IMPLICATIONS OF THE CRYSTALLOGRAPHIC STRUCTURE OF BACTERIORHODOPSIN
Peter Nollert1,
Ehud M. Landau1,
Gabriele Rummel1,
Jurg P. Rosenbusch1,
Eva Pebay-Peyroula2
1Biozentrum, Basel, Switzerland
CH-4056,
2IBS/ESRF, Grenoble, France 38027
Keywords: bacteriorhodopsin; lipidic mesophases; photocycle; proton translocation
An atomic-level understanding of the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. To date, only a few high resolution structures of membrane proteins have been solved, reflecting the major obstacle in this endeavor - the routine production of well-ordered three-dimensional crystals. We have developed a novel concept for the crystallization of membrane proteins by exploiting the properties of lipidic mesophases (1).
Bacteriorhodopsin, an integral membrane protein found in the plasma membrane of Halobacterium salinarium was chosen for its advantageous qualities: stability, color, availability and purity. Hexagonal bacteriorhodopsin microcrystals grown in the matrix of a monoolein mesophase diffracted isotropically to 2.0 A resolution, with a space group P63, and unit cell dimensions of a=b=61.76 A, c=104.16 A, a=b=90° and g=120°, and one monomer per asymmetric unit. The crystal structure was solved at a resolution of 2.5 A by molecular replacement (2) using previous results from electron crystallographic studies as a model.
The earlier structure is confirmed overall, but several
significant differences are revealed. Among those are Arg 82,
which in our structure does not seem to form a salt bridge with
Asp 85, and Glu 204, whose side chain points away from the
putative proton translocation channel. The retinal resides in a
hydrophobic pocket, sandwiched between Trp 86 and Trp 182 and is
highly bent. Our structure identifies the locations of water
molecules and some hydrogen bonds to key residues within this
membrane protein in the ground state, which allows to suggest a
mechanism of proton translocation in the extracellular side of
the protein. In the reprotonation pathway from the cytoplasmic
side, distances between key residues are much too large for
direct proton transfer and an interpretation will have to await a
high resolution structure of a photocycle intermediate.