Jed Lampe (2015-2016)
Assistant Professor, Department of Pharmacology, Toxicology and Therapeutics
University of Kansas Medical Center

Defining the Hydrophobic and Electrostatic Interactions between CYP3A and CYPb5

The project seeks to understand of how protein-protein interactions act to regulate CYP-mediated metabolism.These results will further our understanding of the role protein-protein interactions play in modulating a basic biological process, supporting fundamental knowledge about the nature and behavior of living systems.

Protein-protein interactions are essential to the function of cytochrome P450 (CYP) monooxygenases, yet the details of these interactions are difficult to ascertain due to their transient nature. An example of this is the interaction between the CYP enzyme superfamily and cytochrome b5 (b5), a 17 kDa heme containing protein. Cytochrome b5 can stimulate, inhibit, or have no effect on CYP-mediated metabolism, depending on the specific substrate-CYP pair under consideration. For example, b5 modulates the metabolism of many CYP3A4 substrates, but does not known alter the metabolic profile of many CYP3A7 substrates, despite the high degree of sequence identity (87%) between CYP3A4 and CYP3A7. The objective of this application is to characterize the molecular interactions of b5 with two distinct CYP3A isoforms: CYP3A4 and CYP3A7, using the unnatural amino acid, 13C-p-cyanophenylalaine as an intrinsic spectral reporter. Our hypothesis is that hydrophobic interactions between b5 and the individual CYP3A isoforms mediate the differences in functional activity previously observed between CYP3A4 and CYP3A7. To test our hypothesis, we propose the following specific aim:

Specific Aim 1: Quantify the relative contribution of hydrophobic and electrostatic forces to the interactions between cytochrome b5 and CYP3A enzyme in both the CYP3A4-b5 and CYP3A7-b5 complexes utilizing fluorescence, FTIR, and NMR with the unnatural amino acid 13C-p-cyanophenylalanine as a reporter.
Upon completion of this specific aim, we expect to have quantified the molecular forces involved in the interactions between the b5-CYP3A4 and b5-CYP3A7 complexes. This knowledge will give us a fundamental understanding of how these proteins interact in concert to regulate CYP-mediated metabolism
of both endogenous and xenobiotic substrates.