Genetic selection was exploited in combination with structure-based design to transform an intimately entwined, dimeric chorismate mutase into a monomeric, four-helix-bundle protein with near native activity. Successful re-engineering depended on choosing a themostable starting protein, introducing point mutations that preferentially destabilize the wild-type dimer, and using directed evolution to optimize an inserted interhelical turn. Contrary to expectations based on studies of other four-helix-bundle proteins, only a small fraction of possible turn sequences (fewer than 0.05 percent) yielded well-behaved, monomeric, and highly active enzymes. Selection for catalytic function thus provides an efficient yet stringent method for rapidly assessing correctly folded polypeptides and may prove generally useful for protein design.
CITATION STYLE
MacBeath, G., Kast, P., & Hilvert, D. (1998). Redesigning enzyme topology by directed evolution. Science, 279(5358), 1958–1961. https://doi.org/10.1126/science.279.5358.1958
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