Dynamics of Tryptophan Binding to Escherichia coli Trp Repressor Wild Type and AV77 Mutant: An NMR Study

Abstract

Binding of L-tryptophan to Escherichia coli trp repressor wild type (WT) and AV77 mutant was studied by 1H 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 H4 L-tryptophan proton were monitored as a function of temperature and ligand and protein concentrations. For the WT repressor, the H4 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 H4 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 (upsilon 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Internal Dynamics of the Tryptophan Repressor (TrpR) and Two Functionally Distinct TrpR Variants, L75F-TrpR and A77V-TrpR, in Their l-Trp-Bound Forms

“…Our data support a model in which the primary effect of L-Trp co-repressor binding to TrpR is to contribute to the formation of a well-ordered helix E. In the case of A77V-TrpR, the resulting amino acid substitution triggers such ordering of helix E and co-repressor binding has little effect on this conformational transition. Our 15 N NMR relaxation observations are consistent with a model whereby the reduced flexibility of helix E contributes to the reduced ability of A77V-TrpR to distinguish between closely related DNA operator sequences (22). …”

“…DNA-binding studies showed that the A77V-TrpR variant has a high affinity for operator DNA in its apo-form, similar in magnitude to the DNA binding affinity of holo-WT-TrpR, but interestingly cannot recognize the full complement of operator sequences normally accessible to WT-TrpR (12). These results support the notion that the A to V mutation at residue position 77 results in a more ordered DNA-binding domain in apo-A77V-TrpR, and that this structural ordering leads to the restricted specificity of this variant repressor to a subset of DNA sequences (12, 22). This variant was classified as a “super-repressor” due to its ability to repress gene transcription at low L-Trp concentrations relative to those needed for proper functioning of WT-TrpR (23).…”

The Structure of Eukaryotic Translation Initiation Factor-4E from Wheat Reveals a Novel Disulfide Bond

“…Cross peaks between resonances of the free and bound m 7 GTP indicate that a significant fraction of the free and bound pools exchange during the 200-ms NOE mixing time (Fig. 7); this NOE spectrum is remarkably similar to that of the Trp repressor protein obtained with a similar concentration of excess Trp (Schmitt et al, 1995). The observation that the resonances of the protein and free ligand have the same sign in a two-dimensional NOE spectrum is also consistent with weak ligand binding (Post, 2003 GTP are broadened upon the addition of a relatively small (less than stoichiometric) amount of wheat DN-eIF4E protein (Fig.…”

“…The stated uncertainty in each value is indicative of the estimated uncertainties in the NMR line width measurements, protein and ligand concentrations, and the variability in data obtained using different protein preparations. It therefore appears that there is only a small (1.53) difference in the dissociation rate for the cap analog m 7 GTP for the three forms of the wheat DN-eIF4E (Schmitt et al, 1995) in a similar example of weak ligand binding. This NOE spectrum was obtained using a jump-return method for suppressing the solvent water signal, which tends to attenuate the signals of peaks near 5 ppm.…”

Structural and Dynamic Information on Ligand Binding

“…In fact, while this can be done [11], it is rarely practical, unless the protein is small or specific isotope labelling (or incorporation of a fluorinated amino-acid) makes it possible to isolate a single resonance. Signal overlap is an obvious problem, as is the limiting signalto-noise ratio of the broad signals -in a ligand titration experiment, where populations are unequal, the signal from the species with the lower population will be preferentially broadened and may be undetectable.…”