CONSTRUCTION AND CHARACTERIZATION OF MONOMERIC TRYPTOPHAN REPRESSOR -A MODEL FOR AN EARLY INTERMEDIATE IN THE FOLDING OF A DIMERIC PROTEIN

Tryptophan repressor (TR) from Escherichia coli is a homodimer whose h ighly helical subunits intertwine in a complex fashion. A monomeric ve rsion of Trp repressor has been constructed by introducing a pair of p olar amino acids at the hydrophobic dimer interface. Analytical ultrac entrifugation was used to show that the replacement of leucine at posi tion 39 with glutamic acid results in a monomer/dimer equilibrium whos e dissociation constant is 1.11 x 10(-4) M at 25 degrees C and pH 7.6. Tryptophan fluorescence, both near-and far-UV circular dichroism, and NMR spectroscopies demonstrated that, at the micromolar concentration s where the monomer predominates, secondary and tertiary structure are present. Hydrophobic dye-binding experiments showed that nonpolar sur face is accessible in the monomeric form. The urea-induced equilibrium unfolding of monomeric L39E TR was monitored by circular dichroism, f luorescence, and absorbance spectroscopies. Coincident transitions sho w that the urea denaturation process follows a simple two-state model involving monomeric native and unfolded forms. The free energy at stan dard state in the absence of denaturant was estimated to be 2.37 +/- 0 .15 kcal mol(-1), and the sensitivity of the unfolding transition to d enaturant, the m value, was 0.86 +/- 0.04 kcal mol(-)1 M(urea)(-1) at pH 7.6 and 25 degrees C. The thermal denaturation transition occurred over a broad temperature range, suggesting either that the enthalpy ch ange is small or that intermediates may exist. Kinetic studies showed that both the refolding and unfolding of the monomer were complete in the mixing dead time of stopped-flow CD and fluorescence spectroscopy, 5 ms. These structural, thermodynamic, and kinetic results are very s imilar to those previously reported for an early, monomeric intermedia te in the folding of the wild-type TR dimer [Mann, C. J., & Matthews, C. R. (1993) Biochemistry 32, 5282-5290]. The construction of a stable , monomeric form of TR that strongly resembles a transient folding int ermediate should provide useful insights into the nature of the early events in the folding of TR.