Overall, the project seeks to investigate the structure and dynamics of two P450 enzymes, P450 cam (CYP101) and the microsomal integral membrane protein P450 2B4 (CYP2B4). The first specific aim of the project is to conduct hydrogen/deuterium exchange experiments using nuclear magnetic resonance and mass spectrometry to investigate the structural dynamics involved in cytochrome P450 catalysis. Collaboration with Prof. Jeffery Agar (Brandeis Univ.) will provide crucial training for the researcher in the field of biological mass spectrometry. Multidimensional NMR methods can be used to complement mass spectral studies, and will be used to further localize in a site-specific manner functionally relevant dynamics. The next specific aim is to conduct backbone resonance assignments for investigation of structure and conformational dynamics of CYP2B4. In collaboration with Prof. Lucy Waskell (Univ. of Michigan) the researcher will have access to milligram quantities of isotopically enriched CYP2B4. Backbone assignment is the first step for NMR relaxation measurements, which are an excellent way to investigate both backbone, sidechain, and global dynamics. To accomplish this goal, the researcher will have on-site access to both 800 and 600 MHz spectrometers. The 800 MHz NMR is equipped with a cryoprobe and cold 1H and 13C preamplifiers. The last specific aim is identification of key backbone resonances involved in xenobiotic turnover by mammalian CYP2B4. Effector binding to other P450s has been hypothesized to cause distinct conformational changes that are necessary for turnover, but there have been no investigations of structural fluctuations of CYP2B4. NMR chemical shift perturbation experiments can identify residues involved in binding in and around the active site, and this evidence will be used to direct future dynamic investigation. The first step will be to use heteronuclear multidimensional solution NMR to investigate both oxidation states of the mammalian protein 2B4 bound to an inhibitor. The biologically relevant effectors will be added and the residues that interact with these proteins will be identified. These initial studies will identify key residues and demonstrate the feasibility of using solution NMR as a tool to further investigate the structure and dynamics of CYP2B4. ? ?
. CYP2B4 is a critical integral membrane protein involved in xenobiotic and drug metabolism in mammals and a detailed investigation of dynamics would provide valuable insight into the mechanism. The combination of structural and dynamics studies provides the comprehensive analysis required to elucidate the physical origins of the P450 mechanism. ? ? ?
| Pochapsky, Susan Sondej; Dang, Marina; OuYang, Bo et al. (2009) Redox-dependent dynamics in cytochrome P450cam. Biochemistry 48:4254-61 |
