DYNAMICS OF TRYPTOPHAN BINDING TO ESCHERICHIA-COLI TRP REPRESSOR WILD-TYPE AND AV77 MUTANT - AN NMR-STUDY

Binding of L-tryptophan to Escherichia coli trp repressor wild type (WT) and AV77 mutant was studied by H-1 NMR spectroscopy. Ligand binding to the proteins resulted in changes in line widths and chemical shifts of ligand resonances, but no changes in the coupling constant were observed. Line width and chemical shift changes of the H-4 L-tryptophan proton were monitored as a function of temperature and ligand and protein concentrations. For the WT repressor, the H-4 proton displays slow exchange at low temperatures (20-35 degrees C), while fast exchange occurs in the range from 45 to 65 degrees C. From 35 to 40 degrees C, the range of intermediate exchange, lines are broadened beyond detection. For the AV77 mutant, the intermediate and fast exchange regions are shifted at least 5 degrees C to higher temperatures. Line shapes of L-tryptophan H-4 proton resonances were simulated using a general expression based on McConnell's modified Bloch equations for a two-site exchange. From the simulations, an exchange frequency (nu(exch)) of about 3000 Hz was obtained at 45 degrees C for WT and about 1000 Hz for AV77 mutant. The activation energy for the process is 32.7 kcal K-1 mol(-1) for the WT and 29.1 kcal K-1 mol(-1) for AV77. At 45 degrees C, the dissociation and association rate constants (k-1 and k(+1), respectively) were calculated to be 2.0 x 10(3) s(-1) and 9.9 x 10(6) M(-1) s(-1), for the WT. For the AV77 mutant, k(-1) and k(+1) were 6.4 x 10(2) s(-1) and 2.7 x 10(6) M(-1) s(-1), respectively. The association rates are in the lower border of those values expected for diffusion-controlled processes. Taken together, the data indicate that a sizable barrier of energy is involved in the binding of L-tryptophan to trp repressor WT and AV77 mutant. The biological implications of the binding of L-tryptophan to AV77 mutant as compared to the wild type are discussed.