X-ray Diffraction Analysis of Cytochrome P450 2B4 Reconstituted into Liposomes

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Abstract
Two general models of the membrane topology of microsomal cytochrome P450 have been proposed: (1) deep immersion in the membrane, and (2) a P450cam-like heme domain anchored to the membrane with one or two membrane-spanning helices. Lamellar X-ray diffraction of oriented membrane multilayers was employed to distinguish these alternatives. Cytochrome P450 2B4 was reconstituted into unilamellar phospholipid proteoliposomes (molar protein to lipid ratio 1:90). Sedimentation of the proteoliposomes produced an ordered stack of bilayers with a one-dimensional repeat distance (d) perpendicular to the plane of the bilayer. The stacked multilayers were exposed to an X-ray beam (λ = 1.54 Å) at near grazing incidence, and lamellar diffraction patterns were recorded. With proteoliposome multilayers, up to six diffraction orders could be observed. Their spacing corresponded to a d of 63.6 Å, calculated according to Bragg's Law, comprising the lipid bilayer, the projection of the incorporated protein beyond the bilayer, and the intermembrane water layer. With liposome multilayers containing no P450, the observed d was 59.6 Å. These data suggest that the increase of distance between successive bilayers in the stack due to the presence of P450 2B4 was only about 4 Å. This distance is much less than would be expected with the “N-terminal membrane-anchor” model of the membrane topology, in which the P450 molecules largely extend beyond the surface of the membrane (≥35 Å). Furthermore, the mass distribution deduced from Fourier synthesis confirms that the protein is deeply immersed in the membrane.