Protein Structure Relationships Research Articles

Identification and expression profiling of GAPDH family genes involved in response to Sclerotinia sclerotiorum infection and phytohormones in Brassica napus.

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a crucial enzyme in glycolysis, an essential metabolic pathway for carbohydrate metabolism across all living organisms. Recent research indicates that phosphorylating GAPDH exhibits various moonlighting functions, contributing to plant growth and development, autophagy, drought tolerance, salt tolerance, and bacterial/viral diseases resistance. However, in rapeseed (Brassica napus), the role of GAPDHs in plant immune responses to fungal pathogens remains unexplored. In this study, 28 genes encoding GAPDH proteins were revealed in B. napus and classified into three distinct subclasses based on their protein structural and phylogenetic relationships. Whole-genome duplication plays a major role in the evolution of BnaGAPDHs. Synteny analyses revealed orthologous relationships, identifying 23, 26, and 26 BnaGAPDH genes with counterparts in Arabidopsis, Brassica rapa, and Brassica oleracea, respectively. The promoter regions of 12 BnaGAPDHs uncovered a spectrum of responsive elements to biotic and abiotic stresses, indicating their crucial role in plant stress resistance. Transcriptome analysis characterized the expression profiles of different BnaGAPDH genes during Sclerotinia sclerotiorum infection and hormonal treatment. Notably, BnaGAPDH17, BnaGAPDH20, BnaGAPDH21, and BnaGAPDH22 exhibited sensitivity to S. sclerotiorum infection, oxalic acid, hormone signals. Intriguingly, under standard physiological conditions, BnaGAPDH17, BnaGAPDH20, and BnaGAPDH22 are primarily localized in the cytoplasm and plasma membrane, with BnaGAPDH21 also detectable in the nucleus. Furthermore, the nuclear translocation of BnaGAPDH20 was observed under H2O2 treatment and S. sclerotiorum infection. These findings might provide a theoretical foundation for elucidating the functions of phosphorylating GAPDH.

EncoMPASS: An encyclopedia of membrane proteins analyzed by structure and symmetry

Protein structure determination and prediction, active site detection, and protein sequence alignment techniques all exploit information about protein structure and structural relationships. For membrane proteins, however, there is limited agreement among available online tools for highlighting and mapping such structural similarities. Moreover, no available resource provides a systematic overview of quaternary and internal symmetries, and their orientation relative to the membrane, despite the fact that these properties can provide key insights into membrane protein function and evolution. Here, we describe the Encyclopedia of Membrane Proteins Analyzed by Structure and Symmetry (EncoMPASS), a database for relating integral membrane proteins of known structure from the points of view of sequence, structure, and symmetry. EncoMPASS is accessible through a web interface, and its contents can be easily downloaded. This allows the user not only to focus on specific proteins, but also to study general properties of the structure and evolution of membrane proteins.

Efficient Analysis of Proteome-Wide FPOP Data by FragPipe.

Monitoring protein structure before and after environmental alterations (e.g., different cell states) can give insights into the role and function of proteins. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry (MS) allows for monitoring of structural rearrangements by exposing proteins to OH radicals that oxidize solvent-accessible residues, indicating protein regions undergoing movement. Some of the benefits of FPOP include high throughput and a lack of scrambling due to label irreversibility. However, the challenges of processing FPOP data have thus far limited its proteome-scale uses. Here, we present a computational workflow for fast and sensitive analysis of FPOP data sets. Our workflow, implemented as part of the FragPipe computational platform, combines the speed of the MSFragger search with a unique hybrid search method to restrict the large search space of FPOP modifications. Together, these features enable more than 10-fold faster FPOP searches that identify 150% more modified peptide spectra than previous methods. We hope this new workflow will increase the accessibility of FPOP to enable more protein structure and function relationships to be explored.

Molecular and Structural Analysis of Specific Mutations from Saudi Isolates of SARS-CoV-2 RNA-Dependent RNA Polymerase and their Implications on Protein Structure and Drug-Protein Binding.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has stressed the global health system to a significant level, which has not only resulted in high morbidity and mortality but also poses a threat for future pandemics. This situation warrants efforts to develop novel therapeutics to manage SARS-CoV-2 in specific and other emerging viruses in general. This study focuses on SARS-CoV2 RNA-dependent RNA polymerase (RdRp) mutations collected from Saudi Arabia and their impact on protein structure and function. The Saudi SARS-CoV-2 RdRp sequences were compared with the reference Wuhan, China RdRp using a variety of computational and biophysics-based approaches. The results revealed that three mutations—A97V, P323I and Y606C—may affect protein stability, and hence the relationship of protein structure to function. The apo wild RdRp is more dynamically stable with compact secondary structure elements compared to the mutants. Further, the wild type showed stable conformational dynamics and interaction network to remdesivir. The net binding energy of wild-type RdRp with remdesivir is -50.76 kcal/mol, which is more stable than the mutants. The findings of the current study might deliver useful information regarding therapeutic development against the mutant RdRp, which may further furnish our understanding of SARS-CoV-2 biology.

Identification of transient receptor potential melastatin 3 proteotypic peptides employing an efficient membrane protein extraction method for natural killer cells.

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders, hence they make suitable targets in proteomic studies. However, extraction of membrane proteins is challenging due to their low abundance, stability, and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction, the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research. Methods: Two protocols, internally identified as Protocol 1 and 2, were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation, while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer’s recommendation. Using Protocol 2, protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150–200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples. Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients. Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships, particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states, such as ME/CFS.

Uncovering the Dominant Motion Modes of Allosteric Regulation Improves Allosteric Site Prediction.

Allostery is an important mechanism that biological systems use to regulate function at a distant site. Allosteric drugs have attracted much attention in recent years due to their high specificity and the possibility of overcoming existing drug-resistant mutations. However, the discovery of allosteric drugs remains challenging as allosteric regulation mechanisms are not clearly understood and allosteric sites cannot be accurately predicted. In this study, we analyzed the dominant modes that determine motion correlations between allosteric and orthosteric sites using the Gaussian network model and found that motion correlations between allosteric and orthosteric sites are dominated by either fast or slow vibrational modes. This dependence of modes results from the relative locations of the two sites and local secondary structures. Based on these analyses, we developed CorrSite2.0 to predict allosteric sites by taking the maximum of the Z-scores calculated from using either slow or fast modes. The prediction accuracy of CorrSite2.0 outperformed other commonly used allosteric site prediction methods with prediction accuracy over 90.0%. Our study uncovers the relationship of protein structure, dynamics, and allosteric regulation and demonstrates that using the dominant motion modes greatly improves allosteric site prediction accuracy. CorrSite2.0 has been integrated into the CavityPlus web server, which can be accessed at http://www.pkumdl.cn/cavityplus. CorrSite2.0 provides a powerful and user-friendly tool for allosteric drug and protein design.

Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms.

Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana, to rescue the collapsed vessels, short stems, and low cellulose content of the irx3-1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate "gating loop." A low-to-high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes. Vibrational sum frequency generation microscopy allowed tissue-specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.

A Minireview on Temperature Dependent Protein Conformational Sampling.

In this minireview we discuss the role of the more subtle conformational change-protein conformational sampling and connect it to the classic relationship of protein structure and function. The theory of pre-existing functional states of protein are discussed in context of alternate protein conformational sampling. Last, we discuss how temperature, ligand binding and mutations affect the protein conformational sampling mode which is linked to the protein function regulation. Thereviewincludes several protein systems that showed temperature dependent protein conformational sampling. We also specifically included two enzyme systems, thermophilic alcohol dehydrogenase (ht-ADH) and thermolysin which we previously studied when discussing temperature dependent protein conformational sampling.

Genome-Wide Investigation and Expression Profiling Under Abiotic Stresses of a Soybean Unknown Function (DUF21) and Cystathionine-\u03b2-Synthase (CBS) Domain-Containing Protein Family

Cystathionine-β-synthase (CBS) domain-containing proteins (CDCPs) constitute a large family in plants, and members of this family have been implicated in a variety of biological processes. However, the precise functions and the underlying mechanisms of most members of this family in plants remain to be elucidated. CBSDUF proteins belong to the CDCP superfamily, which contains one domain of unknown function (DUF21) and an N terminus that is adjacent to two intracellular CBS domains. In this study, a comprehensive genome database analysis of soybean was performed to investigate the role(s) of these CBSDUFs and to explore their nomenclature, classification, chromosomal distribution, exon–intron organization, protein structure, and phylogenetic relationships; the analysis identified a total of 18 putative CBSDUF genes. Using specific protein domains and phylogenetic analysis, the CBSDUF gene family was subdivided into eight groups. The soybean CBSDUF genes showed an uneven distribution on 12 chromosomes of Glycine max. RNA-seq transcriptome data from different tissues in public databases revealed tissue-specific and differential expression profiles of the GmCBSDUFs, and qPCR analysis revealed that certain groups of soybean CBSDUFs are likely involved in specific stress responses. In addition, GmCBSDUF3 transgenic Arabidopsis was subjected to phenotypic analysis under NaCl, PEG, and ABA stress treatments. The overexpression of GmCBSDUF3 could enhance tolerance to drought and salt stress in Arabidopsis. This study presents a first comprehensive look at soybean CBSDUF proteins and provides valuable resources for functionally elucidating this protein subgroup within the CBS domain-containing protein family.

John Arthur Joseph Pateman. 18 May 1926—18 May 2011

John Pateman was a distinguished British microbial geneticist. He came from a working-class area of London and a non-academic background. His earliest contribution to genetics was the discovery (together with John Fincham) of intracistronic complementation, an important phenomenon to understand the relationship of genetic information and protein structure; this at a time when the actual coding relationships of DNA and proteins were not yet worked out. Later on, he and his students made a fundamental contribution to the understanding of control of gene expression in eukaryotic microorganisms, providing some of the earliest examples of positive control. This work also led to the discovery of a new enzyme cofactor, the only one discovered through purely genetic evidence. He worked at various universities in Britain and Australia and trained a number of students who further developed the subject.

Trend of hybrid enzyme design in the big data era

The high efficiency and stability of enzymes are the basis for industrial application. Hybrid enzyme suitable for industrial applications could be constructed by many molecular biology technologies including tandem fusion, domain insertion and post-translational protein conjugation. However, the low expression and activity of hybrid enzyme limit its application in industrial production, and multifunctional design of a specific protein domain has been becoming a new trend. With the advent of high-throughput sequencing, biologists are starting to wrestling with massive data sets. Besides, the concept of protein sectors and co-evolution provides novel insight into the relationship of protein structure and function. The residues-covariation of a protein sector displays preference, which imparts functional diversity to different enzymes in the same family. The covariation-residues in specific protein sectors can be located based on the analysis of massive data, and then these functional residues can be assembled in a new enzyme variant using the biotechnology of synthetic biology, thus completing the redesign of natural enzymes. This indicates a new stage of designing hybrid enzyme, as well as the new trend of protein design in the era of biological big data.

Relationships between residue Voronoi volume and sequence conservation in proteins

BackgroundFunctional and biophysical constraints can cause different levels of sequence conservation in proteins. Previously, structural properties, e.g., relative solvent accessibility (RSA) and packing density of the weighted contact number (WCN), have been found to be related to protein sequence conservation (CS). The Voronoi volume has recently been recognized as a new structural property of the local protein structural environment reflecting CS. However, for surface residues, it is sensitive to water molecules surrounding the protein structure. Herein, we present a simple structural determinant termed the relative space of Voronoi volume (RSV); it uses the Voronoi volume and the van der Waals volume of particular residues to quantify the local structural environment. MethodsRSV (range, 0–1) is defined as (Voronoi volume−van der Waals volume)/Voronoi volume of the target residue. The concept of RSV describes the extent of available space for every protein residue. ResultsRSV and Voronoi profiles with and without water molecules (RSVw, RSV, VOw, and VO) were compared for 554 non-homologous proteins. RSV (without water) showed better Pearson's correlations with CS than did RSVw, VO, or VOw values. The mean correlation coefficient between RSV and CS was 0.51, which is comparable to the correlation between RSA and CS (0.49) and that between WCN and CS (0.56). ConclusionsRSV is a robust structural descriptor with and without water molecules and can quantitatively reflect evolutionary information in a single protein structure. Therefore, it may represent a practical structural determinant to study protein sequence, structure, and function relationships.

Revisiting protein structure, function, and evolution in the genomic era

The expansion of genomic data, three-dimensional structures of proteins, and computing power continues to improve our understanding of the evolution of protein structure and function relationships. As of June 2016, publically available databases contain more than 60 million unique protein sequences that group into 16,295 protein families that adopt ∼1400 different three-dimensional folds. This data supports the exploration of evolutionary relationships on protein structure and function to answer a basic question – how do changes in gene sequence lead to alterations in protein structure and to the tailoring of biological and chemical function? This mini-review aims to provide a primer on the basics of protein structure, how evolution of sequence leads to diversity in protein structure and function, how these changes occur, and the role of domains in protein evolution. Understanding how to use the vast amount of sequence and structural information may also aid in assessing if changes in protein sequence and/or structure are relevant for safety assessments of new commercial biotechnology products.

Detecting Non-Trivial Protein Structure Relationships

Detecting Non-Trivial Protein Structure Relationships

Insights into the Immunological Properties of Intrinsically Disordered Malaria Proteins Using Proteome Scale Predictions.

Malaria remains a significant global health burden. The development of an effective malaria vaccine remains as a major challenge with the potential to significantly reduce morbidity and mortality. While Plasmodium spp. have been shown to contain a large number of intrinsically disordered proteins (IDPs) or disordered protein regions, the relationship of protein structure to subcellular localisation and adaptive immune responses remains unclear. In this study, we employed several computational prediction algorithms to identify IDPs at the proteome level of six Plasmodium spp. and to investigate the potential impact of protein disorder on adaptive immunity against P. falciparum parasites. IDPs were shown to be particularly enriched within nuclear proteins, apical proteins, exported proteins and proteins localised to the parasitophorous vacuole. Furthermore, several leading vaccine candidates, and proteins with known roles in host-cell invasion, have extensive regions of disorder. Presentation of peptides by MHC molecules plays an important role in adaptive immune responses, and we show that IDP regions are predicted to contain relatively few MHC class I and II binding peptides owing to inherent differences in amino acid composition compared to structured domains. In contrast, linear B-cell epitopes were predicted to be enriched in IDPs. Tandem repeat regions and non-synonymous single nucleotide polymorphisms were found to be strongly associated with regions of disorder. In summary, immune responses against IDPs appear to have characteristics distinct from those against structured protein domains, with increased antibody recognition of linear epitopes but some constraints for MHC presentation and issues of polymorphisms. These findings have major implications for vaccine design, and understanding immunity to malaria.

PhyreStorm: A Web Server for Fast Structural Searches Against the PDB

The identification of structurally similar proteins can provide a range of biological insights, and accordingly, the alignment of a query protein to a database of experimentally determined protein structures is a technique commonly used in the fields of structural and evolutionary biology. The PhyreStorm Web server has been designed to provide comprehensive, up-to-date and rapid structural comparisons against the Protein Data Bank (PDB) combined with a rich and intuitive user interface. It is intended that this facility will enable biologists inexpert in bioinformatics access to a powerful tool for exploring protein structure relationships beyond what can be achieved by sequence analysis alone. By partitioning the PDB into similar structures, PhyreStorm is able to quickly discard the majority of structures that cannot possibly align well to a query protein, reducing the number of alignments required by an order of magnitude. PhyreStorm is capable of finding 93±2% of all highly similar (TM-score>0.7) structures in the PDB for each query structure, usually in less than 60s. PhyreStorm is available at http://www.sbg.bio.ic.ac.uk/phyrestorm/.

Using homology relations within a database markedly boosts protein sequence similarity search

Inference of homology from protein sequences provides an essential tool for analyzing protein structure, function, and evolution. Current sequence-based homology search methods are still unable to detect many similarities evident from protein spatial structures. In computer science a search engine can be improved by considering networks of known relationships within the search database. Here, we apply this idea to protein-sequence-based homology search and show that it dramatically enhances the search accuracy. Our new method, COMPADRE (COmparison of Multiple Protein sequence Alignments using Database RElationships) assesses the relationship between the query sequence and a hit in the database by considering the similarity between the query and hit's known homologs. This approach increases detection quality, boosting the precision rate from 18% to 83% at half-coverage of all database homologs. The increased precision rate allows detection of a large fraction of protein structural relationships, thus providing structure and function predictions for previously uncharacterized proteins. Our results suggest that this general approach is applicable to a wide variety of methods for detection of biological similarities. The web server is available at prodata.swmed.edu/compadre.

The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines

The hexameric MCM complex is the catalytic core of the replicative helicase in eukaryotic and archaeal cells. Here we describe the first in vivo analysis of archaeal MCM protein structure and function relationships using the genetically tractable haloarchaeon Haloferax volcanii as a model system. Hfx. volcanii encodes a single MCM protein that is part of the previously identified core group of haloarchaeal MCM proteins. Three structural features of the N-terminal domain of the Hfx. volcanii MCM protein were targeted for mutagenesis: the β7-β8 and β9-β10 β-hairpin loops and putative zinc binding domain. Five strains carrying single point mutations in the β7-β8 β-hairpin loop were constructed, none of which displayed impaired cell growth under normal conditions or when treated with the DNA damaging agent mitomycin C. However, short sequence deletions within the β7-β8 β-hairpin were not tolerated and neither was replacement of the highly conserved residue glutamate 187 with alanine. Six strains carrying paired alanine substitutions within the β9-β10 β-hairpin loop were constructed, leading to the conclusion that no individual amino acid within that hairpin loop is absolutely required for MCM function, although one of the mutant strains displays greatly enhanced sensitivity to mitomycin C. Deletions of two or four amino acids from the β9-β10 β-hairpin were tolerated but mutants carrying larger deletions were inviable. Similarly, it was not possible to construct mutants in which any of the conserved zinc binding cysteines was replaced with alanine, underlining the likely importance of zinc binding for MCM function. The results of these studies demonstrate the feasibility of using Hfx. volcanii as a model system for reverse genetic analysis of archaeal MCM protein function and provide important confirmation of the in vivo importance of conserved structural features identified by previous bioinformatic, biochemical and structural studies.

SCOPe: Structural Classification of Proteins—extended, integrating SCOP and ASTRAL data and classification of new structures

Structural Classification of Proteins—extended (SCOPe, http://scop.berkeley.edu) is a database of protein structural relationships that extends the SCOP database. SCOP is a manually curated ordering of domains from the majority of proteins of known structure in a hierarchy according to structural and evolutionary relationships. Development of the SCOP 1.x series concluded with SCOP 1.75. The ASTRAL compendium provides several databases and tools to aid in the analysis of the protein structures classified in SCOP, particularly through the use of their sequences. SCOPe extends version 1.75 of the SCOP database, using automated curation methods to classify many structures released since SCOP 1.75. We have rigorously benchmarked our automated methods to ensure that they are as accurate as manual curation, though there are many proteins to which our methods cannot be applied. SCOPe is also partially manually curated to correct some errors in SCOP. SCOPe aims to be backward compatible with SCOP, providing the same parseable files and a history of changes between all stable SCOP and SCOPe releases. SCOPe also incorporates and updates the ASTRAL database. The latest release of SCOPe, 2.03, contains 59 514 Protein Data Bank (PDB) entries, increasing the number of structures classified in SCOP by 55% and including more than 65% of the protein structures in the PDB.

BBX proteins in green plants: Insights into their evolution, structure, feature and functional diversification

The B-box domain is conserved in a large number of proteins involved in cell growth control, differentiation and transcriptional regulation among animal and plant species. In Arabidopsis thaliana, some works have found that B-box proteins (BBX) play central developmental functions in flowering, light and abiotic stress signaling. Despite the functional importance of this protein family, evolutionary and structural relationships of BBX proteins have not been extensively investigated in the plant kingdom. Using a phylogenetic approach, we conducted a comprehensive evolutionary analysis of the BBX protein family in twelve plant species (four green algae, one moss, one lycophyte, three monocots and three dicots). The analysis classified 214 BBX proteins into five structure groups, which evolved independently at early stages of green plant evolution. We showed that the B-box consensus sequences of each structure groups retained a common and conserved domain topology. Furthermore, we identified seven novel motifs specific to each structure group and a valine–proline (VP) pair conserved at the C-terminus domain in some BBX proteins suggesting that they are required for protein–protein interactions. As it has been documented in mammalian systems, we also found monopartite and bipartite amino acid sequences at the C-terminus domain that could function as nuclear localization signals (NLSs). The five BBX structure groups evolved constrained by the conservation of amino acid sequences in the two B-boxes, but radiating variation into NLSs and novel motifs of each structural group. We suggest that these features are the functional basis for the BBX protein diversity in green plants.