aggregation

PubMed Central

Voynov, Vladimir; Chennamsetty, Naresh; Kayser, Veysel; Helk, Bernhard; Forrer, Kurt; Zhang, Heidi; Fritsch, Cornelius; Heine, Holger; Trout, Bernhardt L.

2009-01-01

Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation. PMID:20037630

Structural basis of reversine selectivity in inhibiting Mps1 more potently than aurora B kinase.

PubMed

Hiruma, Yoshitaka; Koch, Andre; Dharadhar, Shreya; Joosten, Robbie P; Perrakis, Anastassis

2016-12-01

Monopolar spindle 1 (Mps1, also known as TTK) is a protein kinase crucial for ensuring that cell division progresses to anaphase only after all chromosomes are connected to spindle microtubules. Incomplete chromosomal attachment leads to abnormal chromosome counts in the daughter cells (aneuploidy), a condition common in many solid cancers. Therefore Mps1 is an established target in cancer therapy. Mps1 kinase inhibitors include reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine), a promiscuous compound first recognized as an inhibitor of the Aurora B mitotic kinase. Here, we present the 3.0-Å resolution crystal structure of the Mps1 kinase domain bound to reversine. Structural comparison of reversine bound to Mps1 and Aurora B, indicates a similar binding pose for the purine moiety of reversine making three conserved hydrogen bonds to the protein main chain, explaining the observed promiscuity of this inhibitor. The cyclohexyl and morpholinoaniline moieties of reversine however, have more extensive contacts with the protein in Mps1 than in Aurora B. This is reflected both in structure-based docking energy calculations, and in new experimental data we present here, that both confirm that the affinity of reversine towards Mps1 is about two orders of magnitude higher than towards Aurora B. Thus, our data provides detailed structural understanding of the existing literature that argues reversine inhibits Mps1 more efficiently than Aurora B based on biochemical and in-cell assays. Proteins 2016; 84:1761-1766. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

Computational Prediction of Atomic Structures of Helical Membrane Proteins Aided by EM Maps

PubMed Central

Kovacs, Julio A.; Yeager, Mark; Abagyan, Ruben

2007-01-01

Integral membrane proteins pose a major challenge for protein-structure prediction because only ≈100 high-resolution structures are available currently, thereby impeding the development of rules or empirical potentials to predict the packing of transmembrane α-helices. However, when an intermediate-resolution electron microscopy (EM) map is available, it can be used to provide restraints which, in combination with a suitable computational protocol, make structure prediction feasible. In this work we present such a protocol, which proceeds in three stages: 1), generation of an ensemble of α-helices by flexible fitting into each of the density rods in the low-resolution EM map, spanning a range of rotational angles around the main helical axes and translational shifts along the density rods; 2), fast optimization of side chains and scoring of the resulting conformations; and 3), refinement of the lowest-scoring conformations with internal coordinate mechanics, by optimizing the van der Waals, electrostatics, hydrogen bonding, torsional, and solvation energy contributions. In addition, our method implements a penalty term through a so-called tethering map, derived from the EM map, which restrains the positions of the α-helices. The protocol was validated on three test cases: GpA, KcsA, and MscL. PMID:17496035

Preventive role of lens antioxidant defense mechanism against riboflavin-mediated sunlight damaging of lens crystallins.

PubMed

Anbaraki, Afrooz; Khoshaman, Kazem; Ghasemi, Younes; Yousefi, Reza

2016-10-01

The main components of sunlight reaching the eye lens are UVA and visible light exerting their photo-damaging effects indirectly by the aid of endogenous photosensitizer molecules such as riboflavin (RF). In this study, lens proteins solutions were incubated with RF and exposed to the sunlight. Then, gel mobility shift analysis and different spectroscopic assessments were applied to examine the structural damaging effects of solar radiation on these proteins. Exposure of lens proteins to direct sunlight, in the presence of RF, leads to marked structural crosslinking, oligomerization and proteolytic instability. These structural damages were also accompanied with reduction in the emission fluorescence of Trp and Tyr and appearance of a new absorption peak between 300 and 400nm which can be related to formation of new chromophores. Also, photo-oxidation of lens crystallins increases their oligomeric size distribution as examined by dynamic light scattering analysis. The above mentioned structural insults, as potential sources of sunlight-induced senile cataract and blindness, were significantly attenuated in the presence of ascorbic acid and glutathione which are two important components of lens antioxidant defense system. Therefore, the powerful antioxidant defense mechanism of eye lens is an important barrier against molecular photo-damaging effects of solar radiations during the life span. Copyright © 2016 Elsevier B.V. All rights reserved.

Statistical Analysis of Crystallization Database Links Protein Physico-Chemical Features with Crystallization Mechanisms

PubMed Central

Fusco, Diana; Barnum, Timothy J.; Bruno, Andrew E.; Luft, Joseph R.; Snell, Edward H.; Mukherjee, Sayan; Charbonneau, Patrick

2014-01-01

X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis. PMID:24988076

Statistical analysis of crystallization database links protein physico-chemical features with crystallization mechanisms.

PubMed

Fusco, Diana; Barnum, Timothy J; Bruno, Andrew E; Luft, Joseph R; Snell, Edward H; Mukherjee, Sayan; Charbonneau, Patrick

2014-01-01

X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis.

Micro-Raman spectroscopy on oral tissues

NASA Astrophysics Data System (ADS)

Zenone, F.; Lepore, M.; Perna, G.; Carmone, P.; Riccio, R.; Gaeta, G. M.; Capozzi, V.

2006-02-01

Micro-Raman Spectroscopy (μ-RS) provides a unique tool in medicine for a not invasive and real time analysis of biological tissue for biopsy and "in vivo" investigation. Based on the evaluation of molecular vibration frequencies, the μ-RS is able to detect the main molecular bonds of protein constituents, as the C-H and C-C ones. Changes in frequency or in the relative intensity of the vibration modes revealed by μ-RS can be related to changes of chemical bond and of protein structure induced by pathology. The μ-RS has been performed on samples of oral tissue from informed patients, affected by pemphigus vulgaris (an oral pathology) in an advanced regression state. The biopsies were thin slices (about 1mm thick) with 6mm diameter. The sample was measured through a 170 μm thick cover-glass. The experimental set-up was mainly composed by a He-Ne laser and a monochromator equipped with a Peltier cell and with a grating of 1800 grooves/mm. The laser light was focused on the sample surface by means of a long focal length 50X optical objective. The main protein bonds are clearly detectable in the considered samples and this give important information on the integrity and on the state of tissue components (lipids and proteins), and consequently on the occurrence of pathology. The potential application of this method for in vivo analysis is an invaluable alternative to biopsy and pathological examinations for many medical application as screening diagnostic, therapy progress examination, and surgical support.

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

Conformation switching of AIM2 PYD domain revealed by NMR relaxation and MD simulation.

PubMed

Wang, Haobo; Yang, Lijiang; Niu, Xiaogang

2016-04-29

Protein absent in melanoma 2 (AIM2) is a double-strand DNA (ds DNA) sensor mainly located in cytoplasm of cell. It includes one N terminal PYD domain and one C terminal HIN domain. When the ds DNA such as DNA viruses and bacteria entered cytoplasm, the HIN domain of AIM2 will recognize and bind to DNA, and the PYD domain will bind to ASC protein which will result in the formation of AIM2 inflammasome. Three AIM2 PYD domain structures have been solved, but every structure yields a unique conformation around the α3 helix region. To understand why different AIM2 PYD structures show different conformations in this region, we use NMR relaxation techniques to study the backbone dynamics of mouse AIM2 PYD domain and perform molecular dynamics (MD) simulations on both mouse and human AIM2 PYD structures. Our results indicate that this region is highly flexible in both mouse and human AIM2 PYD domains, and the PYD domain may exist as a conformation ensemble in solution. Different environment makes the population vary among pre-existing conformational substrates of the ensemble, which may be the reason why different AIM2 PYD structures were observed under different conditions. Further docking analysis reveals that the conformation switching may be important for the autoinhibition of the AIM2 protein. Copyright © 2016 Elsevier Inc. All rights reserved.

Antigenic Structure of the Human Muscle Nicotinic Acetylcholine Receptor Main Immunogenic Region

PubMed Central

Luo, Jie; Lindstrom, Jon

2009-01-01

The main immunogenic region on the α1 subunits of muscle nicotinic acetylcholine receptors provokes half or more of the autoantibodies in myasthenia gravis and its animal model. Many of these autoantibodies depend on the native conformation of the receptor for their ability to bind with high affinity. We mapped this region and explained the conformation-dependence of its epitopes by making chimeras in which sequences of human muscle α1 subunits were replaced in human neuronal α7 subunits or Aplysia acetylcholine binding protein. These chimeras also revealed that the main immunogenic region can play a major role in promoting conformational maturation, and, consequently, assembly of receptor subunits. PMID:19705087

Understanding the General Packing Rearrangements Required for Successful Template Based Modeling of Protein Structure from a CASP Experiment

PubMed Central

Day, Ryan; Joo, Hyun; Chavan, Archana; Lennox, Kristin P.; Chen, Ann; Dahl, David B.; Vannucci, Marina; Tsai, Jerry W.

2012-01-01

As an alternative to the common template based protein structure prediction methods based on main-chain position, a novel side-chain centric approach has been developed. Together with a Bayesian loop modeling procedure and a combination scoring function, the Stone Soup algorithm was applied to the CASP9 set of template based modeling targets. Although the method did not generate as large of perturbations to the template structures as necessary, the analysis of the results gives unique insights into the differences in packing between the target structures and their templates. Considerable variation in packing is found between target and template structures even when the structures are close, and this variation is found due to 2 and 3 body packing interactions. Outside the inherent restrictions in packing representation of the PDB, the first steps in correctly defining those regions of variable packing have been mapped primarily to local interactions, as the packing at the secondary and tertiary structure are largely conserved. Of the scoring functions used, a loop scoring function based on water structure exhibited some promise for discrimination. These results present a clear structural path for further development of a side-chain centered approach to template based modeling. PMID:23266765

Understanding the general packing rearrangements required for successful template based modeling of protein structure from a CASP experiment.

PubMed

Day, Ryan; Joo, Hyun; Chavan, Archana C; Lennox, Kristin P; Chen, Y Ann; Dahl, David B; Vannucci, Marina; Tsai, Jerry W

2013-02-01

As an alternative to the common template based protein structure prediction methods based on main-chain position, a novel side-chain centric approach has been developed. Together with a Bayesian loop modeling procedure and a combination scoring function, the Stone Soup algorithm was applied to the CASP9 set of template based modeling targets. Although the method did not generate as large of perturbations to the template structures as necessary, the analysis of the results gives unique insights into the differences in packing between the target structures and their templates. Considerable variation in packing is found between target and template structures even when the structures are close, and this variation is found due to 2 and 3 body packing interactions. Outside the inherent restrictions in packing representation of the PDB, the first steps in correctly defining those regions of variable packing have been mapped primarily to local interactions, as the packing at the secondary and tertiary structure are largely conserved. Of the scoring functions used, a loop scoring function based on water structure exhibited some promise for discrimination. These results present a clear structural path for further development of a side-chain centered approach to template based modeling. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

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

1997-09-01

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

Designing small molecules to target cryptic pockets yields both positive and negative allosteric modulators

PubMed Central

Moeder, Katelyn E.; Ho, Chris M. W.; Zimmerman, Maxwell I.; Frederick, Thomas E.; Bowman, Gregory R.

2017-01-01

Allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites, make it possible to target proteins considered “undruggable” and to develop new therapies that circumvent existing resistance. Despite growing interest in allosteric drug discovery, rational design is limited by a lack of sufficient structural information about alternative binding sites in proteins. Previously, we used Markov State Models (MSMs) to identify such “cryptic pockets,” and here we describe a method for identifying compounds that bind in these cryptic pockets and modulate enzyme activity. Experimental tests validate our approach by revealing both an inhibitor and two activators of TEM β-lactamase (TEM). To identify hits, a library of compounds is first virtually screened against either the crystal structure of a known cryptic pocket or an ensemble of structures containing the same cryptic pocket that is extracted from an MSM. Hit compounds are then screened experimentally and characterized kinetically in individual assays. We identify three hits, one inhibitor and two activators, demonstrating that screening for binding to allosteric sites can result in both positive and negative modulation. The hit compounds have modest effects on TEM activity, but all have higher affinities than previously identified inhibitors, which bind the same cryptic pocket but were found, by chance, via a computational screen targeting the active site. Site-directed mutagenesis of key contact residues predicted by the docking models is used to confirm that the compounds bind in the cryptic pocket as intended. Because hit compounds are identified from docking against both the crystal structure and structures from the MSM, this platform should prove suitable for many proteins, particularly targets whose crystal structures lack obvious druggable pockets, and for identifying both inhibitory and activating small-molecule modulators. PMID:28570708

Apolipoprotein ε7 allele in memory complaints: insights through protein structure prediction.

PubMed

Youn, Young Chul; Lim, Yong Kwan; Han, Su-Hyun; Giau, Vo Van; Lee, Mi-Kyung; Park, Kwang-Yeol; Kim, SangYun; Bagyinszky, Eva; An, Seong Soo A; Kim, Hye Ryoun

2017-01-01

APOE ε7 gene is a rare mutant form of APOE ε3 . The mutation occurs in the lipid-binding domain of APOE. Based on the protein's structure, APOE ε7 is expected to function in lipid and β-amyloid metabolism, similar to APOE ε4 . However, unlike that for APOE ε4 , the mechanisms responsible for Alzheimer's disease (AD) cases associated with APOE ε7 expression have not been elucidated. The present study aims to investigate the association between APOE ε7 expression and cognitive impairment. APOE was sequenced in DNA samples collected from 344 memory-complaint patients who visited the memory clinic, and from 345 non-memory-complaint individuals from the health promotion center. The protein structures of ApoE3, ApoE4, and ApoE7 were predicted. Three ε3 / ε7 heterozygote individuals who were all classified under the memory-complaint group were identified. Of these, two subjects were clinically diagnosed with AD with small vessel disease, and the remaining individual was diagnosed with subjective cognitive impairment. This study predicted the protein structures of ApoE3, ApoE4, and ApoE7 and determined the three-dimensional structure of the carboxy terminus of ApoE7, which participates in an electrostatic domain interaction similar to that of APOE ε4 . APOE K244 or K245 mutations for APOE ε7 were not found in the Korean reference genome database, which contains information (http://152.99.75.168/KRGDB/browser/mainBrowser.jsp) from 622 healthy individuals. As verified by the results of structural prediction, APOE ε7 could serve as another risk factor for cognitive impairment and is particularly associated with vascular disease. However, additional studies are required to validate the pathogenic nature of APOE ε7 .

Conformation-dependent backbone geometry restraints set a new standard for protein crystallographic refinement

DOE PAGES

Moriarty, Nigel W.; Tronrud, Dale E.; Adams, Paul D.; ...

2014-06-17

Ideal values of bond angles and lengths used as external restraints are crucial for the successful refinement of protein crystal structures at all but the highest of resolutions. The restraints in common usage today have been designed based on the assumption that each type of bond or angle has a single ideal value independent of context. However, recent work has shown that the ideal values are, in fact, sensitive to local conformation, and as a first step toward using such information to build more accurate models, ultra-high resolution protein crystal structures have been used to derive a conformation-dependent library (CDL)more » of restraints for the protein backbone (Berkholz et al. 2009. Structure. 17, 1316). Here, we report the introduction of this CDL into the Phenix package and the results of test refinements of thousands of structures across a wide range of resolutions. These tests show that use of the conformation dependent library yields models that have substantially better agreement with ideal main-chain bond angles and lengths and, on average, a slightly enhanced fit to the X-ray data. No disadvantages of using the backbone CDL are apparent. In Phenix usage of the CDL can be selected by simply specifying the cdl=True option. This successful implementation paves the way for further aspects of the context-dependence of ideal geometry to be characterized and applied to improve experimental and predictive modelling accuracy.« less

Structural and metabolic transitions of C4 leaf development and differentiation defined by microscopy and quantitative proteomics in maize.

PubMed

Majeran, Wojciech; Friso, Giulia; Ponnala, Lalit; Connolly, Brian; Huang, Mingshu; Reidel, Edwin; Zhang, Cankui; Asakura, Yukari; Bhuiyan, Nazmul H; Sun, Qi; Turgeon, Robert; van Wijk, Klaas J

2010-11-01

C(4) grasses, such as maize (Zea mays), have high photosynthetic efficiency through combined biochemical and structural adaptations. C(4) photosynthesis is established along the developmental axis of the leaf blade, leading from an undifferentiated leaf base just above the ligule into highly specialized mesophyll cells (MCs) and bundle sheath cells (BSCs) at the tip. To resolve the kinetics of maize leaf development and C(4) differentiation and to obtain a systems-level understanding of maize leaf formation, the accumulation profiles of proteomes of the leaf and the isolated BSCs with their vascular bundle along the developmental gradient were determined using large-scale mass spectrometry. This was complemented by extensive qualitative and quantitative microscopy analysis of structural features (e.g., Kranz anatomy, plasmodesmata, cell wall, and organelles). More than 4300 proteins were identified and functionally annotated. Developmental protein accumulation profiles and hierarchical cluster analysis then determined the kinetics of organelle biogenesis, formation of cellular structures, metabolism, and coexpression patterns. Two main expression clusters were observed, each divided in subclusters, suggesting that a limited number of developmental regulatory networks organize concerted protein accumulation along the leaf gradient. The coexpression with BSC and MC markers provided strong candidates for further analysis of C(4) specialization, in particular transporters and biogenesis factors. Based on the integrated information, we describe five developmental transitions that provide a conceptual and practical template for further analysis. An online protein expression viewer is provided through the Plant Proteome Database.

Conformational diversity analysis reveals three functional mechanisms in proteins

PubMed Central

Fornasari, María Silvina

2017-01-01

Protein motions are a key feature to understand biological function. Recently, a large-scale analysis of protein conformational diversity showed a positively skewed distribution with a peak at 0.5 Å C-alpha root-mean-square-deviation (RMSD). To understand this distribution in terms of structure-function relationships, we studied a well curated and large dataset of ~5,000 proteins with experimentally determined conformational diversity. We searched for global behaviour patterns studying how structure-based features change among the available conformer population for each protein. This procedure allowed us to describe the RMSD distribution in terms of three main protein classes sharing given properties. The largest of these protein subsets (~60%), which we call “rigid” (average RMSD = 0.83 Å), has no disordered regions, shows low conformational diversity, the largest tunnels and smaller and buried cavities. The two additional subsets contain disordered regions, but with differential sequence composition and behaviour. Partially disordered proteins have on average 67% of their conformers with disordered regions, average RMSD = 1.1 Å, the highest number of hinges and the longest disordered regions. In contrast, malleable proteins have on average only 25% of disordered conformers and average RMSD = 1.3 Å, flexible cavities affected in size by the presence of disordered regions and show the highest diversity of cognate ligands. Proteins in each set are mostly non-homologous to each other, share no given fold class, nor functional similarity but do share features derived from their conformer population. These shared features could represent conformational mechanisms related with biological functions. PMID:28192432

Investigation of protein folding by coarse-grained molecular dynamics with the UNRES force field.

PubMed

Maisuradze, Gia G; Senet, Patrick; Czaplewski, Cezary; Liwo, Adam; Scheraga, Harold A

2010-04-08

Coarse-grained molecular dynamics simulations offer a dramatic extension of the time-scale of simulations compared to all-atom approaches. In this article, we describe the use of the physics-based united-residue (UNRES) force field, developed in our laboratory, in protein-structure simulations. We demonstrate that this force field offers about a 4000-times extension of the simulation time scale; this feature arises both from averaging out the fast-moving degrees of freedom and reduction of the cost of energy and force calculations compared to all-atom approaches with explicit solvent. With massively parallel computers, microsecond folding simulation times of proteins containing about 1000 residues can be obtained in days. A straightforward application of canonical UNRES/MD simulations, demonstrated with the example of the N-terminal part of the B-domain of staphylococcal protein A (PDB code: 1BDD, a three-alpha-helix bundle), discerns the folding mechanism and determines kinetic parameters by parallel simulations of several hundred or more trajectories. Use of generalized-ensemble techniques, of which the multiplexed replica exchange method proved to be the most effective, enables us to compute thermodynamics of folding and carry out fully physics-based prediction of protein structure, in which the predicted structure is determined as a mean over the most populated ensemble below the folding-transition temperature. By using principal component analysis of the UNRES folding trajectories of the formin-binding protein WW domain (PDB code: 1E0L; a three-stranded antiparallel beta-sheet) and 1BDD, we identified representative structures along the folding pathways and demonstrated that only a few (low-indexed) principal components can capture the main structural features of a protein-folding trajectory; the potentials of mean force calculated along these essential modes exhibit multiple minima, as opposed to those along the remaining modes that are unimodal. In addition, a comparison between the structures that are representative of the minima in the free-energy profile along the essential collective coordinates of protein folding (computed by principal component analysis) and the free-energy profile projected along the virtual-bond dihedral angles gamma of the backbone revealed the key residues involved in the transitions between the different basins of the folding free-energy profile, in agreement with existing experimental data for 1E0L .

Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites.

PubMed

Villoutreix, B O; Härdig, Y; Wallqvist, A; Covell, D G; García de Frutos, P; Dahlbäck, B

1998-06-01

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP alpha-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the alpha-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions.

Hemoglobin bioconjugates with surface-protected gold nanoparticles in aqueous media: The stability depends on solution pH and protein properties.

PubMed

Del Caño, Rafael; Mateus, Lucia; Sánchez-Obrero, Guadalupe; Sevilla, José Manuel; Madueño, Rafael; Blázquez, Manuel; Pineda, Teresa

2017-11-01

The identification of the factors that dictate the formation and physicochemical properties of protein-nanomaterial bioconjugates are important to understand their behavior in biological systems. The present work deals with the formation and characterization of bioconjugates made of the protein hemoglobin (Hb) and gold nanoparticles (AuNP) capped with three different molecular layers (citrate anions (c), 6-mercaptopurine (MP) and ω-mercaptoundecanoic acid (MUA)). The main focus is on the behavior of the bioconjugates in aqueous buffered solutions in a wide pH range. The stability of the bioconjugates have been studied by UV-visible spectroscopy by following the changes in the localized surface resonance plasmon band (LSRP), Dynamic light scattering (DLS) and zeta-potential pH titrations. It has been found that they are stable in neutral and alkaline solutions and, at pH lower than the protein isoelectric point, aggregation takes place. Although the surface chemical properties of the AuNPs confer different properties in respect to colloidal stability, once the bioconjugates are formed their properties are dictated by the Hb protein corona. The protein secondary structure, as analyzed by Attenuated total reflectance infrared (ATR-IR) spectroscopy, seems to be maintained under the conditions of colloidal stability but some small changes in protein conformation take place when the bioconjugates aggregate. These findings highlight the importance to keep the protein structure upon interaction with nanomaterials to drive the stability of the bioconjugates. Copyright © 2017 Elsevier Inc. All rights reserved.

Ultrafast fluorescence upconversion technique and its applications to proteins.

PubMed

Chosrowjan, Haik; Taniguchi, Seiji; Tanaka, Fumio

2015-08-01

The basic principles and main characteristics of the ultrafast time-resolved fluorescence upconversion technique (conventional and space-resolved), including requirements for nonlinear crystals, mixing spectral bandwidth, acceptance angle, etc., are presented. Applications to flavoproteins [wild-type (WT) FMN-binding protein and its W32Y, W32A, E13R, E13K, E13Q and E13T mutants] and photoresponsive proteins [WT photoactive yellow protein and its R52Q mutant in solution and as single crystals] are demonstrated. For flavoproteins, investigations elucidating the effects of ionic charges on ultrafast electron transfer (ET) dynamics are summarized. It is shown that replacement of the ionic amino acid Glu13 and the resulting modification of the electrostatic charge distribution in the protein chromphore-binding pocket substantially alters the ultrafast fluorescence quenching dynamics and ET rate in FMN-binding protein. It is concluded that, together with donor-acceptor distances, electrostatic interactions between ionic photoproducts and other ionic groups in the proteins are important factors influencing the ET rates. In WT photoactive yellow protein and the R52Q mutant, ultrafast photoisomerization dynamics of the chromophore (deprotonated trans-p-coumaric acid) in liquid and crystal phases are investigated. It is shown that the primary dynamics in solution and single-crystal phases are quite similar; hence, the photocycle dynamics and structural differences observed at longer time scales arise mostly from the structural restraints imposed by the crystal lattice rigidity versus the flexibility in solution. © 2014 FEBS.

The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles.

PubMed

Citron, Y Rose; Fagerstrom, Carey J; Keszthelyi, Bettina; Huang, Bo; Rusan, Nasser M; Kelly, Mark J S; Agard, David A

2018-01-01

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results, we suggest an expanded model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix.