SPIN-LABELING STUDIES OF THE CONFORMATIONAL-CHANGES IN THE VICINITY OF D36, D38, T46, AND E161 OF BACTERIORHODOPSIN DURING THE PHOTOCYCLE

Electron paramagnetic resonance (EPR) spectroscopy of site-directed sp in-labeled bacteriorhodopsin mutants is used to study structural chang es during the photocycle. After exchange of the native amino acids D36 and D38 in the A-B loop, E161 in the E-F, loop, and T46 in the putati ve proton channel by cysteines, these positions were modified by a met hanethiosulfonate spin label. Time-resolved EPR spectroscopy reveals s pectral changes during the photocycle for the mutants with spin labels attached to C36, C161, and C46. A comparison of the transient spectra l amplitudes with simulated EPR difference spectra shows that the dete cted signals are due to changes in the spin label mobility and not to possible polarity changes in the vicinity of the attached spin label. The kinetic analysis of the EPR and the visible data with a global fit ting procedure exhibits a structural rearrangement near position 161 i n the E-F loop in the M state. The environmental changes at positions 36 and 46, however, occur during the M-to-N transition. All structural changes reverse with the recovery of the BR ground state. No structur al changes are detected with a spin label attached to C38.