Technology

CSMP involves target selection, membrane protein expression, purification, characterization, crystallization, structure determination, and structure analysis. Target selection aims to identify the different representative classes of membrane proteins that cross the membrane at least 3 times (to eliminate signal directed secreted, single crossing, and membrane anchored proteins) for which a representative will be sought. Guided by Andrej Sali, this will be a dynamic process as structures emerge from efforts to determine membrane protein structures throughout the world.

Expression

Expression of prokaryotic and archaeal membrane proteins will be carried out in bacteria by a completely novel scheme developed and applied by Senyon Choe, termed Mistic, and by standardized procedures in E.coli that have already yielded 4 out of 5 high resolution membrane protein structures in the Stroud laboratory. The expression of all the human and mitochondrial proteins will be attempted first using the Mistic system. A minimal criterion for success is that the protein be assembled in the plasma membrane. Human membrane proteins that are not expressed adequately or correctly folded by the above procedures will also be expressed through the NIH Roadmap Membrane Protein Expression Center (MPEC).

Structures

Following expression, solubilization in detergents, purification, crystallization trials, and protein crystallography will be pursued at beamlines 8.3.1 and 5.0.2 at the Advanced Light Source (ALS) in Berkeley. James Holton has developed state-of-the-art innovative procedures at the ALS beamline 8.3.1. An expert in automation, he developed new robotic systems for rapid evaluation of membrane crystals. Some membrane proteins may not crystallize in three dimensions, but may crystallize in two dimensions within a lipid bilayer. Electron diffraction will be championed by Henning Stahlberg for these cases. Electron crystallography will also be used to validate that the structures of 3-D crystalline samples are congruent with the bilayer form. Some membrane proteins may evade both structure determination schemes. NMR will be applied in these cases under direction of Roland Riek for small size targets.