Characterization of Monomeric Human Cytochrome P450 3A4 and Cytochrome P450 Reductase in Nanoscale Phospholipid Bilayer Discs
The differences that cause the conformational heterogeneity of CYP3A4 in other solubilization systems have complicated the complete biophysical characterization in comparison to similar soluble bacterial counterparts. The use of a discrete soluble nanoscale phospholipid bilayer system with an incorporated CYP3A4 molecule will be described here; it is this homogeneous monomeric population that will give rise to the detailed description of the differences between these systems of study. This Nanodisc technology allows for a homogeneous population of enzyme that is devoid of conformational heterogeneity to describe the binding and catalytic parameters without the use of detergents, whose presence can further complicate these characterizations. The Nanodisc assemblies containing either CYP3A4 or CYP3A4 and its redox partner, cytochrome P450 reductase, as a coincorporated complex are described and further characterized for substrate binding, rates of dithionite dependent reduction, NADPH utilization rates, and testosterone metabolic rates as a function of substrate concentration. This novel methodology of incorporating cytochrome P450s during the self-assembly process to produce soluble monomeric homogeneous preparations of membrane associated cytochrome P450s has led to new insights in the characterization of a single cytochrome P450 in an unrestricted fraction while maintaining a native like phospholipid bilayer environment.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign