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Journal Issue - Volume 6 Issue 4 (April 1997)

Abstract IArchitecture of GroEL and GroES and the reaction pathway A.Architecture of the chaperonins B.Reaction pathway of GroEL‐GroES‐mediated folding II.Polypeptide binding A.A parallel network of chaperones binding polypeptides in vivo ...

Abstract The three‐dimensional structures of the copper‐containing enzymes ascorbate oxidase, ceruloplasmin, and nitrite reductase, comprised of multiple domains with a cupredoxin fold, are consistent with having evolved from a common ancestor. The presence or absence of copper sites has complicated ascertaining the structural and evolutionary relationship among these and related proteins. Simultaneous structural superposition of...

Abstract The high‐resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, f1, fd) has been solved recently for the wild‐type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP‐ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4:187–197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic...

Abstract An anti‐p185HER2/anti‐CD3 humanized bispecific diabody was previously constructed from two cross‐over single‐chain Fv in which VH and VL domains of the parent antibodies are present on different polypeptides. Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers. A disulfide‐stabilized diabody was obtained by introducing two cysteine...

Abstract The effect of xylose on the rates of folding and unfolding of staphylococcal nuclease (nuclease) have been investigated using fluorescence‐detected pressure‐jump relaxation kinetics in order to establish the kinetic basis for the observed stabilization of nuclease by this sugar (Frye KJ, Perman CS, Royer CA, 1996, Biochemistry 35:10234–10239). The activation volumes for both folding and unfolding and the equilibrium volume change for...

Abstract Ca2+‐activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM‐regulated proteins: phosphorylase kinase, CaM‐dependent protein kinase 11, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca2+‐ATPase. The...

Abstract We have analyzed the structure of mitochondrial cytochrome c oxidase in terms of general characteristics thought to be important for describing the architecture of helix bundle membrane proteins. Many aspects of the structure are similar to what has previously been found for the photosynthetic reaction center and bacteriorhodopsin. Our results lead to a considerably more precise general picture of membrane protein architecture than ...

Abstract The application of mass spectrometry for determining the topography of integral membrane proteins has focused primarily on the mass determination of fragments that do not reside in the lipid bilayer. In this work, we present the accurate mass determination of transmembrane tryptic peptides of bovine rhodopsin using matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. The ability to determine the...

Abstract The eight amino acid sequence, Asp‐Tyr‐Lys‐Asp‐Asp‐Asp‐Asp‐Lys, representing the FLAG peptide, was inserted after codons 22 or 88 of the mouse (Mo) prion protein (PrP) gene. Inclusion of the FLAG sequence at these locations interfered neither with the cellular processing of PrPc nor its conversion into PrPSc. Inclusion of the FLAG epitope at residue 22 but not at residue 88 facilitated immunodetection of tagged PrP by anti‐FLAG ...

Abstract Two mutant forms of fumarase C from E. coli have been made using PCR and recombinant DNA. The recombinant form of the protein included a histidine arm on the C‐terminal facilitating purification. Based on earlier studies, two different carboxylic acid binding sites, labeled A‐ and B‐, were observed in crystal structures of the wild type and inhibited forms of the enzyme. A histidine at each of the sites was mutated to an...

Abstract Recoverin is a calcium‐binding protein that regulates the vertebrate photoresponse by inhibiting rhodopsin kinase in response to high calcium concentrations. It is heterogeneously N‐acylated by myristoyl and related fatty acyl residues that are thought to act as “calcium‐myristoyl switches,” whereby, in the presence of Ca2+, the N‐terminal acyl group is extended away from recoverin and, in the absence of calcium, it is more closely...

Abstract A new attractive interaction in metalloprotein structures, between the thiolate anion of a metal‐bound cysteine (acting as a nucleophile) and a carbonyl carbon of a peptide group (an electrophile), has been identified. From 82 cases extracted from 23 metalloprotein structures, the interacting S and C atoms are found to be at a distance of 3.2 (±2) Å, such that the angle S···C — O is 109° (±15°). Usually, the interacting...

Abstract We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE‐resistant...

Abstract The glutathione S‐transferase (GST) isozyme A1‐1 contains at its active site a catalytic tyrosine, Tyr 9, which hydrogen bonds to, and stabilizes, the thiolate form of glutathione, GS−. In the substrate‐free GST A1‐1, the Tyr 9 has an unusually low pKa, ∼8.2, for which the ionization to tyrosinate is monitored conveniently by UV and fluorescence spectroscopy in the tryptophan‐free mutant, W21F. In addition, a short α‐helix, residues...

Abstract Previous studies have suggested that the carboxy‐terminal peptide (residues 401–415) and interdomain helix (residues 185–199) of yeast phosphoglycerate kinase, a two‐domain enzyme, play a role in the folding and stability of the amino‐terminal domain (residues 1–184). A deletion mutant has been created in which the carboxy‐terminal peptide is attached to the amino‐terminal domain (residues 1–184) plus interdomain helix...

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