Time-resolved x-ray diffraction reveals multiple conformations in the M–N transition of the bacteriorhodopsin photocycle

We measured the M–N transition of wild-type bacteriorhodopsin (pH 9, 10°C) by time-resolved x-ray diffraction study at SPring8 BL45XU-A. We confirmed the accumulation of M and N intermediates by absorbance measurements, and we found that the time resolution of x-ray diffraction experiments (244 ms) was sufficient to resolve the M–N transition. From the x-ray diffraction data, three components were decomposed by singular value decomposition analysis. The existence of three components in the M→N→BR reaction revealed that BR changes its structure during the M–N transition. Moreover, the difference Fourier maps of reconstituted fast and slow decay components clearly showed that the electron density distributions of the F helix changes in the M–N transition. The observed structural change at the F helix will increase access of the Schiff base and D96 to the cytoplasmic surface and facilitate the proton transfer steps that begin with the decay of the M state.