Sans Studies of Bacteriorhodopsin Incorporation and Crystallization in Cubic Phase

Abstract

The direct crystallization of membrane proteins from lipidic mesophases has become an increasingly important, and even routinely used, tool in the field of membrane protein structural biology. Despite the method's practical successes, however, the exact molecular mechanisms for crystallization remain poorly understood. It has been proposed that protein molecules first align in transient, locally lamellar regions of lipid, forming small 2D crystals that initiate growth. These crystals could then stack to form the 3D crystal, which is fed laterally by lamellar-phase membrane extensions from the bulk cubic phase. Nevertheless, the initial formation of 2D crystal “patches” and initiation of lamellar transition has not been experimentally observed. Other mechanisms of crystal formation can also be envisioned, and the possibility of multiple crystallization mechanisms exists. A clear understanding of these mechanisms would allow for the rational optimization of crystallization conditions for different proteins, including the use of different lipids and additives.Using Small-Angle Neutron Scattering (SANS), we have begun a systematic study of cubic phase crystallization using the membrane protein Bacteriorhodopsin (bR) in monoolein cubic phase as a model system, alone and in the presence of precipitants. SANS with contrast variation is uniquely suited for the study of proteins in cubic phase, since the scattering contribution of the large excess of lipid can be eliminated, allowing the scattering from embedded protein to be directly measured without interference. This allows us to study bR embedded in the monoolein cubic phase.