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Journal Issue - Volume 9 Issue 12 (2000)

Abstract Because of the simplicity and regularity of the α‐helical coiled coil relative to other structural motifs, it can be conveniently used to clarify the molecular interactions responsible for protein folding and stability. Here we describe the de novo design and characterization of a two heptad‐repeat peptide stabilized by a complex network of inter‐ and intrahelical salt bridges. Circular dichroism spectroscopy and analytical...

Abstract Polymerization of metallothioneins is one of the usually encountered puzzles during the research process of metallothioneins' structure and function. Our work focuses on the cysteine independently occurred polymerization from metallothioneins monomers in different milieus, while it leaves out the aggregation caused by the oxidation of cysteine, because the latter circumstance is the result of purification lapsus. After the...

Abstract Structural genomic projects envision almost routine protein structure determinations, which are currently imaginable only for small proteins with molecular weights below 25,000 Da. For larger proteins, structural insight can be obtained by breaking them into small segments of amino acid sequences that can fold into native structures, even when isolated from the rest of the protein. Such segments are autonomously folding...

Abstract Residue Asn47 at position L1 of a type II′ β‐turn of the α‐spectrin SH3 domain is located in a disallowed region of the Ramachandran plot (ϕ = 56 ± 12, ± = −118 ± 17). Therefore, it is expected that replacement of Asn47 by Gly should result in a considerable stabilization of the protein. Thermodynamic analysis of the N47G and N47A mutants shows that the change in free energy is small (∼0.7 kcal/mol; ∼3 kJ/mol) and...

Abstract The Ntn‐hydrolases (N‐terminal nucleophile) are a superfamily of diverse enzymes that has recently been characterized. All of the proteins in this family are activated autocatalytically; they contain an N‐terminally located catalytic nucleophile, and they cleave an amide bond. In the present study, the structures of four enzymes of this superfamily are compared in more detail. Although the amino acid sequence homology is...

Abstract A right‐handed parallel β‐helix of 400 residues in 13 tightly packed coils is a major motif of the chains forming the trimeric P22 tailspike adhesin. The β‐helix domains of three identical subunits are side‐by‐side in the trimer and make predominantly hydrophilic inter‐subunit contacts (Steinbacher S et al., 1994, Science 265:383‐386). After the 13th coil the three individual β‐helices terminate and the chains wrap around each other to...

Abstract Orthologs typically retain the same function in the course of evolution. Using β‐decarboxylating dehydrogenase family as a model, we demonstrate that orthologs can be confidently identified. The strategy is based on our recent findings that substitutions of only a few amino acid residues in these enzymes are sufficient to exchange substrate and coenzyme specificities. Hence, the few major specificity determinants can serve...

Abstract Backbone dynamics of the basic/helix‐loop‐helix domain of Pho4 from Saccharomyces cerevisae have been probed by NMR techniques, in the absence of DNA, nonspecifically bound to DNA and bound to cognate DNA. Alpha proton chemical shift indices and nuclear Overhauser effect patterns were used to elucidate the secondary structure in these states. These secondary structures are compared to the co‐crystal complex of Pho4 bound to a cognate...

Abstract The WW domain is an approximately 38 residue peptide‐binding motif that binds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one‐third of its three‐stranded antiparallel β‐sheet. Improved binding to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three different concentrations of proteinase K to simultaneously...

Abstract X‐linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B‐cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C‐γ phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B‐cell linker protein (BLNK)...

Abstract The immunosuppressant drug cyclosporin A (CsA) inhibits T‐cell function by blocking the phosphatase activity of calcineurin. This effect is mediated by formation of a complex between the drug and cyclophilin (CyP), which creates a composite surface able to make high‐affinity contacts with calcineurin. In vitro, the CyPB/CsA complex is more effective in inhibiting calcineurin than the CyPA/CsA and CyPC/CsA complexes,...

Abstract The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain...

Abstract One of the proposed roles of the GroEL‐GroES cavity is to provide an “infinite dilution” folding chamber where protein substrate can fold avoiding deleterious off‐pathway aggregation. Support for this hypothesis has been strengthened by a number of studies that demonstrated a mandatory GroES requirement under nonpermissive solution conditions, i.e., the conditions where proteins cannot spontaneously fold. We have found that...

Abstract To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 °C,...

Abstract We have used X‐ray fiber diffraction to probe the structure of fibers of tau and tau fragments. Fibers of fragments from the microtubule binding domain had a cross β‐structure that closely resembles that reported both for neurofibrillary tangles found in Alzheimer's disease brain and for fibrous lesions from other protein folding diseases. In contrast, fibers of full‐length tau had a different, more complex structure....

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