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 H2O/(H2O)-H-2 exchange, was used to co mpare 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 humid ity (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 o f 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 t he 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 hyd rogen 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 t o certain models, the structural changes in the M-state are not correl ated with major hydration changes in the proton channel projection. (C ) 1998 Academic Press Limited.