Structural Studies of Biological Membrane Proteins 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, due to the difficulties in purifying sufficient amounts of membrane proteins, especially for those of eukaryotic origin, and in obtaining membrane protein crystals. My lab 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. (1) Advancing our understanding of how bc1 complexes work has been our focus; we are doing so by studying bc1 in complex with substrate and with various inhibitors; we have been successful in obtaining bovine mitochondrial bc1 crystals that diffracted X-rays to higher resolution for native, substrate- and various inhibitor-bound bc1. We found that the network of aromatic-aromatic (Ar-Ar) interactions is both effective and specific for inhibitor binding to the hydrophobic active sites of bc1; we 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. We are refining structures of bc1 with various bound inhibitors that are known to induce conformational switch to the iron-sulfur protein (ISP) subunit. We believe 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. (2) We have been working on the expression, purification and crystallization of P-gp and its prokaryotic and eukaryotic homologs. 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, LmrA and Pdr5p. Both proteins have been purified to homogeneity and crystallization experiments are underway. Structural Studies of the ATP-dependent Clp Protease Intracellular protein degradation is a major post-translational regulatory mechanism and plays a crucial role in many vital cellular functions; it also serves to remove damaged, denatured, and other abnormal proteins. The Clp proteases, which we have chosen to study crystallographically, are essential in many organisms and highly conserved. ClpA belongs to the family of AAA+ proteins, a broad class of protein conformation-transducing ATPases involved in a plethora of vital cellular functions. Clp and other ATP-dependent proteases are structurally and mechanistically complex proteins, whose structure/function relationships reflect important biochemical principles that need to be understood at the sub-molecular level and would be beneficial to understanding functions of other ATP utilizing enzymes such as ABC transporters. We have determined the crystal structure of the full-length ClpA, the regulatory component of the ClpAP complex. As the first AAA+ structure with two AAA+ modules, the ClpA structure has provided insights into the structural basis for functional difference of the two AAA+ modules. We have also obtained the structure of the isolated N-domain of ClpA in complex with ClpS that is known to alter the substrate selectivity in ClpA, much like the adaptor proteins used by many other AAA+ proteins. Having obtained structures of all components in the ClpAPS system, we are now focusing on obtaining structures of ClpA in various conformations induced by bound nucleotide, and on crystallizing binary complexes of ClpA with either ClpS or ClpP, as well as with various substrates.
