Ubiquitin function studied by disulfide engineering.
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.
Ecker
D J
DJ
Department of Molecular Pharmacology, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406-0939.
Butt
T R
TR
Marsh
J
J
Sternberg
E
E
Shatzman
A
A
Dixon
J S
JS
Weber
P L
PL
Crooke
S T
ST
D003198:Computer Simulation; D003553:Cystine; D004220:Disulfides; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011487:Protein Conformation; D014452:Ubiquitins / Q000235:genetics*
D004220:Disulfides; D014452:Ubiquitins; D003553:Cystine