Structural investigation of Tet repressor loop 154-167: a time-resolved fluorescence study of three single Trp mutants.
We have studied the time-resolved fluorescence of three engineered Tet repressor (TetR) mutants bearing a single Trp residue at positions 162, 163, and 165 in the C-terminal part of the loop joining helices 8 and 9. Detailed analysis indicates that, at 20 degrees, the fluorescence 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 wavelength 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 loop, at least in the 162-165 range, is not altered with temperature. The equilibrium between the rotamers is characterized by an enthalpy-entropy compensation effect which strongly suggests the involvement of background structural regions of TetR in the thermodynamics of the process. The very high deltaH degrees and TdeltaS degrees observed (up to 18 kcal/ mol) should reflect the temperature-dependent conformational change of a large part of the protein which would alter the rotamer distribution of the Trp residues. Taken together, our results are consistent with the existence of (at least) two conformations of the loop and suggest a model for loop motion.
Alberti
P
P
Dipartimento di Fisica, Università degli Studi di Milano, Italy.
Bombarda
E
E
Kintrup
M
M
Hillen
W
W
Lami
H
H
Piémont
E
E
Doglia
S M
SM
Chabbert
M
M
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012044:Regression Analysis; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D014364:Tryptophan