John Hackett, Ph.D.
Research program member: Developmental Therapeutics
1101 E. Marshall St.
Richmond, VA 23298-0133
Associate Professor, Physiology and Biophysics, School of Medicine
PhD, Ohio State University (2004)
CYPs involved in the synthesis of steroid hormones serve essential endocrine functions and have been successfully targeted for the treatment of hormone-dependent cancers. There is minimal understanding of the CYP mechanisms used for substrate recognition and discrimination or their ability to catalyze highly selective oxidations. It is well-recognized that the membrane environment can dramatically alter function of membrane proteins, including CYPs. Properties of lipids that preferentially interact with the catalytic domain, how they affect substrate access, and how they perturb the functional ensemble of CYP conformations are unknown. To fill these voids, we have adopted the enzyme that catalyzes the terminal step in estrogen biosynthesis, 19A1, as a model. It is representative of other steroidogenic CYPs insofar that it also catalyzes a sequential oxidation and shares structural features at the putative membrane interface. The central hypotheses are that 1) 19A1 captures substrates and intermediate products through the membrane utilizing a conformational selection (CS) mechanism, 2) lipid composition alters the 19A1 conformational ensemble and thereby modulates the efficiency of substrate recruitment, and 3) cytochrome b5 (b5) regulates 19A1 activity through allosteric networks. We propose to approach these holes in our current understanding and overcome the limitations of existing methods using a suite of computational and experimental approaches. The significance of this set of studies is the promise of unprecedented insight into the nature of 19A1-lipid interactions and their functional impact on ligand binding, detailed descriptions of ligand dependent changes in protein dynamics, definitive assignment of ligand binding mechanisms, and identification of the protein interaction networks that are involved in the regulation by b5.
Aromatase, membrane proteins, estrogens, cytochrome P450, spectroscopy