We have chosen as a model the truncated (residues 20-83) form of the chymotrypsin inhibitor-2 (CI-2), one of the shortest sequences known to adopt a stable native conformation. Which residues and combinations of side chain interactions are critical for specifying the native and stable fold of CI-2? In an attempt to answer to this question, we propose to develop a phage display and a colony screening procedures that would provide a means to select variants of CI-2 that fold from large libraries. The premise underlying the selection is that binding of CI-2 to chymotrypsin requires the protein be properly folded. We will construct two independent mutant libraries using doped oligodeoxyribonucleotides mutagenesis but holding constant the residues involved in binding. Phages corresponding to combinations of mutations that restore the CI-2 fold will be retrieved by affinity selection from a known CI-2 variant with disrupted folding. Residues critical for specifying the fold of CI-2 will be identified by randomizing the remaining "scaffold" residues of CI-2 wild-type. Colonies that lose the binding activity then allow recovery of non viable scaffold sequences. The structure of folded CI-2 variants will be analysed using biophysical methods. The folding pathway of CI-2 variants will be compared to that of I CI-2. Characterization of the thermodynamics and kinetics of these mutants should yield insights into the sequence determinants of protein folding.
Key words: chymotrypsin inhibitor-2, protein folding-stability, protein engineering combinatorial mutagenesis, phage display.
The aim of this project is a better understanding of the relationships between protein folding and amino acid sequence by varying the sequence and examining the effect of these changes on both structure and folding pathway. Enhanced understanding of the folding problem requires further applications of protein engineering strategies that combine the tools of recombinant DNA technology with more classical protein chemistry methods. Our protein engineering strategy should be a powerful tool to aid in deciphering the rules governing protein folding. A better understanding of how amino acid sequences specify structure, activity and stability is important to design proteins with rationally devised novel properties. Futhermore, the identification of the sequence determinants of protein folding may help to avoid problems of aggregation or proteolysis, ultimately leading to improved yields of products for biotechnology industry.