| Abstract
| - Two general models of the membrane topology ofmicrosomal cytochrome P450 have beenproposed: (1) deep immersion in the membrane, and (2) aP450cam-like heme domain anchored to themembrane with one or two membrane-spanning helices. Lamellar X-raydiffraction of oriented membranemultilayers was employed to distinguish these alternatives.Cytochrome P450 2B4 was reconstitutedinto unilamellar phospholipid proteoliposomes (molar protein to lipidratio 1:90). Sedimentation of theproteoliposomes produced an ordered stack of bilayers with aone-dimensional repeat distance (d)perpendicular to the plane of the bilayer. The stacked multilayerswere exposed to an X-ray beam (λ =1.54 Å) at near grazing incidence, and lamellar diffraction patternswere recorded. With proteoliposomemultilayers, up to six diffraction orders could be observed. Theirspacing corresponded to a d of 63.6 Å,calculated according to Bragg's Law, comprising the lipid bilayer, theprojection of the incorporatedprotein beyond the bilayer, and the intermembrane water layer.With liposome multilayers containing noP450, the observed d was 59.6 Å. These data suggestthat the increase of distance between successivebilayers in the stack due to the presence of P450 2B4 was only about 4Å. This distance is much lessthan would be expected with the “N-terminalmembrane-anchor” model of the membrane topology, inwhich the P450 molecules largely extend beyond the surface of themembrane (≥35 Å). Furthermore,the mass distribution deduced from Fourier synthesis confirms that theprotein is deeply immersed in themembrane.
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