Crystal structure of the D85S mutant of bacteriorhodopsin: Model of an O-like photocycle intermediate

Crystal structures are reported for the D85S and D85S/F219L mutants of the light-driven proton/hydroxyl-pump bacteriorhodopsin. These mutants crystall ize in the orthorhombic C222(1) spacegroup, and provide the first demonstra tion that monoolein-based cubic lipid phase crystallization can support the growth of well-diffracting crystals in non-hexagonal spacegroups. Both str uctures exhibit similar and substantial differences relative to wild-type b acteriorhodopsin, suggesting that they represent inherent features resultin g from neutralization of the Schiff base counterion Asp85. We argue that th ese structures provide a model for the last photocycle intermediate (O) of bacteriorhodopsin, in which Asp85 is protonated, the proton release group i s deprotonated, and the retinal has reisomerized to all-traps. Unlike for t he M and N photointermediates, where structural changes occur mainly on the cytoplasmic side, here the large-scale changes are confined to the extrace llular side. As in the M intermediate, the side-chain of Arg82 is in a down ward configuration, and in addition, a TE-cloud hydrogen bond forms between Trp189 NE1 and Trp138. On fine cytoplasmic side, there is increased hydrat ion near the surface, suggesting how Asp96 might communicate with the bulk during the rise of the O intermediate. (C) 2001 Academic Press.