(335i) Understanding Gradient-Driven Diffusion of Membrane Proteins in Lipidic Mesophases

Understanding of membrane-associated processes in the cell relies on availability of high-resolution X-ray crystal structures of membrane proteins. However, crystallization of membrane proteins has a notoriously low success rate and presents a major bottleneck for membrane protein studies.

The in meso approach to membrane protein crystallization takes advantage of the rich phase behavior exhibited by some lipid-water systems to create a membrane-like environment for crystallization trials. Here the protein is incorporated into a cubic mesophase of bicontinuous water channels and highly curved lipid bilayers, thus maintaining the membrane protein in its native environment. A number of membrane proteins, such as bacteriorhodopsin, photosynthetic reaction centers, and, recently, human β-2 adrenergic receptor, have been successfully crystallized in meso. The mechanism of the process, however, remains poorly understood, which prevents optimization of the method.

Gradient-driven diffusion is an important part of the crystal growth process, as the depleted layer in the vicinity of the crystal must be replenished by diffusion of protein from the bulk mesophase for further growth of the crystal. In meso crystallization systems present a particular challenge for understanding diffusion effects: the region of the mesophase adjacent to the protein crystal is lamellar as opposed to the bulk of the mesophase that retains its cubic microstructure. Mobility of the protein in highly curved bilayers of the cubic phase is expected to be significantly different from that in flat bilayer sheets of the lamellar phase.

As a first step toward understanding these phenomena we are investigating gradient-driven diffusion of a model membrane protein, bacteriorhodopsin, in lamellar and cubic lipidic mesophases. We are also analyzing the effects of various components of crystallization cocktails on mesophase geometry and the resultant protein mobility in these mesophases. For the study we are employing microfluidic platforms that allow us to create concentration gradients in a highly controlled manner and enable formation of planar interfaces between highly viscous mesophases.