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Deconvoluting the Dance: Cytochrome P450 Interaction Mapping via Super‐Resolution Imaging
Author(s) -
Humphreys Sara,
Barden Adam,
Barnaba Carlo,
Brozik James,
Jones Jeffrey
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.778.2
Subject(s) - lipid bilayer , cytochrome p450 , biophysics , chemistry , biological system , biology , biochemistry , membrane , enzyme
Generate a lipid bilayer on a hydrophilically treated borosilicate glass surface. Insert cytochrome P450 (CYP) and P450 reductase (CPR). Add NADPH regenerating system. Introduce substrate. Observe, measure and map the ‘dance' in near real‐time at low nanometer resolution using fluorescence microscopy. Variations on this theme are what constitutes our experimental system. By fluorescently labeling one component at a time, we are literally building – molecule by molecule – a working spatiotemporal model of the P450 machine in a lipid‐bilayer environment. To this end, we aim to contribute to knowledge of the biophysical mechanisms underlying P450 drug metabolism. Starting with simple lipid‐lipid interactions and expanding to encompass protein‐lipid and protein‐protein interactions, we have applied single molecule techniques to characterize planar diffusion, determine residence times and monitor intensity – thus obtaining a rudimentary understanding of the behavioral distribution of individual proteins and lipid molecules. By overlaying many thousands of 10 ms images, we have generated super‐resolution interaction maps that highlight (1) the trajectories of thousands of CPRs (2) the influence of CYPs on localization and motion of CPRs, and (3) the heterogeneous nature of lipid domain organization and how the CYPs and CPRs move with respect to them. By direct observation, this system delivers a structural road‐map to monitor lipid phase heterogeneity, dimensionality and segregation of proteins within and between domains. We have detected distinct patterns of spatiotemporal protein confinement and postulate that this may be related to the transfer of electrons between CPR and CYP, and the subsequent positioning of CYP for monooxygenation of its substrates.