Membrane proteins participate in many vital cellular functions, yet atomic resolution structural data for membrane proteins are being obtained rather slowly and the membrane protein structural biology is widely perceived as an research area of high impact with high risk, due to the difficulties in purifying sufficient amounts of membrane proteins, especially for those of eukaryotic origin, and in obtaining membrane protein crystals. My unit has been studying structures and functions of a few selected families of membranes proteins: those involved in cellular multidrug resistance (MDR) such as the P-glycoprotein (P-gp) and its homologs and orthologs, the respiratory components such as the cytochrome bc1 complexes (bc1) of mitochondria and bacteria, and the P-type ion-transporting ATPases such as the bacterial copper transporter CopB. Over the past few years and in collaboration of Dr. C.-A. Yu of Oklahoma State University, we have made significant progress in obtaining bovine mitochondrial bc1 complex crystals that diffracted X-rays to high resolution (2.2 ), and in understanding the mechanism of function of the enzyme by analyzing both native and inhibitor bound structures. Based on these analyses, we put forward a modified classification scheme for bc1 inhibitors and proposed mechanisms for quinone reduction at the Qn (Qi) site and quinol oxidation at the Qp (Qo) site. We described the relationship between the motion switch of Iron-sulfur-protein (ISP) subunit and conformational changes in the cytochrome b subunit upon binding of different types of inhibitors, and extended this relationship to the mechanism of electron bifurcation at the Qo site. Recently, we successfully determined the crystal structures of the wild type and mutant cytochrome bc1 complex from the photosynthetic bacteria Rhodobacter sphaeroides in complex with various bc1 inhibitors in the resolution range between 2.4 - 3.0 , demonstrating our ability to obtain atomic resolution structural information of the bacterial bc1 in various forms reproducibly and our perseverance in pursuing difficult projects. This work accomplishes one of our goals in establishing a model system to systematically study the bc1 complex combining structural, genetic, and biochemical techniques.A major focus of our research has been on the expression, purification and crystallization of P-gp and its prokaryotic and eukaryotic homologs. In collaboration with Drs. S. Ambudkar and M. Gottesman of LCB, we have made considerable progress toward eventual structural solution of the P-gp. In addition to being able to produce mg amount of protein consistently, we have also achieved P-gp preparation at high concentration in various detergents; we have tested tens of thousands of crystallization conditions and obtained valuable knowledge of behaviors of P-gp under these conditions. Several interesting hits have been pursued but so far none produced crystals suitable for structure determination. While continuing our effort in crystallizing P-gp, we have been exploring other venues to increase our chance for P-gp crystallization. We have been working on producing monoclonal antibodies for co-crystallization experiments. We are also working on crystallization of the yeast MDR protein pdr5 and on bacterial ortholog LmrA from L. lactice. In addition to the crystallization effort, we have been working with Dr. S. Bates of NCI to produce the full-length ABCG2 model and with Dr. S. Ambudkar to make models of NBD of Pgp in different states to facilitate our studies of the mechanisms of function of ABC transporters.My unit has also been working on structural studies of P-type ATPases, especially those of Type-I or soft-metal ATPases for which structural information is not available. We have made progress in producing crystals of a transmembrane fragment of CopB, which diffracted X-ray to about 6 resolution.
