STRUCTURAL INVESTIGATION OF TET REPRESSOR LOOP-154-167 - A TIME-RESOLVED FLUORESCENCE STUDY OF 3 SINGLE TRP MUTANTS

We have studied the time-resolved fluorescence of three engineered Tet repressor (TetR) mutants bearing a single Trp residue at positions 16 2, 163, and 165 in the C-terminal part of the loop joining helices 8 a nd 9. Detailed analysis indicates that, at, 20 degrees, the fluorescen ce decay of each Trp can be described as the sum of three exponential components with lifetimes in the 1-, 3-, and 6-ns range. Emission wave length and temperature dependence studies are consistent with a model in which these components are due to the existence of three classes of Trp residues non-interconverting on the nanosecond timescale. Within the framework of the rotamer model, the weak temperature dependence of the lifetimes strongly suggests that the secondary structure of the l oop, at least ill the 162-65 range, is not altered with temperature. T he equilibrium between the rotamers is characterized by an enthalpy-en tropy compensation effect which strongly suggests the involvement of b ackground structural regions of TetR in the thermodynamics of the proc ess. The very high Delta H degrees and T Delta S degrees observed (up to 18 kcal/mol) should reflect the temperature-dependent conformationa l change of a large part of the protein which would alter the rotamer distribution of the Trp residues. Taken together, our results are cons istent with the existence of (at least) two conformations of the loop and suggest a model for loop motion. (C) 1997 Academic Press.