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 confomation 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. Recently, we have 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. 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 and its homologues. 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 P-gp 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, 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.
