Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor

A mutant tryptophan repressor (TrpR) protein containing the substitution of phenylalanine for leucine 75 has been isolated following a genetic screen for temperature-sensitive mutations. Two-dimensional (2D) H-1 NMR spectra i ndicate an overall very similar fold for the purified mutant and wild-type proteins. Circular dichroism spectropolarimetry indicates an increased heli x content relative to the wild-type protein, and a slightly higher urea den aturation midpoint for the mutant protein, although there is no difference in thermal stability. Fluorescence spectra indicate a more buried environme nt for one or both tryptophan residues in the mutant protein. The rate of p roton-deuterium exchange-out for the resolved indole ring protons of the tw o tryptophan residues was quantified from NMR spectra of mutant and wild-ty pe proteins and found to be approximately 50% faster in the wild-type prote in. The mutant protein binds the corepressor L-tryptophan (L-Trp) approxima tely ten times more weakly than does the wild-type protein, but in L-Trp ex cess its DNA-binding affinity is only two to fivefold weaker. Taken togethe r the results imply that, despite its conservative chemical character and s urface location at the C terminus of helix one in the helix-turn-helix DNA recognition motif, this mutational change confers long-range effects on the dynamics of the protein's secondary and tertiary structure without substan tially altering its fold, and with relatively minor effects on protein func tion. (C) 1999 Academic Press.