AFM Study of Membrane Proteins, Cytochrome P450 2B4, and NADPH–Cytochrome P450 Reductase and Their Complex Formation

The application of the AFM technique for visualization of membrane proteins and for measuring their dimensions was demonstrated. The AFM images of the microsomal monooxygenase system components—cytochrome P450 2B4 and NADPH–cytochrome P450 reductase—were obtained by using two types of supports—hydrophobic, highly oriented pyrolytic graphite (HOPG) and hydrophilic mica. It was shown that hemo- and flavoprotein monomers and oligomers can be adsorbed to and visualized on HOPG. On the negatively charged mica matrix, flavoprotein oligomers dissociated to monomers while hemoprotein oligomers dissociated into less aggregated particles. The images of cytochrome P450 2B4 and NADPH–cytochrome P450 reductase monomers were about 3 and 5 nm high, respectively, while the images of oligomeric forms of these proteins were about 10 and 8 nm high, respectively. We were able to observe the binary complexes composed of monomeric proteins, cytochrome P450 2B4 and its reductase and to measure the heights of these complexes (7 nm). The method is applicable for visualization of not only individual proteins but also their complexes.