Structure and hydration of the M-state of the bacteriorhodopsin mutant D96N studied by neutron diffraction

Neutron diffraction from oriented purple membrane fragments at various hydration levels, coupled with H(2)O/(2)H(2)O exchange, was used to compare the structure and hydration of the light-adapted initial state (B-state) and the M photointermediate of bacteriorhodopsin mutant D96N. Diffraction patterns were recorded at 86%, 75% and 57% relative humidity (r.h.). Structural changes observed at 86% and 75% r.h. are absent at 57% r.h., showing that they are uncoupled from the deprotonation of the Schiff base during formation of the M-state. in a current model, the M-state consists of two substates, M(1) and M(2). Our data suggest that the state trapped at 57% r.h. is M(1) and that M(2) is trapped at the higher r.h. values. The observed structural changes are, therefore, associated with the M(1) --> M(2) transition, which can only take place at higher r.h. The difference Fourier projections of exchangeable hydrogen atoms and water molecules in the membrane plane are very similar for the B and M-states at 75% and 86% r.h. This shows that contrary to certain models, the structural changes in the M-state are not correlated with major hydration changes in the proton channel projection. (C) 1998 Academic Press Limited.