REDESIGNING ENZYME TOPOLOGY BY DIRECTED EVOLUTION

Genetic selection was exploited in combination with structure-based de sign to transform an intimately entwined, dimeric chorismate mutase in to a monomeric, four-helix-bundle protein with near native activity. S uccessful reengineering depended on choosing a thermostable starting p rotein, introducing point mutations that preferentially destabilize th e wild-type dimer, and using directed evolution to optimize an inserte d interhelical turn, Contrary to expectations based on studies of othe r four-helix-bundle proteins, only a small fraction of possible turn s equences (fewer than 0.05 percent) yielded well-behaved, monomeric, an d highly active enzymes, Selection for catalytic function thus provide s an efficient yet stringent method for rapidly assessing correctly fo lded polypeptides and may prove generally useful for protein design.