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Journal Issue - Volume 7 Issue 10 (October 1998)

Abstract Cluster analysis is presented as a technique for analyzing the conservation and chemistry of water sites from independent protein structures, and applied to thrombin, trypsin, and bovine pancreatic trypsin inhibitor (BPTI) to locate shared water sites, as well as those contributing to specificity. When several protein structures are superimposed, complete linkage cluster analysis provides an objective technique for...

Abstract DsbA is the strongest protein disulfide oxidant yet known and is involved in catalyzing protein folding in the bacterial periplasm. Its strong oxidizing power has been attributed to the lowered pKa of its reactive active site cysteine and to the difference in thermodynamic stability between the oxidized and the reduced form. However, no structural data are available for the reduced state. Therefore, an NMR study of DsbA in its two...

Abstract Despite its relatively low pH and temperature optimum, the xylanase from Penicillium simplicissimum performs exceedingly well under conditions of paper bleaching. We have purified and characterized this enzyme, which belongs to family 10 of glycosyl hydrolases. Its gene was cloned, and the sequence of the protein was deduced from the nucleotide sequence. The xylanase was crystallized from ammonium sulfate at pH 8.4, and X‐ray data were...

Abstract Point mutations at the dimer interface of the homodimeric enzyme ascorbate peroxidase (APx) were constructed to assess the role of quaternary interactions in the stability and activity of APx. Analysis of the APx crystal structure shows that Glu 112 forms a salt bridge with Lys20 and Arg24 of the opposing subunit near the axis of dyad symmetry between the subunits. Two point mutants, E112A and E112K, were made to determine...

Abstract The crystal structure of plastocyanin from spinach has been determined using molecular replacement, with the structure of plastocyanin from poplar as a search model. Successful crystallization was facilitated by site‐directed mutagenesis in which residue Gly8 was substituted with Asp. The region around residue 8 was believed to be too mobile for the wild‐type protein to form crystals despite extensive screening. The current...

Abstract The three‐dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 Å resolution by the X‐ray crysallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH...

Abstract The binding of two 5‐substituted‐l,3,4‐thiadiazole‐2‐thione inhibitors to the matrix metalloproteinase stromelysin (MMP‐3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1‐P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond...

Abstract The transforming growth factor β (TGF‐β) binding protein‐like (TB) domain is found principally in proteins localized to extracellular matrix fibrils, including human fibrillin‐1, the defective protein in the Marfan syndrome. Analysis of the nuclear magnetic resonance (NMR) data for the sixth TB module from human fibrillin‐1 has revealed the existence of two stable conformers that differ in the isomerization states of two...

Abstract We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the αI/βB loop, of the N‐domain that are critical for in vivo folding of the maltose‐binding protein (MalE31). The tertiary context‐dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent αβ loop of the C‐domain to the same amino acid substitutions (MaIE219). Moving the...

Abstract Efficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single‐chain recombinant form of the protease has been constructed in which NS4A residues 21‐32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3ndash181) through a...

Abstract For echidna and canine milk lysozymes, which were presumed to be the calcium‐binding lysozymes by their amino acid sequences, we have quantitated their calcium‐binding strength and examined their guanidine unfolding profiles. The calcium‐binding constants of echidna and canine lysozymes were determined to be 8.6 × 106 M−1 and 8.9 X 106 M−1 in 0.1 M KCl at pH 7.1 and 20 °C, respectively. The unfolding of decalcified...

Abstract Transcarboxylase (TC) from Propionibacterium shermanii, a biotin‐dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl‐CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier. The 1.3S is a 123‐amino acid polypeptide (12.6 kDa), to which biotin is covalently attached at Lys 89. We have expressed 1.3S in Escherichia coli with...

Abstract The RNase H domain from HIV‐1 (HIV RNase H) encodes an essential retroviral activity. Refolding of the isolated HIV RNase H domain shows a kinetic intermediate detectable by stopped‐flow far UV circular dichroism and pulse‐labeling H/D exchange. In this intermediate, strands 1, 4, and 5 as well as helices A and D appear to be structured. Compared to its homolog from Escherichia coli, the rate limiting step in refolding of...

Abstract Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat‐stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10,...

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