Increasing thermal stability of subtilisin from mutations suggested by strongly interacting side-chain clusters

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Abstract
Experimental and computational analyses were performed on the corepressor (L-tryptophan) binding site of the trp-repressor of Escherichia coli to investigate the ligandprotein interactions. Gly 85, one of the residues forming the hydrophobic pocket of the binding site, was systematically replaced with Ala, Val, Leu and Trp by cassette mutagenesis. Biochemical characterization showed that all these mutations caused significant decreases in tryptophan binding activity. Free energy perturbation calculations were performed for the mutants and were consistent with the experimental results. The lack of a side chain at position 85 was concluded to be essential for binding the corepressor; the structure of the binding pocket was suggested to be tight in the vicinity of Gly85.

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