Redesigning enzyme topology by directed evolution.
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.
MacBeath
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G
The Scripps Research Institute, Department of Chemistry, 10550 North Torrey Pines Road, La Jolla, California, 92037, USA.
Kast
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P
Hilvert
D
D
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002826:Chorismate Mutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D002826:Chorismate Mutase