Determining Molecular Structures of Cytochrome P450 and Its Complex Associated with Model Cell Membranes

Abstract

The lipid membrane assisted interaction between cytochrome P450 (CYP450) and its reductase (CPR) plays a significant role in many biochemical reactions and drug metabolism.Previous solution and solid-state studies have shown the importance of lipid bilayer on the structural folding of CYP450 and the electrostatic interactions between the soluble domains and the hydrophobic interactions between the transmembrane helical domains of CYP450 and CPR using bicelles and nanodiscs. In this study we report an effective way to determine both the conformation and orientation of the truncated microsomal CYP450 2B4 associated with lipid membranes, and its orientation transition when binding to a truncated flavin mononucleotide binding domain of CPR. The developed methodology combines measurements using sum frequency generation vibrational spectroscopy and attenuated total reflectance -Fourier transform infrared spectroscopy, and computational data interpretation, which is powerful and generally applicable to reveal the interfacial structures of protein complexes in physiologically relevant environments.