Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment
Section snippets
Chemicals
Imidazole, sodium cholate, testosterone, 6β-OH testosterone, and androstenedione were purchased from Sigma Chemical (St. Louis, MO), Emulgen 913 was from Karlan Research Products (Santa Rosa, CA), palmitoyl-oleoyl-phosphatidylcholine (POPC) was from Avanti Polar Lipids (Alabaster, AL), and hydrogen peroxide was from Fisher Chemical (Pittsburgh, PA).
Cytochrome P450 3A4 expression and purification
Cytochrome P450 3A4 was expressed from the NF-14 construct in the PCWori+ vector with a C-terminal pentahistidine tag generously provided by Dr. F.
Results
To guarantee monomeric cytochrome P450 3A4 in a native like phospholipid bilayer environment, we utilized Nanodisc technology. This methodology uses a MSP which self-assembles phospholipid and a membrane protein target into soluble discoidal bilayers ∼10 nm in diameter. The length of the encircling “belt” defines the size of the solubilized bilayer domain and hence can control the oligomerization state of incorporated targets. Self-assembly of cytochrome P450 3A4 was as described in Experimental
Discussion
Human cytochrome P450 3A4 was self-assembled into soluble Nanodiscs, in a clean 1:1 complex. These CYP 3A4:Nanodiscs allow the study of individual cytochrome P450 molecules in a system which mimics the native biological membrane, and is free of artifacts caused by aggregation. SAXS shows that these complexes have a monomeric CYP 3A4 molecule in a nanoscale phospholipid bilayer environment. Analysis of the possible models of CYP3A4 incorporated into the Nanodisc bilayer as illustrated at the
Acknowledgments
We gratefully acknowledge Dr. F.P. Guengerich for the clone of cytochrome P450 3A4. We also thank Aretta Weber for her excellent editorial help as well as a special thanks to Dr. Timothy Bayburt and Yelena Grinkova for their helpful discussion and expertise. Portions of this work were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) Synchrotron Research Center located at Sector 5 of the Advanced Photon Source. DND-CAT is supported by the E.I. DuPont de Nemours & Co.,
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