The Distribution of Cytochrome P450 Reductase and Cytochrome 2C9 Into Different
Phase Domains of Planar Biomimetic Lipid Assembly
David J. Wright, Dr. James A. Brozik, Dr. Jeff Jones
Washington State University, Department of Chemistry, Pullman, WA 99164
wrightdj@whitman.edu
Introduction
A series of experiments observing Fluorescence Recovery After Photobleaching (FRAP) microscopy were used to find the recovery rates in lipid bilayers formed with dilauroylphosphatidylserine (DLPS), dilauroylglycerophosphocholine (DLPC),
dioleoylglycerophosphocholine (DOPC), and a fluorescently labeled dimyrsitoylglycerophosphoethanolamine formed in a high ionic strength buffer solution containing HEPES, magnesium chloride with varying concentrations of CHAPS detergent. These experiments were
run with 0X, 1X, and 5X CHAPS detergent initially. In each of these samples, there was a presence of round domains sporadically located on the bilayer, and FRAP techniques were used on both the bilayer and the round domains in each sample. Research was also done in
the absence of the fluorescently labeled lipid, using the same procedures as before, but then laying down a 50μL layer of 100nM Cytochrome P450 Reductase (a membrane protein) labeled with AlexaFluor 555 in HEPES, sodium chloride, and calcium chloride. Again, using
0X, 1x, and 5X concentrations of CHAPS detergent, FRAP techniques were used to note the phase segregation of the protein in the different domains and to measure the varying diffusion coefficients. Further experiments were conducted with labeled 2C9 Cytochrome P450
to determine co-localization with Cytochrome P450 Reductase within the heterogeneous biomimetic lipid assembly.
Goals
FRAP Results
1. Observe diffusion coefficients of round domains using FRAP techniques
2. Observe diffusion coefficients of lipid bilayer using FRAP techniques.
3. Observe behavior of Cytochrome 2C9 using FRET microscopy
FRAP
Cytochrome 2C9 FRET Sample
• Cytochrome 2C9 colocalized on the
DLPS, DOPC, and DLPC lipid bilayer
•Cytochrome 2C9 after being
rinsed 7 times with 50 μL of
FRAP Microscopy Setup
5X HEPES and magnesium
chloride and 1X CHAPS
detergent on the same sample
as above
References
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Acknowledgments
Special thanks to the National Science Foundation, Adam Barden, Elsa Silva-Lopez, Angela Rudolph, and Adam Goler
for their support.