SEMINAR: Application of nanodiscs for cytochrome P450 studies

Abstract:

Nanodiscs represent the most stable, versatile and biologically relevant model system for solubilization and functional studies of membrane proteins. In addition Nanodiscs provide an unprecedented opportunity for isolation and biophysical characterization of functional complexes of membrane proteins with controlled stoichiometry. Using Nanodisc technology we pursue the detailed mechanistic studies of steroidogenic human CYPs, such as CYP17A1 and CYP19A1, as well as the main xenobiotic metabolizing CYP in humans, CYP3A4. The oxygen activation and generation of active catalytic intermediates in P450 enzymes is studied using cryoradiolytic reduction at 77 K of frozen oxy-complexes. Protonation of peroxo- and hydroperoxo-ferric intermediates can be monitored spectroscopically using controlled annealing of unstable intermediates in H2O and D2O buffers. Kinetic solvent isotope effects in the steady-state turnover at ambient conditions allows to distinguish between alternative catalytic pathways through peroxo-ferric or Compound  1 driven catalysis. We also developed an efficient method for the detection and quantitation of drug-drug interactions mediated by CYP3A4 and applied it to several drugs to decipher the molecular details of heterotropic cooperativity in human cytochromes P450.