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Journal Issue - Volume 6 Issue 7 (July 1997)

Abstract Insight into the dynamic properties of α‐lytic protease (αP) has been obtained through the use of low‐temperature X‐ray crystallography and multiple‐conformation refinement. Previous studies of αLP have shown that the residues around the active site are able to move significantly to accommodate substrates of different sizes. Here we show a link between the ability to accommodate ligands and the dynamics of the binding...

Abstract The hydrogen‐deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50°C at pD 5.6 and at 40 and 45 °C at pD 6.6. The hydrogen exchange rate constants of the hydrogen‐bonded residues varied over eight orders of magnitude at 25°C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in...

Abstract As a model for analyzing the role of charge repulsion in proteins and its shielding by the solvent, we designed a peptide of 27 amino acid residues that formed a homodimeric coiled‐coil. The interface between the coils consisted of hydro‐phobic Leu and Val residues, and 10 Lys residues per monomer were incorporated into the positions exposed to solvent. During the preparation of a disulfide‐linked dimer in which the two...

Abstract Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine, and tyrosine have been studied extensively, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins was discovered originally in prokaryotic organisms, but also has been found recently in...

Abstract The crystal structure of human α‐thrombin in complex with LY178550, a nonpeptidyl, active site‐directed inhibitor, has been solved to 2.07 α resolution by the method of X‐ray crystallography. The final model of the complex has a crystallographic R‐value of 21.5% (Rfree = 23.1%) with 0.014 Å and 2.4° standard deviation from ideal bond lengths and angles, respectively. Well‐defined electron density was observed for the...

Abstract A crystal structure of the serine protease, mouse glandular kallikrein 13 (mGK‐13) has been determined at 2.6‐Å resolution. This enzyme, isolated from the mouse submandibular gland, is also known as prorenin‐converting enzyme and cleaves submandibular gland Ren‐2 prorenin to yield active renin. The mGK‐13 structure is similar to other members of the mammalian serine protease family, having five conserved disulfide bonds and...

Abstract A hydrophobic folding unit cutting algorithm, originally developed for dissecting single‐chain proteins, has been applied to a dataset of dissimilar two‐chain protein‐protein interfaces. Rather than consider each individual chain separately, the two‐chain complex has been treated as a single chain. The two‐chain parsing results presented in this work show hydrophobicity to be a critical attribute of two‐state versus...

Abstract Unliganded bovine α‐thrombin and prethrombin‐2 have been co‐crystallized, in space group P21212, using either ammonium sulfate or polyethylene glycol 2000 (PEG2K), and their structures determined at 2.2 Å and 2.3 Å, respectively. Initial phases were determined by molecular replacement and refined using XPLOR to final R factors of 0.187 (Rfree = 0.255) and 0.190 (Rfree = 0.282) for the salt and PEG2K models, respectively. The apo‐enzyme...

Abstract The B‐domain of protein A has one of the simplest protein topologies, a three‐helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C‐terminal helical hairpin could be a potential folding intermediate. Kinetic...

Abstract Human cathepsin D is a lysosomal aspartic protease that has been implicated in breast cancer metastasis and Alzheimer's disease. Based on a crystal structure of a human cathepsin D‐pepstatin A complex, a series of statine‐containing inhibitors was designed, synthesized, and tested for inhibitory activity toward the enzyme in vitro. The compounds were modified systematically at individual positions (P4, P3, P2, P1, and P'2)...

Abstract Statistical potentials based on pairwise interactions between Cα atoms are commonly used in protein threading/fold‐recognition attempts. Inclusion of higher order interaction is a possible means of improving the specificity of these potentials. Delaunay tessellation of the Cα‐atom representation of protein structure has been suggested as a means of defining multi‐body interactions. A large number of parameters are...

Abstract Human placental S‐adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited papain digestion. The multiple cleavage sites in the enzyme were identified to be Lys94—Ala95, Tyr100‐Ala101, Glu243‐Ile244, Met367‐Ala368, Gln369‐Ile370, and Gly382‐Val383. Despite multiple cleavage sites in the backbone of the protein, the digested enzyme was able to maintain its quaternary structure and retain its full catalytic activity. The enzyme...

Abstract The folding of heat‐denatured ovalbumin, a non‐inhibitory serpin with a molecular size of 45 kDa, was examined. Ovalbumin was heat‐denatured at 80°C under nonreducing conditions at pH 7.5 and then cooled either slowly or rapidly. Slow cooling allowed the heat‐denatured ovalbumin to refold to its native structure with subsequent resistance to digestion by trypsin. Upon rapid cooling, by contrast, the heat‐denatured molecules...

Abstract The conformationally sensitive epitope for monoclonal antibody (mAb) 4B1, which uncouples lactose from H+ trans‐location in the lactose permease of Escherichia coli, is localized in the periplasmic loop between helices VII and VIII (loop VII/VIII) on one face of a short helical segment (Sun J, et al., 1996, Biochemistry35:990‐998). Comparison of sequences in the region corresponding to loop VII/VIII in members of Cluster 5 of the Major...

Abstract Tetranectin, a plasminogen‐binding protein belonging to the family of C‐type lectins, was expressed in E. coli and converted to its native form by in vitro refolding and proteolytic processing. Recombinant tetranectin—as well as natural tetranectin from human plasma—was shown by chemical cross‐linking analysis and SDS‐PAGE to be a homo‐trimer in solution as are other known members of the collectin family of C‐type lectins. Biochemical...

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