Backbone dynamics of Tet repressor alpha 8 boolean AND alpha 9 loop

A set of single Trp mutants of class B Tet repressor (TetR), in which Trp r esidues are located from positions 159 to 167, has been engineered to inves tigate the dynamics of the loop joining the alpha-helices 8 and 9. The fluo rescence anisotropy decay of most mutants can be described by the sum of th ree exponential components. The longest rotational correlation time, 30 ns at 10 degrees C, corresponds to the overall rotation of the protein. The sh ortest two components, on the subnanosecond and nanosecond time scale, are related to internal motions of the protein. The initial anisotropy, in the 0.16-0.22 range, indicates the existence of an additional ultrafast motion on the picosecond time scale. Examination of physical models for underlying motions indicates that librational motions of the Trp side chain within th e rotameric chi(1) x chi(2) potential wells contribute to the picosecond de polarization process, whereas the subnanosecond and nanosecond depolarizati on processes are related to backbone dynamics. In the absence of inducer, t he order parameters of these motions, about 0.90 and 0.80 for most position s, indicate limited flexibility of the loop backbone. Anhydrotetracycline b inding to TetR induces an increased mobility of the loop on the nanosecond time scale. This suggests that entropic factors might play a role in the me chanism of allosteric transition.