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 the corepressor (L-tryptophan) binding site of the trp-repressor of Escherichia coli to investigate the ligand-protein interactions. Gly85, 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.