PMID:9506949
| Citation |
MacBeath, G, Kast, P and Hilvert, D (1998) Redesigning enzyme topology by directed evolution. Science 279:1958-61 |
|---|---|
| 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. |
| Links | |
| Keywords |
Amino Acid Sequence; Binding Sites; Catalysis; Chorismate Mutase/chemistry; Chorismate Mutase/genetics; Chorismate Mutase/metabolism; Circular Dichroism; Cloning, Molecular; Dimerization; Directed Molecular Evolution; Escherichia coli/genetics; Models, Molecular; Molecular Sequence Data; Protein Conformation; Protein Engineering; Protein Folding; Protein Structure, Secondary; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Transformation, Bacterial |
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