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Journal Issue - Volume 5 Issue 4 (April 1996)

Abstract Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of factor D activity is instead attained by a novel mechanism that depends on reversible conformational changes for expression and control of catalytic activity. These conformational changes are believed to be induced by the single natural...

Abstract The three‐dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1, 091 NOE‐derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (ϕ, ψ, χ1) derived from NOEs and 3J coupling constants. The global fold involving...

Abstract The new functionality of the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137–168; Bassolino‐Klimas D et al., 1996, Protein Sci 5:593–603) has been applied for energy refinement of two previously determined solution NMR structures, murine epidermal growth factor (mEGF) and human type‐α transforming growth factor (hTGFα). A summary of considerations used in converting experimental NMR data into distance constraints for CONGEN is...

Abstract A new functional representation of NMR‐derived distance constraints, the flexible restraint potential, has been implemented in the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137–168) for molecular structure generation. In addition, flat‐bottomed restraint potentials for representing dihedral angle and vicinal scalar coupling constraints have been introduced into CONGEN. An effective simulated annealing (SA) protocol...

Abstract A PCR‐based random mutagenesis procedure was employed to identify several thermosensitive mutants of the MPTP enzyme, the murine homologue of the human T‐cell PTPase and rat PTP‐S enzymes. Four mutants with varying degrees of thermosensitivity were characterized for their thermostability and refolding properties following incubation at the nonpermissive temperature. Structure analysis of these mutations based on the hPTP1B...

Abstract A long sought goal in the physical chemistry of macromolecular structure, and one directly relevant to understanding the molecular basis of biological recognition, is predicting the geometry of bimolecular complexes from the geometries of their free monomers. Even when the monomers remain relatively unchanged by complex formation, prediction has been difficult because the free energies of alternative conformations of the...

Abstract Multidimensional NMR methods were used to obtain 1H‐15N correlations and 15N resonance assignments for amide and side‐chain nitrogens of oxidized and reduced putidaredoxin (Pdx), the Fe2S2 ferredoxin, which acts as the physiological reductant of cytochrome P‐450cam (CYP101). A model for the solution structure of oxidized Pdx has been determined recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry...

Abstract The three‐dimensional structure of a secreted aspartic protease from Candida albicans complexed with a potent inhibitor reveals variations on the classical aspartic protease theme that dramatically alter the specificity of this class of enzymes. The structure presents: (1) an 8‐residue insertion near the first disulfide (Cys 45‐Cys 50, pepsin numbering) that results in a broad flap extending toward the active site; (2) a 7‐residue...

Abstract The pH dependence of amide proton exchange rates have been measured for trp‐repressor. One class of protons exchanges too fast to be measured in these experiments. Among the protons that have measurable hydrogen‐deuterium exchange rates, two additional classes may be distinguished. The second class of protons are in elements of secondary structure that are mostly on the surface of the protein, and exchange linearly with increasing base...

Abstract The structural framework of cod liver alcohol dehydrogenase is similar to that of horse and human alcohol dehydrogenases. In contrast, the substrate pocket differs significantly, and main differences are located in three loops. Nevertheless, the substrate pocket is hydrophobic like that of the mammalian class I enzymes and has a similar topography in spite of many main‐chain and side‐chain differences. The structural...

Abstract A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl‐terminal end, was generated from a cDNA encoding the glycophospholipid‐linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE...

Abstract Phosphatidylinositide‐specific phospholipase Cs (PI‐PLCs) catalyze the calcium‐dependent hydrolysis of phosphatidylinositides in response to diverse stimuli in higher eukaryotes. Mammalian PI‐PLCs contain divergent regulatory regions, but all share three conserved regions: an N‐terminal pleckstrin homology (PH) domain, X, and Y. We report the high‐level expression and characterization of a recombinant “catalytic core” of...

Abstract Acidic conditions within the endosomal lumen induce the T domain of receptor‐bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH 5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans...

Abstract Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved...

Abstract Xylanase has been used in wood pulp bleaching in an effort to reduce chlorine release into the environment and pollution associated with paper production. The three‐dimensional structure of xylanase is important to enable better understanding of the enzyme mechanism and to help design a more thermostable xylanase mutant. At the time this work was begun, there was no sequence homologous protein available for traditional...

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