SINGLE-TRYPTOPHAN MUTANTS OF MONOMERIC TRYPTOPHAN REPRESSOR - OPTICALSPECTROSCOPY REVEALS NONNATIVE STRUCTURE IN A MODEL FOR AN EARLY FOLDING INTERMEDIATE

A monomeric version of the dimeric tryptophan repressor from Escherich ia coli, L39E TR, has previously been shown to resemble a transient in termediate that appears in the first few milliseconds of folding [Shao , X., Hensley, P., and Matthews, C. R. (1997) Biochemistry 36, 9941-99 49]. In the present study, the optical properties of the two intrinsic tryptophans were used to compare the structure and dynamics of the mo nomeric form with those of the native, dimeric form. The urea-induced unfolding equilibria of Trp19/L39E TR (Trp99 replaced with Phe) and Tr p99/L39E TR (Trp19 replaced with Phe) mutants were monitored by circul ar dichroism and fluorescence spectroscopies at pH 7.6 and 25 degrees C. Coincident normalized transitions show that the urea denaturation p rocess for each single-tryptophan mutant follows a two-state model inv olving monomeric native and unfolded forms. The free energies at stand ard state in the absence of denaturant for Trp19/L39E TR and Trp99/L39 E TR are less than that for L39E TR, indicating that both tryptophans are involved in stabilizing the monomer. Fluorescence and near-UV circ ular dichroism spectroscopies indicate that the tryptophan side chains in monomeric Trp19/L39E TR and Trp99/L39E TR occupy hydrophobic, well -structured environments that are distinctively different from those f ound in their dimeric counterparts. Acrylamide quenching experiments s how that both Trp19 and Trp99 are partially exposed to solvent in the native state, with Trp99 having a slightly greater degree of exposure. Measurements of the steady-state anisotropies of Trp19/L39E and Trp99 /L39E TR demonstrate that the motions of both tryptophan side chains a re restricted in the folded conformation. On the basis of these data, it can be concluded that this monomeric form of the tryptophan repress or adopts a well-folded, stable conformation with nonnative tertiary s tructure. When combined with previous results, the current findings de monstrate that the development of higher order structure during the fo lding of this intertwined dimer does not follow a simple hierarchical model.