Crystal structures are reported for the D85S and D85S/F219L mutants of the light-driven proton/hydroxyl-pump bacteriorhodopsin. These mutants crystallize in the orthorhombic C222(1) spacegroup, and provide the first demonstration that monoolein-based cubic lipid phase crystallization can support the growth of well-diffracting crystals in non-hexagonal spacegroups. Both structures exhibit similar and substantial differences relative to wild-type bacteriorhodopsin, suggesting that they represent inherent features resulting from neutralization of the Schiff base counterion Asp85. We argue that these structures provide a model for the last photocycle intermediate (O) of bacteriorhodopsin, in which Asp85 is protonated, the proton release group is deprotonated, and the retinal has reisomerized to all-trans. Unlike for the M and N photointermediates, where structural changes occur mainly on the cytoplasmic side, here the large-scale changes are confined to the extracellular side. As in the M intermediate, the side-chain of Arg82 is in a downward configuration, and in addition, a pi-cloud hydrogen bond forms between Trp189 NE1 and Trp138. On the cytoplasmic side, there is increased hydration near the surface, suggesting how Asp96 might communicate with the bulk during the rise of the O intermediate.
Copyright 2001 Academic Press.