DECONVOLUTING THE DANCE OF THE CYTOCHROME P450 METABOLON AT THE LIPID BILAYER

Sara Charlotte Humphreys

A fundamental postulate of statistical mechanics—the ergodic hypothesis—states that the time-average of a physical quantity along the trajectory of a member of the ensemble is equivalent to the average of that quantity at a given time over the ensemble. In simpler terms, we should be able to reconcile the observed spatiotemporal dynamics of an individual molecule with the behavior of the ensemble. Much of our understanding of biological processes has been derived from ensemble measurements. In this dissertation, the ergodic hypothesis was examined from an experimental perspective using the hepatic cytochrome P450 metabolon as a model system. Single molecule measurements of cytochrome P450 2C9 (CYP2C9) and cytochrome P450 reductase (CPR) interacting with lipid bilayers were obtained in space over time. Findings were discussed in the context of the substantial body of literature describing the cytochrome P450 metabolon, which derives largely from ensemble experiments. The significance of this work is two-fold. First, cytochromes P450 are responsible for metabolizing ∼90% of FDA-approved drugs. Drug metabolism is significantly affected by dynamic interactions between the individual proteins that constitute this endoplasmic reticulum-bound complex. Therefore, a molecular understanding of these interactions is crucial for development of pharmaceuticals. Second, life is rife with transient protein-protein and protein-lipid interactions, which are difficult to characterize using classical ensemble methods. Here, experimental procedures have been developed, and analytical foundations laid out, to collectively obtain unprecedented insight into these processes. The exposition of the work is divided into five chapters. Chapter 1 is a review of cytochrome P450 metabolon literature, emphasizing protein-protein and protein-lipid interactions. Chapter 2 describes a single molecule total internal reflectance (SM-TIRF) spectroscopy study performed to characterize 2D diffusion of CPR in supported lipid bilayers, and perform frequency-based mapping of CPR-SLB interactions. In Chapter 3, fluorescence correlation spectroscopy (FCS) was used to quantify the equilibrium partitioning of CPR and CYP2C9 between lipid bilayers and bulk solution. In Chapter 4, previously uncharacterized native P450 luminescence was exploited to follow substrate binding to CYP in SLBs using confocal microscopy. Finally, in Chapter 5, the works are summarized and compared in a brief conclusion.

DECONVOLUTING THE DANCE OF THE CYTOCHROME P450 METABOLON AT THE LIPID BILAYER

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