Summary and General Perspectives

Nature has solved the problem of protein design through the mechanism of Darwinian evolution. Every one of the proteins in our cells, from enzymes and receptors to structural proteins, has arisen by this process. As the examples presented here with chor-ismate mutase amply demonstrate, evolutionary strategies can also be successfully exploited in the laboratory to study the structure and function of existing proteins and to engineer new ones. 

Site-directed mutagenesis, by which one amino acid in a protein is substituted for another, is an established and invaluable tool in protein chemistry. However, the size and complexity of proteins often precludes unambiguous interpretation of the results of such experiments. This is due to the non-uniform distribution of structural information within a protein sequence - the fact that some residues are tolerant to substitution, whereas others cannot be replaced without deleterious consequences - and to the many energetically similar conformational states available to any polypeptide. These factors, and the extreme sensitivity of catalytic activity to seemingly modest structural perturbation, make characterization and (re)design of enzymes in the laboratory difficult. 

In contrast to methods involving single substitutions, combinatorial strategies are ideally suited for evaluating the complex network of interactions in proteins that ultimately determine form and function [73]. As shown in the mechanistic and structural investigations of chorismate mutases, degenerate protein ensembles can illuminate subtle trends that might be missed in analyses of individual amino acid replace- 

The authors wish to thank Kinya Hotta and Gavin MacBeath for assistance in the preparation of graphics. The financial support of the ETH Zurich, the Schweizer-ischer Nationalfonds, and Novartis Pharma are gratefully acknowledged. 

Edited by Susanne Brakmann and Kai Johnsson Copyright 2002 Wiley-VCH Verlag GmbH Co. KGaA ISBNs 3-527-30423-1 (Hardback) 3-527-60064-7 (Electronic) 

---