My group has been working on the mechanisms of function of three integral membrane proteins: proton transporting cyt bc1 complexes, multidrug ABC transporters and putative cisplatin resistance (CP-r) associated protein TMEM205. These projects are currently at different stages with respect to our understanding of their mechanisms of function. For the bc1 complex, we have a very good understanding of its mechanism of coupling. We have determined the crystal structures of bc1 complexes from the bovine mitochondria (Mtbc1) and from the photosynthetic bacterium Rhodobacter sphaeroides (Rsbc1) in both apo and inhibitor-bound forms. We have identified critical structural elements that are essential for the coupling of electron transfer and proton translocation. We have proposed a surface-affinity modulated iron-sulfur protein (ISP) motion control hypothesis to explain the bifurcated electron transfer in bc1. For the multidrug ABC transporters, in particular human P-glycoprotein (hP-gp), advances are being made towards its structure solution with respect to its over-expression in a number of eukaryotic systems, its purification and complex formation with monoclonal antibodies (mAb). We also made significant progress in obtain diffraction quality crystals of TMEM205 and its structure solution is on the way. Over the past few years, we have made significant progress in understanding the mechanism of function of the bc1 at atomic resolution by analyzing both native- and inhibitor-bound structures. We proposed a scheme for bc1 inhibitor classification and put forward mechanisms for quinone reduction at the QN site and quinol oxidation at the QP site. Most importantly, we have obtained experimental evidence to support our surface modulated conformation switch model for the electron bifurcation at the quinol oxidation site, which is the key to the high proton translocation efficiency in the bc1 complex. We have also successfully determined the crystal structures of the wild type and mutant bc1 complex from the photosynthetic bacterium R. sphaeroides (Rsbc1) in complex with various inhibitors, demonstrating our ability to reproducibly obtain atomic resolution structural information on the bacterial bc1 in various forms 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 by combining structural, genetic, and biochemical techniques;it marks another milestone in the study of bc1 complex and in the field of membrane protein structural biology. Recently, we have achieved a long-sought after structure of ubiquinol, the substrate, bound at the quinol oxidation site of bc1, which is a one step forward toward experimental verification of our hypothesis on bifurcated electron transfer under physiological conditions. Development of CP-r in cancer cells appears to be a consequence of multifaceted alterations involved in various cellular processes. Recently, it was found that expression of the hypothetical transmembrane protein TMEM205 is associated with cisplatin resistance, first detected by functional cloning from a retroviral cDNA library made from human CP-r cells. Using a polyclonal antibody, it was found that TMEM205 expression is increased in our CP-r cell lines. Stable transfection of the TMEM205 gene confers resistance to cisplatin by approximately 2.5-fold. Recombinant over-expression of TMEM205 in yeast directs the protein to cytoplasmic membrane, which was purified to homogeneity and in large quantity suitable for crystallographic studies. Crystals of TMEM205 were grown and diffracted X-rays to 3.4 resolution. Structural solution is in progress and it is expected that it will help to unveil the structural basis of TMEM205 involvement in cisplatin resistant. The development of methodology for membrane protein expression, purification, and crystallization has been an integral part of our research on structure determinations of P-gp, its homologues and TMEM205. To this end, we have been exploring various expression systems to achieve consistent high-level protein expression for a few membrane proteins;those include yeast systems such as S. cerevisiae and P. pastoris expression systems, bacterial systems such as E. coli and L. lactis expression systems, and photosynthetic bacterum R. sphaeroides. We have extended the use of Blue-Native techniques to detecting monodispersity of membrane protein preparations. We have also developed and refined a multi-parameter kit to screen for conditions for stabilizing membrane proteins in solution. We have achieved high-level expressions for a number of integral membrane proteins. In addition to the bacterial bc1 complex, the human P-glycolprotein, human TMEM205, bacterial ABC transporter LmrA, and bacterial CopB were purified in large quantities. To obtain monodispersed protein samples, we have been using the Blue-Native technique developed in house to screen for various detergents, which is very successful. For conformationally flexible membrane proteins such as ABC transporters, we tested mutants and Fab-complexed P-gp in crystallization experiments. Although we have yet to reach our goal of structure solutions of these membrane proteins, the methods developed here will be useful for other membrane proteins.
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