CRYSTALLIZATION OF BACTERIORHODOPSIN IN LIPIDIC MESOPHASES
Peter Nollert1, Martin Caffrey2, Ehud M. Landau1, Eva Pebay-Peyroula3, Gabriele Rummel1, Jurg P. Rosenbusch1
1Biozentrum,
Basel, Switzerland CH-4056,
2Ohio
State University, Columbus, Ohio, USA 43210,
3IBS/ESRF,
Grenoble, France 38027
Keywords: bacteriorhodopsin; lipidic mesophases; cubic phase; membrane protein crystallization mechanism.
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 mesophases1.
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 micro crystals grown in the matrix of a monoolein mesophase diffracted isotropically to 2.0 A resolution, with a space group P 63, 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 replacement2 using previous results from electron crystallographic studies as a model. The earlier structure is confirmed overall, but several significant differences are revealed. Our structure identifies the locations of water molecules and key residues within this membrane protein in the ground state. Thus, the structural basis for elucidating the mechanism of the proton translocation pathway is set.
A detailed understanding of the
processes involved in bacteriorhodopsin crystallization in
lipidic mesophases will presumably help to apply this method to
other membrane proteins. We hypothesize that the formation of
bacteriorhodopsin crystals in the lipidic mesophase involves
first an insertion of the membrane protein into and then an
expulsion from the curved membrane. Initially bacteriorhodopsin
is reconstituted into the monoolein/water matrix and may diffuse
laterally in the curved bilayer. In the presence of salt a
transition of the lipid phase and a contraction of the cubic unit
cells is observed by small angle x-ray diffraction. We envisage
that the accompanying increase in membrane curvature provides a
less favorable environment for the membrane protein and thus
induces a well-organized phase separation: the formation of
well-ordered three-dimensional bacteriorhodopsin crystals in a
lipid crystal.