Application of Nanodisc Technology to a Membrane-Bound P450: Functional Studies and Resonance Raman Studies on CYP51A1
Date of Award
Doctor of Philosophy (PhD)
Membrane-bound cytochrome P450s are highly insoluble and tend to form aggregations in aqueous solution due to its hydrophobic N-terminal transmembrane anchor. The traditional methodology of studying membrane-bound proteins depends on the use of detergent; however, detergent solubilization of membrane P450s does not satisfy many criteria of biochemical and biophysical studies on membrane proteins. A well-developed Nanodisc technology has been proven to be an extraordinarily efficient method of studying membrane-bound P450s. The human sterol 14-demethylases (CYP51A1) are membrane-bound P450 enzymes that catalyze oxidative removal of the C32 methyl group of lanosterol by first forming an alcohol, then an aldehyde, and finally initiating the C-C bond cleavage. The mechanism of the third-step C-C bond cleavage is still under debate. The present study on CYP51A1 performs functional studies to reveal the difference in catalytic activity between detergent-solubilized CYP51A1 and nanodisc-incorporated CYP51A1. Resonance Raman spectroscopy coupled with nanodisc technology reveals the structural changes in the heme pocket induced by physiological substrates of CYP51A1. An enhancement in ligand affinity, NADPH oxidation, and product formation is observed for Nanodisc-incorporated CYP51A1. Ligand-binding induced partial low-to-high-spin conversion is observed by applying electronic absorption spectroscopy and Resonance Raman (RR) spectroscopy. This low degree of spin conversion of CYP51 is contributed by the retention of the water ligand coordinated to the heme iron as well as direct interaction between the hydroxyl group of lyase substrate and the iron center. No significant changes in active site structure are found between detergent-stabilized CYP51 and nanodisc-incorporated CYP51, nevertheless, it is demonstrated that nanodisc-incorporated assemblies provide much more well-defined active site RR spectroscopic responses, which induces a larger conversion from the low-to-high-spin state in presence of the substrates. Moreover, a positive polar environment around the exogenous diatomic ligand is detected, providing insight into the mechanism of this essential C-C bond cleavage reaction.