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Redesigning Enzyme Topology by Directed Evolution

Science  20 Mar 1998:
Vol. 279, Issue 5358, pp. 1958-1961
DOI: 10.1126/science.279.5358.1958

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Abstract

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 reengineering depended on choosing a thermostable 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.

  • * To whom correspondence should be addressed at Department of Chemistry, Swiss Federal Institute of Technology (ETH), CH-8092 Zürich, Switzerland. E-mail: hilvert{at}org.chem.ethz.ch

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