Evidence for coupling of folding and function in trp repressor: physical characterization of the superrepressor...

The fine-control of gene expression in the trp repressor system is achieved through the thermodynamic linkage of multiple equilibria involving the trp repressor protein (TR), tryptophan (L-Trp) and DNA. We have undertaken studies of superrepressor mutants of TR as a means of dissecting the coupled equilibria that contribute to repressor function. Unlike all the other tested super-repressors that exhibit differences from wild-type TR DNA binding affinity or stoichiometry, the AV77 superrepressor (an alanine to valine substitution at position 77: AV77TR) has been indistinguishable from TR in vitro. The present studies using a variety of biophysical measurements comparing TR and AV77TR provide strong evidence that the helix-turn-helix (HTH) region of apoTR exists in a partially folded conformation. Far UV CD spectra of the two proteins reveal a 10% increase in helical content for the apoAV77TR compared to apoTR. Moreover, urea denaturation studies demonstrate that apoAV77TR is more stable to denaturation than apoTR. ApoTR binds large amounts of 1,8-ANS, a hydrophobic fluorescence probe used to detect protein folding intermediates, with high affinity, where apoAV77TR exhibits only marginal binding of this ligand. While the tryptophan affinities of the two proteins as measured by titration calorimetry are quite similar, the thermodynamic signatures are distinct, with a much reduced unfavorable entropic contribution for AV77TR. Finally, the allosteric effect of L-Trp on oligomerization is abolished by the AV77 mutation. Taken together these data support previous calorimetric studies implicating coupling of folding and L-Trp binding for TR. Moreover, they are consistent with NMR observations indicating partial disorder in the HTH region of apoTR. Based upon the distinct biophysical properties of TR and AV77TR, we propose a model in which folding of the HTH region accompanies ligand binding in TR. In this model distinct protein-protein interactions of the apo- and holoTR link this conformational change to apparent operator affinities, thereby modulating TR function in vivo.