Over the past few years, we have made significant progress in obtaining better diffracting bovine mitochondrial cytochrome bc1 (cyt bc1, bc1) crystals and in understanding the mechanism of function 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 Qi site and quinol oxidation at the Qo site. 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 crystallization has been an integral part of our research on structure determinations of P-gp and its homologues. We have explored various expression systems to achieve consistent high-level protein expression. We have developed and refined a multi-parameter kit to screen for conditions for stabilizing P-gp in solution. As a result, we achieved P-gp preparations at high concentration. To achieve conformation uniformity, we tested mutants and Fab-complexed P-gp in crystallization experiments. We designed and tested tens of thousands crystallization conditions and obtained valuable first-hand knowledge of the behavior of P-gp under these conditions. Although we have yet to reach our goal of structure solution of P-gp, the methods developed have been used for expression, purification, and crystallization of P-gp homologues such as the yeast Pdr5p and the bacterial LmrA from L. lactice. These methods have also been successfully applied to the crystallization of a different class of membrane transporters, the P-type ATPases, including the CopB ion transporter from E. coli and proton transporter Pma1p of yeast, both diffracted X-ray at synchrotron. In addition, my unit has developed procedures to model full-length ABC transporter structures.
