title: Ubiquitin function studied by disulfide engineering.
abstract: Disulfide engineering was used to probe the role of conformational mobility in ubiquitin-mediated proteolysis. Six genes that encode cysteine-containing mutants of ubiquitin were constructed, expressed in Escherichia coli and the proteins purified. Single cysteine-containing mutants and a 4/14 disulfide were active in degradation of a substrate protein in vitro, while the 4/66 disulfide, which cross-links the NH2- and COOH-terminal strands of the protein, was only 20-30% active. The solution structure of the 4/66 mutant was solved: the disulfide is left-handed with no perturbations in the backbone from that of wild type ubiquitin. The results suggest that conformational mobility is required for the activity of ubiquitin in signaling proteolysis.
authors.affiliation: Department of Molecular Pharmacology, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406-0939.
authors.forename: D J
authors.initials: DJ
authors.lastname: Ecker
authors.forename: T R
authors.initials: TR
authors.lastname: Butt
authors.forename: J
authors.initials: J
authors.lastname: Marsh
authors.forename: E
authors.initials: E
authors.lastname: Sternberg
authors.forename: A
authors.initials: A
authors.lastname: Shatzman
authors.forename: J S
authors.initials: JS
authors.lastname: Dixon
authors.forename: P L
authors.initials: PL
authors.lastname: Weber
authors.forename: S T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D003198:Computer Simulation; D003553:Cystine; D004220:Disulfides; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011487:Protein Conformation; D014452:Ubiquitins / Q000235:genetics*
chemical_list: D004220:Disulfides; D014452:Ubiquitins; D003553:Cystine