Recent advances in genome research have provided new insights into the importance of membrane proteins in cellular functions. In eukaryotes such as yeast, over 14% of open reading frames are predicted to be integral membrane proteins with more than three trans-membrane (TM) segments (25% for two or more TM). Membrane proteins participate in many vital cellular functions; the demand for structural knowledge of membrane proteins has increased more than ever in light of the increased number of these proteins for which important functions have been identified. However structural data for membrane proteins at atomic resolution are only being obtained rather slowly (fewer than 50 unique membrane protein structures available in the Protein Data Bank). The picture gets even more depressing for eukaryotic membrane proteins. Excluding those of mitochondrial origin, only a couple of eukaryotic membrane protein structures were determined to date at atomic resolution; there is not a single recombinant eukaryotic membrane protein structure determined crystallographically! my group has been studying the structure and function of a few selected families of membranes proteins: those involved in cellular multidrug resistance such as P-glycoprotein (P-gp) and its homologs, and the respiratory component cytochrome bc1 complexes (bc1) of mitochondria and bacteria. In collaboration with C. A. Yu (OSU), we have have been successful in obtaining bovine mitochondrial bc1 crystals that diffracted X-rays to higher resolution for native, substrate- and various inhibitor-bound bc1. Our work found that the network of aromatic-aromatic interactions is both effective and specific for inhibitor binding to the hydrophobic active sites of bc1, and provided explanations at atomic resolution for bc1 inhibition by various inhibitors. Moreover the refined structures unveiled rich structural information that suggests mechanisms for substrate reduction and protonation at the quinone reduction site of the cyt. b subunit. Currently my group is refining structures of bc1 with various bound inhibitors that are known to induce conformational switch to the iron-sulfur protein (ISP) subunit. It is believed that correlating structural changes to inhibitor binding and to variations in redox potential may hold the key to understanding the relationship between quinol oxidation and the ISP conformational switch and to providing an explanation for the electron bifurcation at the quinol oxidation site. A major focus of our crystallography unit has been on the expression, purification and crystallization of ABCB1 (P-glycoprotein, P-gp) and its prokaryotic and eukaryotic homologs in collaboration with S. Ambudkar (LCB) and M. Gottesman (LCB). Efforts have been made to purify P-gp from different expression systems such as the baculovirus infected insect cells and the P. pastoris yeast expression system. We have also dedicated resources to expressing, refolding and purifying monoclonal antibodies in the hope of facilitating P-gp purification and crystallization. More recently, we initiated purification and crystallization of the P-gp homologs from the gram-positive bacterium L. lactis (LmrA) and from S. cerevisiae (Pdr5p). Both proteins have been purified to homogeneity and crystallization experiments are underway.Technically, to obtain a membrane protein structure, four obstacles must be overcome: (1) to achieve high-level protein expression, (2) to obtain pure and mono-dispersed proteins in large quantity, (3) to grow diffraction quality crystals, and (4) to solve crystallographic phase problems often at relatively low resolutions for membrane proteins. All these difficulties are due to the fact that membrane proteins have large hydrophobic surface. Membrane proteins are difficult to express in large quantities and in active forms, especially for eukaryotic membrane proteins, in commonly available expression systems. Currently, the most used approach for expressing large amounts of active membrane protein is to screen for high-level expression of a large number of homologs, mostly those of prokaryotes. The concept of high-level expression for membrane protein is perhaps one or two orders of magnitude different from that of high expression of soluble proteins. Even for a very high-level expression, the total amount of membrane protein is rarely over 30% of total membrane proteins in cell membrane (2% for P-gp, 5% for LmrA, and 2% for Pdr5p), making purification of large amount of proteins impossible without large-scale fermentation and cell disruption facilities. Furthermore, success in achieving high-level expression in test tubes does not guarantee success when the production is scaled up. Membrane proteins are often associated with each other in a non-specific manner when purified, which are detrimental to successful crystallization. The problem of polydispersity can be eliminated by screen for different detergents and solvent conditions. When purified, membrane proteins exist in solution as protein-detergent complexes; the available hydrophilic surface that is useful for specific crystal contact is limited. It is quite common to screen for over 30,000 conditions before a diffraction quality crystal form can be found. It has been successful in a few cases to artificially increase the hydrophilic surface by attaching conformational sensitive monoclonal antibodies to target membrane proteins. Our group is also working toward improving existing or devise new technologies to facilitate crystallization of membrane proteins.
