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SPINE bioinformatics and data-management aspects of high-throughput structural biology.
SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.
Gene polymorphism in Netherton and common atopic disease.
Atopic dermatitis (AD) and asthma are characterized by IgE-mediated atopic (allergic) responses to common proteins (allergens), many of which are proteinases. Loci influencing atopy have been localized to a number of chromosomal regions, including the chromosome 5q31 cytokine cluster. Netherton disease is a rare recessive skin disorder in which atopy is a universal accompaniment. The gene underlying Netherton disease (SPINK5) encodes a 15-domain serine proteinase inhibitor (LEKTI) which is expressed in epithelial and mucosal surfaces and in the thymus. We have identified six coding polymorphisms in SPINK5 (Table 1) and found that a Glu420-->Lys variant shows significant association with atopy and AD in two independent panels of families. Our results implicate a previously unrecognized pathway for the development of common allergic illnesses.
Solution structure of a HNA-RNA hybrid.
BACKGROUND: Synthetic nucleic acid analogues with a conformationally restricted sugar-phosphate backbone are widely used in antisense strategies for biomedical and biochemical applications. The modified backbone protects the oligonucleotides against degradation within the living cell, which allows them to form stable duplexes with sequences in target mRNAs with the aim of arresting their translation. The biologically most active antisense oligonucleotides also trigger cleavage of the target RNA through activation of endogenous RNase H. Systematic studies of synthetic oligonucleotides have also been conducted to delineate the origin of the chirality of DNA and RNA that are both composed of D-nucleosides. RESULTS: Hexitol nucleic acids (HNA) are the first example of oligonucleotides with a six-membered carbohydrate moiety that can bind strongly and selectively to complementary RNA oligomers. We present the first high resolution nuclear magnetic resonance structure of a HNA oligomer bound to a complementary RNA strand. The HNA-RNA complex forms an anti-parallel heteroduplex and adopts a helical conformation that belongs to the A-type family. Possibly, due to the rigidity of the rigid chair conformation of the six-membered ring both the HNA and RNA strand in the duplex are well defined. The observed absence of end-fraying effects also indicate a reduced conformational flexibility of the HNA-RNA duplex compared to canonical dsRNA or an RNA-DNA duplex. CONCLUSIONS: The P-P distance across the minor groove, which is close to A-form, and the rigid conformation of the HNA-RNA complex, explain its resistance towards degradation by Rnase H. The A-form character of the HNA-RNA duplex and the reduced flexibility of the HNA strand is possibly responsible for the stereoselectivity of HNA templates in non-enzymatic replication of oligonucleotides, supporting the theory that nucleosides with six-membered rings could have existed at some stage in molecular evolution.
Recording information on protein complexes in an information management system.
The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.
xtalPiMS: a PiMS-based web application for the management and monitoring of crystallization trials.
A major advance in protein structure determination has been the advent of nanolitre-scale crystallization and (in a high-throughput environment) the development of robotic systems for storing and imaging crystallization trials. Most of these trials are carried out in 96-well (or higher density) plates and managing them is a significant information management challenge. We describe xtalPiMS, a web-based application for the management and monitoring of crystallization trials. xtalPiMS has a user-interface layer based on the standards of the Protein Information Management System (PiMS) and a database layer which links the crystallization trial images to the meta-data associated with a particular crystallization trial. The user interface has been optimized for the efficient monitoring of high-throughput environments with three different automated imagers and work to support a fourth imager is in progress, but it can even be of use without robotics. The database can either be a PiMS database or a legacy database for which a suitable mapping layer has been developed.
Modulation of fibroblast growth factor-2 receptor binding, signaling, and mitogenic activity by heparin-mimicking polysulfonated compounds.
Basic fibroblast growth factor (FGF-2) interacts with high-affinity tyrosine-kinase fibroblast growth factor receptors (FGFRs) and low-affinity heparan sulfate proteoglycans (HSPGs) in target cells. Both interactions are required for FGF-2-mediated biological responses. Here we report the FGF-2 antagonist activity of novel synthetic sulfonic acid polymers with distinct chemical structures and molecular masses (MMs). PAMPS [poly(2-acrylamido-2-methyl-1-propanesulfonic acid)], (MM approximately 7,000-10,000), PAS [poly(anetholesulfonic acid)], (MM approximately 9,000-11,000), PSS [poly(4-styrenesulfonic acid)], (MM = 70,000), and poly(vinylsulfonic acid) (MM = 2,000), inhibited FGF-2 binding to HSPGs and FGFRs in fetal bovine aortic endothelial GM 7373 cells. They also abrogated the formation of the HSPG/FGF-2/FGFR ternary complex, as evidenced by their capacity to prevent FGF-2-mediated cell-cell attachment of FGFR-1-overexpressing, HSPG-deficient Chinese hamster ovary cells to wild-type HSPG-bearing cells. Direct interaction of the polysulfonates with FGF-2 was demonstrated by their ability to protect the growth factor from proteolytic cleavage. Accordingly, molecular modeling, based on the crystal structure of the interaction of FGF-2 with a heparin hexamer, showed the feasibility of docking PAMPS into the heparin-binding domain of FGF-2. In agreement with their FGF-2-binding capacity, PSS, PAS, and PAMPS inhibited FGF-2-induced cell proliferation in GM 7373 cells and murine brain microvascular endothelial cells. The antiproliferative activity of these compounds was associated with the abrogation of FGF-2-induced tyrosine phosphorylation of FGFR-1. Moreover, the polysulfonates PSS and PAS inhibited FGF-2-induced activation of mitogen-activated protein kinase-1/2, involved in FGF-2 signal transduction. In conclusion, sulfonic acid polymers bind FGF-2 by mimicking heparin interaction. These compounds may provide a tool to inhibit FGF-2-induced endothelial cell proliferation in angiogenesis and tumor growth.
A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization.
Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.
A proline-to-histidine substitution at position 225 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) sensitizes HIV-1 RT to BHAP U-90152.
Two mutant virus strains in which the novel P225H mutation appeared in a V106A reverse transcriptase (RT)-mutated genetic background upon treatment of human immunodeficiency virus type 1 (HIV-1) with quinoxaline S-2720 were isolated. Surprisingly, the addition of the P225H mutation to the V106A RT mutant genetic background resensitized the V106A RT mutant virus to the non-nucleoside RT inhibitor (NNRTI) BHAP U-90152, but not to other NNRTIs. Construction of both recombinant viruses and recombinant RTs containing the V106A, P225H and V106A+P225H mutations revealed that P225H was indeed responsible for the marked potentiation of the antiviral activity of BHAP against the P225H single-mutant virus and the V106A+P225H double-mutant virus when compared to wild-type and V106A single-mutant viruses, respectively. An explanation for the markedly increased sensitivity of the P225H mutant HIV-1 RT to BHAP and not to the other NNRTIs was provided by the unique features of the X-ray structure of the RT-BHAP complex.
Lack of susceptibility of bicyclic nucleoside analogs, highly potent inhibitors of varicella-zoster virus, to the catabolic action of thymidine phosphorylase and dihydropyrimidine dehydrogenase.
The susceptibility of the bicyclic nucleoside analogs (BCNAs), highly potent and selective inhibitors of varicella-zoster virus (VZV), to the enzymes involved in nucleoside/nucleobase catabolism has been investigated in comparison with the established anti-VZV agent (E)-5-(2-bromovinyl)-2'-deoxyuridine [BVDU; brivudine (Zostex)]. Whereas human and bacterial thymidine phosphorylases (TPases) efficiently converted BVDU to its antivirally inactive free base (E)-5-(2-bromovinyl)uracil (BVU), BCNAs showed no evidence of conversion to the free base in the presence of these enzymes. The lack of substrate affinity of TPase for the BCNAs could be rationalized by computer-assisted molecular modeling of the BCNAs in the TPase active site. Moreover, in contrast with BVU, which is a potent and selective inhibitor of dihydropyrimidine dehydrogenase (DPD) (50% inhibitory concentration; 10 microM in the presence of a 25 microM concentration of the natural substrate thymine), the free base (Cf 1381; 6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) of BCNA (Cf 1368; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) and the free base Cf 2200 [6-(4-n-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one] of BCNA (Cf 1743; 3-(2'-deoxy-beta-D-ribofuranosyl)-6-(4-n-pentylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one) did not inhibit the DPD-catalyzed catabolic reaction of pyrimidine bases (i.e., thymine) and pyrimidine base analogs [i.e., 5-fluorouracil (FU)] at a concentration of 250 microM. Consequently, whereas BVU caused a dramatic rise of FU levels in FU-treated mice, the BCNAs did not affect FU levels in such mice. From our data it is evident that BCNAs represent highly stable anti-VZV compounds that are not susceptible to breakdown by nucleoside/nucleobase catabolic enzymes and are not expected to interfere with cellular catabolic processes such as those involved in FU catabolism.
Mutational analysis of trp-229 of human immunodeficiency virus type 1 reverse transcriptase (RT) identifies this amino acid residue as a prime target for the rational design of new non-nucleoside RT inhibitors.
Trp-229 is part of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-binding pocket of HIV type 1 (HIV-1) reverse transcriptase (RT), and is also part of the "primer grip" of HIV-1 RT. Using site-directed mutagenesis, seven RT mutants were constructed bearing the mutations 229Phe, 229Tyr, 229Ile, 229His, 229Lys, 229Cys, and 229Gln. We found that all of the mutants showed severely compromised RNA- and DNA-dependent DNA polymerase activities (<2% of wild-type activity). The recombinant 229Phe and 229Tyr RT enzymes were among the mutant enzymes with the highest activity (0.7 and 1.1% of wild-type activity, respectively) and we evaluated these for resistance against several NNRTIs. No resistance was found for the 229Phe RT, but the 229Tyr RT showed a approximately 20-fold resistance against UC-781 and lower resistance against emivirine and nevirapine. Attempts to make recombinant virus strains bearing the single 229Phe or 229Tyr RT mutation failed. Experiments in which we varied the pentenyl ether substituent of the thiocarboxanilide UC-781 revealed that Trp-229 can be specifically targeted by NNRTIs and that an alkenyloxy group length of five atoms assures an optimal interaction of the thiocarboxanilides with Trp-229. Our findings indicate that Trp-229, when combined with other crucial immutable amino acids (i.e., Tyr-318), is an appropriate candidate for the targeted design of new NNRTIs.
The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory.
The techniques used in protein production and structural biology have been developing rapidly, but techniques for recording the laboratory information produced have not kept pace. One approach is the development of laboratory information-management systems (LIMS), which typically use a relational database schema to model and store results from a laboratory workflow. The underlying philosophy and implementation of the Protein Information Management System (PiMS), a LIMS development specifically targeted at the flexible and unpredictable workflows of protein-production research laboratories of all scales, is described. PiMS is a web-based Java application that uses either Postgres or Oracle as the underlying relational database-management system. PiMS is available under a free licence to all academic laboratories either for local installation or for use as a managed service.
Missense mutations in the homeodomain of HOXD13 are associated with brachydactyly types D and E.
HOXD13, the most 5' gene of the HOXD cluster, encodes a homeodomain transcription factor with important functions in limb patterning and growth. Heterozygous mutations of human HOXD13, encoding polyalanine expansions or frameshifts, are believed to act by dominant negative or haploinsufficiency mechanisms and are predominantly associated with synpolydactyly phenotypes. Here, we describe two mutations of HOXD13 (923C-->G encoding Ser308Cys and 940A-->C encoding Ile314Leu) that cause missense substitutions within the homeodomain. Both are associated with distinctive limb phenotypes in which brachydactyly of specific metacarpals, metatarsals, and phalangeal bones is the most constant feature, exhibiting overlap with brachydactyly types D and E. We investigated the binding of synthetic mutant proteins to double-stranded DNA targets in vitro. No consistent differences were found for the Ser308Cys mutation compared with the wild type, but the Ile314Leu mutation (which resides at the 47th position of the homeodomain) exhibited increased affinity for a target containing the core recognition sequence 5'-TTAC-3' but decreased affinity for a 5'-TTAT-3' target. Molecular modeling of the Ile314Leu mutation indicates that this mixed gain and loss of affinity may be accounted for by the relative positions of methyl groups in the amino acid side chain and target base.
Modeling study of human renal chloride channel (hCLC-5) mutations suggests a structural-functional relationship.
BACKGROUND: Dent's disease, a renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, and nephrolithiasis, is due to inactivating mutations in the X-linked renal-specific chloride channel, hCLC-5. The x-ray crystal structures of two bacterial chloride channels (CLCs) have recently been established, thereby allowing us to construct a model for hCLC-5 and further examine the role of its mutations. METHODS: The data regarding 49 hCLC-5 mutations were reviewed. Thirty-four mutations that predicted absent or truncated channels were excluded. The remaining 15 mutations (one in-frame insertion and 14 missense mutations), 12 of which have been studied electrophysiologically, were assessed. The hCLC-5 sequence was aligned with the Salmonella typhimurium and Escherichia coli sequences and used to map the hCLC-5 mutations onto a three-dimensional model. RESULTS: hCLC-5 is a homodimeric protein, with each subunit consisting of 18 helices. None of the missense mutations involved the chloride (Cl-) selectivity filter, but 12 of the 15 mutations were found to be clustered at the interface of the two subunits. Six of these mutations occurred in two of the helices that either form part of the interface or lie in close proximity to the interface, and three other mutations that did not lead to complete loss of Cl- conductance were at the edge of the interface. CONCLUSION: These results demonstrate a crucial role for the interaction between the two subunits at the interface of the homodimeric hCLC-5.
Further additions to MolScript version 1.4, including reading and contouring of electron-density maps.
MolScript is one of the most popular programs for the generation of publication-quality figures and the recent re-working of the program should ensure its continued popularity. However, some functionality of particular interest to crystallographers is not part of the standard program. A modified MolScript version 1.4 has been described previously, with more flexible colouring schemes among its new features. This report describes further enhancements to MolScript version 1.4, including facilities for drawing rods for helices and ribbons for oligonucleotides and allowing several formats of electron-density maps to be read and contoured using either lines or smoothed triangulated surfaces.
An extensively modified version of MolScript that includes greatly enhanced coloring capabilities.
Owing to its flexibility, MolScript has become one of the most widely used programs for generating publication-quality molecular graphics. Integration with the Raster3D package, to allow the production of photorealistic rendered images, has increased its popularity still further. However, this intensive use has shown the need for enhancement of some areas of the program, especially for controlling the coloring of atoms, bonds, and molecules. This work describes a heavily modified version of MolScript that has added syntax for describing complicated coloring schemes and also has new graphics commands. Enhancements include drawing split-bond ball-and-stick models, smoothly varying the color of molecules (color ramping), abrupt color changes within secondary structural units, and the creation of dashed bonds. Making use of these added features is simple because all MolScript syntax is still supported and one typically needs only to add a few control commands. The final section of this article suggests some uses for this modified MolScript and provides illustrative examples.
Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors.
The structure of unliganded HIV-1 reverse transcriptase has been determined at 2.35 A resolution and refined to an R-factor of 0.219 (for all data) with good stereochemistry. The unliganded structure was produced by soaking out a weak binding non-nucleoside inhibitor, HEPT, from pregrown crystals. Comparison with the structures of four different RT and non-nucleoside inhibitor complexes reveals that only minor domain rearrangements occur, but there is a significant repositioning of a three-stranded beta-sheet in the p66 subunit (containing the catalytic aspartic acid residues 110, 185 and 186) with respect to the rest of the polymerase site. This suggests that NNIs inhibit RT by locking the polymerase active site in an inactive conformation, reminiscent of the conformation observed in the inactive p51 subunit.
High resolution structures of HIV-1 RT from four RT-inhibitor complexes.
We have determined the structures of four complexes of HIV-1 reverse transcriptase with non-nucleoside inhibitors, three fully refined at high resolution. The highest resolution structure is of the RT-nevirapine complex which has an R-factor of 0.186 and a root-mean-square bond length deviation of 0.015 A for all data to 2.2 A. The structures reveal a common mode of binding for these chemically diverse compounds. The common features of binding are largely hydrophobic interactions and arise from induced shape complementarity achieved by conformational rearrangement of the enzyme and conformational/configurational rearrangement of the compounds.
Crystal structure of a soluble CD28-Fab complex.
Naive T cell activation requires signaling by the T cell receptor and by nonclonotypic cell surface receptors. The most important costimulatory protein is the monovalent homodimer CD28, which interacts with CD80 and CD86 expressed on antigen-presenting cells. Here we present the crystal structure of a soluble form of CD28 in complex with the Fab fragment of a mitogenic antibody. Structural comparisons redefine the evolutionary relationships of CD28-related proteins, antigen receptors and adhesion molecules and account for the distinct ligand-binding and stoichiometric properties of CD28 and the related, inhibitory homodimer CTLA-4. Cryo-electron microscopy-based comparisons of complexes of CD28 with mitogenic and nonmitogenic antibodies place new constraints on models of antibody-induced receptor triggering. This work completes the initial structural characterization of the CD28-CTLA-4-CD80-CD86 signaling system.