Crystal Structure of the M Intermediate of Bacteriorhodopsin: Allosteric Structural Changes Mediated by Sliding Movement of a Transmembrane Helix

Structural changes in the proton pumping cycle of wild-type bacteriorhodopsin were investigated by using a 3D crystal (space group P622) prepared by the membrane fusion method. Protein–protein contacts in the crystal elongate the lifetime of the M intermediate by a factor of ∼100, allowing high levels of the M intermediate to accumulate under continuous illumination. When the M intermediate generated at room temperature was exposed to a low flux of X-rays (∼1014 photons/mm2), this yellow intermediate was converted into a blue species having an absorption maximum at 650 nm. This color change is suggested to accompany a configuration change in the retinal-Lys216 chain. The true conformational change associated with formation of the M intermediate was analyzed by taking the X-radiation-induced structural change into account. Our result indicates that, upon formation of the M intermediate, helix G moves towards the extra-cellular side by, on average, 0.5 Å. This movement is coupled with several reactions occurring at distal sites in the protein: (1) re-orientation of the side-chain of Leu93 contacting the C13 methyl group of retinal, which is accompanied by detachment of a water molecule from the Schiff base; (2) a significant distortion in the F–G loop, triggering destruction of a hydrogen bonding interaction between a pair of glutamate groups (Glu194 and Glu204); (3) formation of a salt bridge between the carboxylate group of Glu204 and the guanidinium ion of Arg82, which is accompanied by a large distortion in the extra-cellular half of helix C; (4) noticeable movements of the AB loop and the cytoplasmic end of helix B. But, no appreciable change is induced in the peptide backbone of helices A, D, E and F. These structural changes are discussed from the viewpoint of translocation of water molecules.