Glycine 85 of the trp-repressor of E.coli is important in forming the hydrophobic tryptophan binding pocket: experimental and computational approaches

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