Scientific Abstract Cytochrome P450s (cytP450s) are one of the most important proteins in xenobiotic metabolism. More than ~70% of the drugs in clinical use are metabolized by cytP450s, and the activity of thousands of molecules as substrates for this powerful enzyme is routinely investigated. CytP450s, for instance, are the primary targets for the treatment of prostate and breast cancer. Cytochrome P450 reductase (CPR) plays a crucial role by donating two electrons to cytochrome P450 which is essential for the completion of substrate turnover. However, high-resolution structural information on the protein-protein interaction of the cytP450-CPR complex is still lacking, preventing an in-depth understanding of the fundamental electron-transfer mechanism between the two redox partners. The main objective of the proposed research is to obtain an in-depth understanding of the molecular basis for the effects of CPR on cytP450 activity by determining the structural interactions between the membrane-bound full-length rabbit cytP4502B4 and the membrane-bound FMN binding domain (FBD) of CPR. The structure of the ~90-kDa cytP450-FBD complex embedded in a membrane environment will provide insights into the mechanism by which CPR regulates the enzymatic function of cytP450 and the electron-transfer process between the two proteins. We propose to use a combination of a variety of cutting- edge solution and solid-state NMR experiments to determine the high-resolution structure, dynamics, and membrane orientation of ~30-kDa membrane-bound FMN binding domain of CPR in bicelles first. Then, we will determine the structural and dynamic interactions of the cytP450-FBD complex embedded in bicelles and lipid vesicles. Mutational and functional studies will also be carried out to identify the residues on the interaction interface of the cytP450-FBD complex. The effect of selected substrates on the cytP450-FBD complex will be investigated using NMR and functional measurements. After completing our investigation on rabbit cytP4502B4, if time permitting, we will use other cytochrome P450s (including cytP4503A4, cytP4502D6 and cytP45017A1) to further elucidate the role of the membrane-bound FMN binding domain of CPR on the function of cytochrome P450.
to the public health Cytochrome P450s (cytP450s) are a ubiquitous superfamily of mixed-function oxygenases, which are found in all kingdoms of life but are especially abundant in eukaryotes. In fact, it has been discovered that humans have been found to possess 57 different membrane-bound cytP450s. They are found in all tissues of the body and are responsible for influencing a dazzling array of biochemical and physiological processes, including embryonic development, blood coagulation, and the metabolism of carcinogens and environmental toxins, over 70% of the drugs in clinical use today along with other exogenous and endogenous compounds. Human cytP450s are the target for the treatment of various diseases including prostate and breast cancer. The outcome of the proposed structural studies on the full-length membrane-bound cytochrome P450-FBD (FMN binding domain of cytochrome P450 reductase) complex will provide powerful insights into the molecular mechanism by which cytochrome P450 reductase influences the catalytic activity of cytP450 that is responsible for the metabolism of more than 70% of the current-day pharmaceuticals.
| Damron, Joshua T; Ma, Jialiu; Kurz, Ricardo et al. (2018) The Influence of Chemical Modification on Linker Rotational Dynamics in Metal-Organic Frameworks. Angew Chem Int Ed Engl 57:8678-8681 |
| Ravula, Thirupathi; Hardin, Nathaniel Z; Bai, Jia et al. (2018) Effect of polymer charge on functional reconstitution of membrane proteins in polymer nanodiscs. Chem Commun (Camb) 54:9615-9618 |
| Ravula, Thirupathi; Hardin, Nathaniel Z; Ramadugu, Sudheer Kumar et al. (2018) Formation of pH-Resistant Monodispersed Polymer-Lipid Nanodiscs. Angew Chem Int Ed Engl 57:1342-1345 |
| Barnaba, Carlo; Sahoo, Bikash Ranjan; Ravula, Thirupathi et al. (2018) Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs. Angew Chem Int Ed Engl 57:3391-3395 |
| Gentry, Katherine A; Zhang, Meng; Im, Sang-Choul et al. (2018) Substrate mediated redox partner selectivity of cytochrome P450. Chem Commun (Camb) 54:5780-5783 |
| Prade, Elke; Mahajan, Mukesh; Im, Sang-Choul et al. (2018) A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs. Angew Chem Int Ed Engl 57:8458-8462 |
| Barnaba, Carlo; Ravula, Thirupathi; Medina-Meza, Ilce G et al. (2018) Lipid-exchange in nanodiscs discloses membrane boundaries of cytochrome-P450 reductase. Chem Commun (Camb) 54:6336-6339 |
| Naito, Akira; Matsumori, Nobuaki; Ramamoorthy, Ayyalusamy (2018) Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy. Biochim Biophys Acta Gen Subj 1862:307-323 |
| Zhang, Rongchun; Mroue, Kamal H; Ramamoorthy, Ayyalusamy (2017) Proton-Based Ultrafast Magic Angle Spinning Solid-State NMR Spectroscopy. Acc Chem Res 50:1105-1113 |
| Ravula, Thirupathi; Ramadugu, Sudheer Kumar; Di Mauro, Giacomo et al. (2017) Bioinspired, Size-Tunable Self-Assembly of Polymer-Lipid Bilayer Nanodiscs. Angew Chem Int Ed Engl 56:11466-11470 |
Showing the most recent 10 out of 94 publications
