Unraveling photoexcited conformational changes of bacteriorhodopsin by time resolved electron paramagnetic resonance spectroscopy

By means of time-resolved electron paramagnetic resonance (EPR) spectroscop y, the photoexcited structural changes: of site-directed spin-labeled bacte riorhodopsin are studied, a complete set of cysteine mutants of the C-D loo p, positions 100-107, and of the E-F loop, including the first alpha-helica l turns of helices E and F, positions 154-171, was modified with a methanet hiosulfonate spin label. The EPR spectral changes occurring during the phot ocycle are consistent with a small movement of helix C and an outward tilt of helix F. These helix movements are accompanied by a rearrangement of the E-F loop and of the C-terminal turn of helix E. The kinetic analysis of th e transient EPR data and the absorbance changes in the visible spectrum rev eals that the conformational change occurs during the lifetime of the M int ermediate. Prominent rearrangements of nitroxide side chains in the vicinit y of D96 may indicate the preparation of the reprotonation of the Schiff ba se. All structural changes reverse with the recovery of the bacteriorhodops in initial state.