Ubiquitin function studied by disulfide engineering |
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Authors: | D J Ecker T R Butt J Marsh E Sternberg A Shatzman J S Dixon P L Weber S T Crooke |
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Institution: | Department of Molecular Pharmacology, Smith Kline and French Laboratories, King of Prussia, Pennsylvania 19406-0939. |
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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. |
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