共查询到20条相似文献,搜索用时 15 毫秒
1.
Joanne L. Parker Simon Newstead 《Protein science : a publication of the Protein Society》2013,22(11):1664-1668
In this report we highlight the latest trends in phasing methods used to solve alpha helical membrane protein structures and analyze the use of heavy atom metals for the purpose of experimental phasing. Our results reveal that molecular replacement is emerging as the most successful method for phasing alpha helical membrane proteins, with the notable exception of the transporter family, where experimentally derived phase information still remains the most effective method. To facilitate selection of heavy atoms salts for experimental phasing an analysis of these was undertaken and indicates that organic mercury salts are still the most successful heavy atoms reagents. Interestingly the use of seleno‐l ‐methionine incorporated protein has increased since earlier studies into membrane protein phasing, so too the use of SAD and MAD as techniques for phase determination. Taken together this study provides a brief snapshot of phasing methods for alpha helical membrane proteins and suggests possible routes for heavy atom selection and phasing methods based on currently available data. 相似文献
2.
Goulding CW Perry LJ Anderson D Sawaya MR Cascio D Apostol MI Chan S Parseghian A Wang SS Wu Y Cassano V Gill HS Eisenberg D 《Biophysical chemistry》2003,105(2-3):361-370
The growing list of fully sequenced genomes, combined with innovations in the fields of structural biology and bioinformatics, provides a synergy for the discovery of new drug targets. With this background, the TB Structural Genomics Consortium has been formed. This international consortium is comprised of laboratories from 31 universities and institutes in 13 countries. The goal of the consortium is to determine the structures of over 400 potential drug targets from the genome of Mycobacterium tuberculosis and analyze their structures in the context of functional information. We summarize the efforts of the UCLA consortium members. Potential drug targets were selected using a variety of bioinformatics methods and screened for certain physical and species-specific properties to yield a starting group of protein targets for structure determination. Target determination methods include protein phylogenetic profiles and Rosetta Stone methods, and the use of related biochemical pathways to select genes linked to essential prokaryotic genes. Criteria imposed on target selection included potential protein solubility, protein or domain size, and targets that lack homologs in eukaryotic organisms. In addition, some protein targets were chosen that are specific to M. tuberculosis, such as PE and PPE domains. Thus far, the UCLA group has cloned 263 targets, expressed 171 proteins and purified 40 proteins, which are currently in crystallization trials. Our efforts have yielded 13 crystals and eight structures. Seven structures are summarized here. Four of the structures are secreted proteins: antigen 85B; MPT 63, which is one of the three major secreted proteins of M. tuberculosis; a thioredoxin derivative Rv2878c; and potentially secreted glutamate synthetase. We also report the structures of three proteins that are potentially essential to the survival of M. tuberculosis: a protein involved in the folate biosynthetic pathway (Rv3607c); a protein involved in the biosynthesis of vitamin B5 (Rv3602c); and a pyrophosphatase, Rv2697c. Our approach to the M. tuberculosis structural genomics project will yield information for drug design and vaccine production against tuberculosis. In addition, this study will provide further insights into the mechanisms of mycobacterial pathogenesis. 相似文献
3.
Phasing of lysozyme crystals using co-crystallized barium ions was performed using single-wavelength anomalous diffraction (SAD) method using Cu Ka radiation with in-house source of data collection. As the ion binding sites vary with respect to the pH of the buffer during crystallization, the highly isomorphic forms of lysozyme crystals grown at acidic and alkaline pH were used for the study. Intrinsic sulphur anomalous signal was also utilized with anomalous signal from lower occupancy ions for phasing. The study showed that to solve the structure by SAD technique, 2.8-fold data redundancy was sufficient when barium was used as an anomalous marker in the in-house copper X-ray radiation source for data collection. Therefore, co-crystallization of proteins with barium containing salt can be a powerful tool for structure determination using lab source. 相似文献
4.
Thomas G. Flower James H. Hurley 《Protein science : a publication of the Protein Society》2021,30(4):728
The majority of crystal structures are determined by the method of molecular replacement (MR). The range of application of MR is limited mainly by the need for an accurate search model. In most cases, pre‐existing experimentally determined structures are used as search models. In favorable cases, ab initio predicted structures have yielded search models adequate for MR. The ORF8 protein of SARS‐CoV‐2 represents a challenging case for MR using an ab initio prediction because ORF8 has an all β‐sheet fold and few orthologs. We previously determined experimentally the structure of ORF8 using the single anomalous dispersion (SAD) phasing method, having been unable to find an MR solution to the crystallographic phase problem. Following a report of an accurate prediction of the ORF8 structure, we assessed whether the predicted model would have succeeded as an MR search model. A phase problem solution was found, and the resulting structure was refined, yielding structural parameters equivalent to the original experimental solution. 相似文献
5.
The crystal structures of two proteins, a putative pyrazinamidase/nicotinamidase from the dental pathogen Streptococcus mutans (SmPncA) and the human caspase-6 (Casp6), were solved by de novo arsenic single-wavelength anomalous diffraction (As-SAD) phasing method. Arsenic (As), an uncommonly used element in SAD phasing, was covalently introduced into proteins by cacodylic acid, the buffering agent in the crystallization reservoirs. In SmPncA, the only cysteine was bound to dimethylarsinoyl, which is a pentavalent arsenic group (As (V)). This arsenic atom and a protein-bound zinc atom both generated anomalous signals. The predominant contribution, however, was from the As anomalous signals, which were sufficient to phase the SmPncA structure alone. In Casp6, four cysteines were found to bind cacodyl, a trivalent arsenic group (As (III)), in the presence of the reducing agent, dithiothreitol (DTT), and arsenic atoms were the only anomalous scatterers for SAD phasing. Analyses and discussion of these two As-SAD phasing examples and comparison of As with other traditional heavy atoms that generate anomalous signals, together with a few arsenic-based de novo phasing cases reported previously strongly suggest that As is an ideal anomalous scatterer for SAD phasing in protein crystallography. 相似文献
6.
7.
Loren Baugh Larry A. Gallagher Rapatbhorn Patrapuvich Matthew C. Clifton Anna S. Gardberg Thomas E. Edwards Brianna Armour Darren W. Begley Shellie H. Dieterich David M. Dranow Jan Abendroth James W. Fairman David Fox III Bart L. Staker Isabelle Phan Angela Gillespie Ryan Choi Steve Nakazawa-Hewitt Mary Trang Nguyen Alberto Napuli Lynn Barrett Garry W. Buchko Robin Stacy Peter J. Myler Lance J. Stewart Colin Manoil Wesley C. Van Voorhis 《PloS one》2013,8(1)
Background
The genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite.Methodology/Principal Findings
We experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an “ortholog rescue” strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail.Conclusions/Significance
This collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request. 相似文献8.
Patching SG 《Molecular membrane biology》2011,28(6):370-397
Membrane proteins represent up to 30% of the proteins in all organisms, they are involved in many biological processes and are the molecular targets for around 50% of validated drugs. Despite this, membrane proteins represent less than 1% of all high-resolution protein structures due to various challenges associated with applying the main biophysical techniques used for protein structure determination. Recent years have seen an explosion in the number of high-resolution structures of membrane proteins determined by NMR spectroscopy, especially for those with multiple transmembrane-spanning segments. This is a review of the structures of polytopic integral membrane proteins determined by NMR spectroscopy up to the end of the year 2010, which includes both β-barrel and α-helical proteins from a number of different organisms and with a range in types of function. It also considers the challenges associated with performing structural studies by NMR spectroscopy on membrane proteins and how some of these have been overcome, along with its exciting potential for contributing new knowledge about the molecular mechanisms of membrane proteins, their roles in human disease, and for assisting drug design. 相似文献
9.
Recent advances in ab initio direct methods have enabled the solution of crystal structures of small proteins from native X-ray data alone, that is, without the use of fragments of known structure or the need to prepare heavy-atom or selenomethionine derivatives, provided that the data are available to atomic resolution. These methods are also proving to be useful for locating the selenium atoms or other anomalous scatterers in the multiple wavelength anomalous diffraction phasing of larger proteins at lower resolution. 相似文献
10.
《Molecular membrane biology》2013,30(6):370-397
AbstractMembrane proteins represent up to 30% of the proteins in all organisms, they are involved in many biological processes and are the molecular targets for around 50% of validated drugs. Despite this, membrane proteins represent less than 1% of all high-resolution protein structures due to various challenges associated with applying the main biophysical techniques used for protein structure determination. Recent years have seen an explosion in the number of high-resolution structures of membrane proteins determined by NMR spectroscopy, especially for those with multiple transmembrane-spanning segments. This is a review of the structures of polytopic integral membrane proteins determined by NMR spectroscopy up to the end of the year 2010, which includes both β-barrel and α-helical proteins from a number of different organisms and with a range in types of function. It also considers the challenges associated with performing structural studies by NMR spectroscopy on membrane proteins and how some of these have been overcome, along with its exciting potential for contributing new knowledge about the molecular mechanisms of membrane proteins, their roles in human disease, and for assisting drug design. 相似文献
11.
We have determined the X-ray crystal structure of the Mycobacterium tuberculosis (Mtb) gene product encoded by the open reading frame Rv0760c at 1.50 A resolution by single-wavelength anomalous dispersion (SAD) phasing of diffraction data from crystals of the selenomethionine-substituted protein. Refinement against diffraction data from the native protein resulted in R(work)=19.5% and R(free)=21.4%. The X-ray crystal structure shows that the homodimeric Rv0760c polypeptide has an alpha + beta conical barrel fold placing it among many structural neighbors of the nuclear transport factor 2 family (NTF2). This family is highly conserved in terms of structure; however the substrates and individual protein functions are diverse. The structures of native Rv0760c in several different crystal forms and Rv0760c bound to 17beta-estradiol 17-hemisuccinate (EH) have also been solved and analyzed. 相似文献
12.
Sabine Wenzel Tsuyoshi Imasaki Yuichiro Takagi 《Protein science : a publication of the Protein Society》2019,28(4):808-822
The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60–90% compared with native protein expression. 相似文献
13.
The high-resolution structure of the DNA (5′-GTGTACA-C-3′) with the selenium derivatization at the 2′-position of T2 was determined via MAD and SAD phasing. The selenium-derivatized structure (1.28 Å resolution) with the 2′-Se modification in the minor groove is isomorphorous to the native structure (2.0 Å). To directly compare with the conventional bromine derivatization, we incorporated bromine into the 5-postion of T4, determined the bromine-derivatized DNA structure at 1.5 Å resolution, and found that the local backbone torsion angles and solvent hydration patterns were altered in the structure with the Br incorporation in the major groove. Furthermore, while the native and Br-derivatized DNAs needed over a week to form reasonable-size crystals, we observed that the Se-derivatized DNAs grew crystals overnight with high-diffraction quality, suggesting that the Se derivatization facilitated the crystal formation. In addition, the Se-derivatized DNA sequences crystallized under a broader range of buffer conditions, and generally had a faster crystal growth rate. Our experimental results indicate that the selenium derivatization of DNAs may facilitate the determination of nucleic acid X-ray crystal structures in phasing and high-quality crystal growth. In addition, our results suggest that the Se derivatization can be an alternative to the conventional Br derivatization. 相似文献
14.
Glycosylation of proteins in prokaryotes has been known for the last few decades. Glycan structures and/or the glycosylation pathways have been experimentally characterized in only a small number of prokaryotes. Even this has become possible only during the last decade or so, primarily due to technological and methodological developments. Glycosylated proteins are diverse in their function and localization. Glycosylation has been shown to be associated with a wide range of biological phenomena. Characterization of the various types of glycans and the glycosylation machinery is critical to understand such processes. Such studies can help in the identification of novel targets for designing drugs, diagnostics, and engineering of therapeutic proteins. In view of this, the experimentally characterized pgl system of Campylobacter jejuni, responsible for N-linked glycosylation, has been used in this study to identify glycosylation loci in 865 prokaryotes whose genomes have been completely sequenced. Results from the present study show that only a small number of organisms have homologs for all the pgl enzymes and a few others have homologs for none of the pgl enzymes. Most of the organisms have homologs for only a subset of the pgl enzymes. There is no specific pattern for the presence or absence of pgl homologs vis-à-vis the 16S rRNA sequence-based phylogenetic tree. This may be due to differences in the glycan structures, high sequence divergence, horizontal gene transfer or non-orthologous gene displacement. Overall, the presence of homologs for pgl enzymes in a large number of organisms irrespective of their habitat, pathogenicity, energy generation mechanism, etc., hints towards the ubiquity of N-linked glycosylation in prokaryotes. 相似文献
15.
Puri M Robin G Cowieson N Forwood JK Listwan P Hu SH Guncar G Huber T Kellie S Hume DA Kobe B Martin JL 《Biomolecular engineering》2006,23(6):281-289
The flood of new genomic sequence information together with technological innovations in protein structure determination have led to worldwide structural genomics (SG) initiatives. The goals of SG initiatives are to accelerate the process of protein structure determination, to fill in protein fold space and to provide information about the function of uncharacterized proteins. In the long-term, these outcomes are likely to impact on medical biotechnology and drug discovery, leading to a better understanding of disease as well as the development of new therapeutics. Here we describe the high throughput pipeline established at the University of Queensland in Australia. In this focused pipeline, the targets for structure determination are proteins that are expressed in mouse macrophage cells and that are inferred to have a role in innate immunity. The aim is to characterize the molecular structure and the biochemical and cellular function of these targets by using a parallel processing pipeline. The pipeline is designed to work with tens to hundreds of target gene products and comprises target selection, cloning, expression, purification, crystallization and structure determination. The structures from this pipeline will provide insights into the function of previously uncharacterized macrophage proteins and could lead to the validation of new drug targets for chronic obstructive pulmonary disease and arthritis. 相似文献
16.
Marassi FM Das BB Lu GJ Nothnagel HJ Park SH Son WS Tian Y Opella SJ 《Methods (San Diego, Calif.)》2011,55(4):363-369
Rotational Alignment (RA) solid-state NMR provides the basis for a general method for determining the structures of membrane proteins in phospholipid bilayers under physiological conditions. Membrane proteins are high priority targets for structure determination, and are challenging for existing experimental methods. Because membrane proteins reside in liquid crystalline phospholipid bilayer membranes it is important to study them in this type of environment. The RA solid-state NMR approach we have developed can be summarized in five steps, and incorporates methods of molecular biology, biochemistry, sample preparation, the implementation of NMR experiments, and structure calculations. It relies on solid-state NMR spectroscopy to obtain high-resolution spectra and residue-specific structural restraints for membrane proteins that undergo rotational diffusion around the membrane normal, but whose mobility is otherwise restricted by interactions with the membrane phospholipids. High resolution spectra of membrane proteins alone and in complex with other proteins and ligands set the stage for structure determination and functional studies of these proteins in their native, functional environment. 相似文献
17.
Secretome analysis is important in pathogen studies. A fundamental and convenient way to identify secreted proteins is to first predict signal peptides, which are essential for protein secretion. However, signal peptides are highly complex functional sequences that are easily confused with transmembrane domains. Such confusion would obviously affect the discovery of secreted proteins. Transmembrane proteins are important drug targets, but very few transmembrane protein structures have been determined experimentally; hence, prediction of the structures is essential. In the field of structure prediction, researchers do not make assumptions about organisms, so there is a need for a general signal peptide predictor.To improve signal peptide prediction without prior knowledge of the associated organisms, we present a machine-learning method, called SVMSignal, which uses biochemical properties as features, as well as features acquired from a novel encoding, to capture biochemical profile patterns for learning the structures of signal peptides directly.We tested SVMSignal and five popular methods on two benchmark datasets from the SPdb and UniProt/Swiss-Prot databases, respectively. Although SVMSignal was trained on an old dataset, it performed well, and the results demonstrate that learning the structures of signal peptides directly is a promising approach. We also utilized SVMSignal to analyze proteomes in the entire HAMAP microbial database. Finally, we conducted a comparative study of secretome analysis on seven tuberculosis-related strains selected from the HAMAP database. We identified ten potential secreted proteins, two of which are drug resistant and four are potential transmembrane proteins.SVMSignal is publicly available at http://bio-cluster.iis.sinica.edu.tw/SVMSignal. It provides user-friendly interfaces and visualizations, and the prediction results are available for download. 相似文献
18.
Lundstrom K 《Journal of cellular and molecular medicine》2007,11(2):224-238
Structure determination has already proven useful for lead optimization and direct drug design. The number of high-resolution structures available in public databases today exceeds 30,000 and will definitely aid in structure-based drug design. Structural genomics approaches covering whole genomes, topologically similar proteins or gene families are great assets for further progress in the development of new drugs. However, membrane proteins representing 70% of current drug targets are poorly characterized structurally. The problems have been related to difficulties in obtaining large amount of recombinant membrane proteins as well as their purification and structure determination. Structural genomics has proven successful in developing new methods in areas from expression to structure determination by studying a large number of target proteins in parallel. 相似文献
19.
Structural analysis of a set of proteins resulting from a bacterial genomics project 总被引:1,自引:0,他引:1
Badger J Sauder JM Adams JM Antonysamy S Bain K Bergseid MG Buchanan SG Buchanan MD Batiyenko Y Christopher JA Emtage S Eroshkina A Feil I Furlong EB Gajiwala KS Gao X He D Hendle J Huber A Hoda K Kearins P Kissinger C Laubert B Lewis HA Lin J Loomis K Lorimer D Louie G Maletic M Marsh CD Miller I Molinari J Muller-Dieckmann HJ Newman JM Noland BW Pagarigan B Park F Peat TS Post KW Radojicic S Ramos A Romero R Rutter ME Sanderson WE Schwinn KD Tresser J Winhoven J Wright TA Wu L Xu J Harris TJ 《Proteins》2005,60(4):787-796
The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB. 相似文献
20.
The notorious difficulty of elucidating structures of membrane transporters by crystallography has long prevented our understanding of active transport mechanism coupled with ion/proton transport. The determination of the first crystal structure of the drug/H+ antiporter AcrB was a breakthrough for structure-based understanding of drug/H+ antiport. However, although AcrB is a major multidrug exporter in Gram-negative organisms, the majority of bacterial drug exporters are major facilitator superfamily (MFS) drug transporters. As no crystal structures have been solved for MFS transporters, the alternative protein-engineering methods are still very useful for estimating structures and functions of drug/H+ antiporters. This review describes this alternative approach for investigating the structure and function of tetracycline/H+ antiporters. 相似文献