GLYCINE-85 OF THE TRP-REPRESSOR OF ESCHERICHIA-COLI IS IMPORTANT IN FORMING THE HYDROPHOBIC TRYPTOPHAN BINDING POCKET - EXPERIMENTAL AND COMPUTATIONAL APPROACHES

Experimental and computational analyses were performed on the corepres sor (L-tryptophan) binding site of the trp-repressor of Escherichia co li to investigate the ligand protein interactions. Gly85, one of the r esidues forming the hydrophobic pocket of the binding site, was system atically replaced with Ala, Val, Leu and Trp by cassette mutagenesis. Biochemical characterization showed that all these mutations caused si gnificant decreases in tryptophan binding activity. Free energy pertur bation calculations were performed for the mutants and were consistent with the experimental results. The lack of a side chain at position 8 5 was concluded to be essential for binding the corepressor; the struc ture of the binding pocket was suggested to be tight in the vicinity o f Gly85.