Protein Engineering vol. 7 no. 10 pp. 1239-1247, 1994
© 1994 Oxford University Press
RESEARCH-ARTICLE |
Glycine 85 of the trp-repressor of E.coli is important in forming the hydrophobic tryptophan binding pocket: experimental and computational approaches
Molecular Physics Section, Electrotechnical Laboratory Umezono, Tsukuba-shi, Ibaraki 305 1Department of Biological Science and Technology, Science University of Tokyo Yamazaki, Noda-shi, Chiba 278, Japan 2Mitsubishi Kasei Co. Ltd Kamoshida-cho, Midori-ku, Yokohama-shi, Kanagawa 227, Japan 3Kissei Pharmaceutical Co. Ltd Yoshino, Matsumoto-shi, Nagano 399, Japan 4Taisho Pharmaceutical Co. Ltd Yoshino-cho, Ohmiya-shi, Saitama 330, Japan
5To whom correspondence should be addressed
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.
Keywords: cassette mutagenesis/computational design/free energy perturbation/ligand/trp-repressor
Received March 18, 1994; revised June 20, 1994; accepted July 6, 1994.