Summary
Recently reported X-ray structures for large core fragments derived from human fibrinogen and fibrin make it possible to correlate structural and functional anomalies of known genetic ...variants. Here we examine a variety of amino acid replacements previously reported for hereditary dysfibrinogenemias, most of which are associated with impaired fibrin polymerization. For many of these we have modeled in the mutant amino acid and considered the structural consequences. We have also examined the cases of a small deletion and a large insertion, as well as the impact of substitutions in the GPRPam ligand that was co-crystallized with fragment double-D.
Computational analysis of crystallization trials Spraggon, Glen; Lesley, Scott A.; Kreusch, Andreas ...
Acta crystallographica. Section D, Biological crystallography.,
November 2002, Letnik:
58, Številka:
11
Journal Article
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A system for the automatic categorization of the results of crystallization experiments generated by robotic screening is presented. Images from robotically generated crystallization screens are ...taken at preset time intervals and analyzed by the computer program Crystal Experiment Evaluation Program (CEEP). This program attempts to automatically categorize the individual crystal experiments into a number of simple classes ranging from clear drop to mountable crystal. The algorithm first selects features from the images via edge detection and texture analysis. Classification is achieved via a self‐organizing neural net generated from a set of hand‐classified images used as a training set. New images are then classified according to this neural net. It is demonstrated that incorporation of time‐series information may enhance the accuracy of classification. Preliminary results from the screening of the proteome of Thermotoga maritima are presented showing the utility of the system.
Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium ...tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F420 deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F420 and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.
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► Crystal structures of Ddn, the reductase for antitubercular drug PA-824 ► Active site residues and F420-binding mode described ► The N terminus is important for PA-824 binding but could not be fully characterized ► An active homolog from Nocardia farcinica was identified and studied
NOD2 (nucleotide-binding oligomerization domain-containing protein 2) is an internal pattern recognition receptor that recognizes bacterial peptidoglycan and stimulates host immune responses. ...Dysfunction of NOD2 pathway has been associated with a number of autoinflammatory disorders. To date, direct inhibitors of NOD2 have not been described due to technical challenges of targeting the oligomeric protein complex. Receptor interacting protein kinase 2 (RIPK2) is an intracellular serine/threonine/tyrosine kinase, a key signaling partner, and an obligate kinase for NOD2. As such, RIPK2 represents an attractive target to probe the pathological roles of NOD2 pathway. To search for selective RIPK2 inhibitors, we employed virtual library screening (VLS) and structure based design that eventually led to a potent and selective RIPK2 inhibitor 8 with excellent oral bioavailability, which was used to evaluate the effects of inhibition of RIPK2 in various in vitro assays and ex vivo and in vivo pharmacodynamic models.
A recently developed method makes it possible to genetically encode unnatural amino acids with diverse physical, chemical or biological properties in Escherichia coli and yeast. We now show that this ...technology can be used to efficiently and site-specifically incorporate p-iodo-L-phenylalanine (iodoPhe) into proteins in response to an amber TAG codon. The selective introduction of the anomalously scattering iodine atom into proteins should facilitate single-wavelength anomalous dispersion experiments on in-house X-ray sources. To illustrate this, we generated a Phe153 → iodoPhe mutant of bacteriophage T4 lysozyme and determined its crystal structure using considerably less data than are needed for the equivalent experiment with cysteine and methionine. The iodoPhe residue, although present in the hydrophobic core of the protein, did not perturb the protein structure in any meaningful way. The ability to selectively introduce this and other heavy atom-containing amino acids into proteins should facilitate the structural study of proteins.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Photochemie P.B. Alper und Mitarbeiter zeigen in der Zuschrift auf S.916, wie lichtinduzierte Umlagerungen einfacher 2,5-Dienone sowie des Naturstoffs Santonin durch die Wahl des ...Amin-Reaktionspartners umgelenkt werden können.
Photochemie P. B. Alper und Mitarbeiter zeigen in der Zuschrift auf S. 916, wie lichtinduzierte Umlagerungen einfacher 2,5‐Dienone sowie des Naturstoffs Santonin durch die Wahl des ...Amin‐Reaktionspartners umgelenkt werden können.
Fibrinogen is a 340 kDa glycoprotein found in the blood plasma of all vertebrates. It is transformed into a fibrin clot by the action of thrombin. Recent X-ray structures of core fragments of both ...fibrinogen and fibrin have revealed many details about this polymerization event. These include structures of a 30 kDa recombinant γC domain, an 86 kDa fragment D from human fibrinogen and a cross-linked double-D fragment from fibrin.
The crystal structure of a recombinant αEC domain from human fibrinogen-420 has been determined at a resolution of 2.1 angstrom. The protein, which corresponds to the carboxyl domain of the αEchain, ...was expressed in and purified from Pichia pastoris cells. Felicitously, during crystallization an amino-terminal segment was removed, apparently by a contaminating protease, allowing the 201-residue remaining parent body to crystallize. An x-ray structure was determined by molecular replacement. The electron density was clearly defined, partly as a result of averaging made possible by there being eight molecules in the asymmetric unit related by noncrystallographic symmetry (P1 space group). Virtually all of an asparagine-linked sugar cluster is present. Comparison with structures of the β - and γ -chain carboxyl domains of human fibrinogen revealed that the binding cleft is essentially neutral and should not bind Gly-Pro-Arg or Gly-His-Arg peptides of the sort bound by those other domains. Nonetheless, the cleft is clearly evident, and the possibility of binding a carbohydrate ligand like sialic acid has been considered.
Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed ...to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.
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