W0087

In cubo Membrane Protein Crystallization. Peter Nollert, Dept. of Biochemistry and Biophysics, School of Medicine, University of California, San Francisco, San Francisco CA 94143-0448 and Emerald BioStructures, 7869 NE Day Rd., W., Bainbridge Island, WA 98110.

Several membrane proteins have been crystallized in a lipidic cubic phase; the crystal architecture, growth mechanism and recent advances of this crystallization method will be discussed. Membrane proteins are difficult to crystallize and consequently only a small number of high-resolution structures are available. Crystals of transmembrane proteins for X-ray diffraction experiments may be grown either employing detergent micelles, or in a matrix of liquid crystalline membraneous material forming a lipidic cubic phase (in cubo). Several structures of bacteriorhodopsin in the ground state and various photocycle intermediate states of the wild type as well as those of mutated bacteriorhodopsins have been determined utilizing crystals grown in cubo.

So far the methodology has been successfully applied to five membrane proteins. Throughout, the crystals consist of layers in which the membrane proteins dwell in a lipid environment, similar to their biological membranes. The crystallization of bacteriorhodopsin in lipidic cubic phases was investigated and a mechanism for crystal nucleation and growth is proposed. Since its conception, several technical advances facilitate crystallographers to use this crystallization methodology for routine crystallization trials. Particularly, a micromethod for in cubo crystallization was developed that requires fewer handling steps, consumes less material for a single crystallization setup and that allows the use of standard multiwell plates.