Advances in membrane-protein crystallization: From detergent-free crystallization to in situ approaches
14:00 - 15:00
University of Toronto, Canada
CFEL (Bldg. 99)
Seminar Room IV, O1.111
R. J. Dwayne Miller
Three-dimensional structures of membrane proteins are of paramount value for understanding protein function on a molecular level. However, in vitro studies such as structure determination are impaired by the necessity to purify membrane proteins with the aid of detergents that often compromise protein stability and function.
We report the detergent-free crystallization of a membrane protein that has been in a lipid bilayer at every stage since its production in the cell. To do so, we combined styrene maleic acid (SMA) nanodiscs with crystallization in lipidic cubic phases (LCP). The 2.0-Å structure of an α-helical 7-transmembrane microbial rhodopsin thus obtained is of excellent quality and virtually identical to the 2.2-Å LCP structure obtained from a traditional detergent-based approach. This study is of fundamental interest, because it may allow to get structural insight into proteins, which so far have not been amenable to solubilization in detergents.
Membrane-protein structure determination is also impaired by the difficulties associated with harvesting small membrane-protein crystals from the highly viscous LCP. Recently, an in situ approach has been introduced, in which crystals are not harvested and flash-frozen but placed in the X-ray beam within the LCP. We introduce novel in situ plates that show significantly less background scattering, are cheaper, and easier to handle. Moreover, we developed a variety of holders, which are suited for measurements at room temperature and/or under cryogenic conditions. Some allow for storage and shipping of entire wells (with typically several dozens of crystals) in liquid nitrogen and are compatible with auto-mounting at synchrotrons, while others are perfect for rapid and automated screening of different crystallization conditions at room temperature. We validated the new setups using water-soluble hen egg lysozyme and myoglobin, as well as the membrane protein bacteriorhodopsin. In conjunction with the current developments at synchrotrons like smaller beams, faster detectors, and software for multi-crystal strategies, this approach promises high-resolution structural studies of membrane proteins to become faster and more routine.