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首页> 外文期刊>Biochemistry >X-RAY DIFFRACTION ANALYSIS OF CYTOCHROME P450 2B4 RECONSTITUTED INTO LIPOSOMES
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X-RAY DIFFRACTION ANALYSIS OF CYTOCHROME P450 2B4 RECONSTITUTED INTO LIPOSOMES

机译：重构为脂质体的细胞色素P450 2B4的X射线衍射分析

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摘要

Two general models of the membrane topology of microsomal cytochrome P450 have been proposed: (1) deep immersion in the membrane, and (2) a P450(cam)-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 (lambda = 1.54 Angstrom) 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 Angstrom, 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 Angstrom. 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 Angstrom. 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 (greater than or equal to 35 Angstrom). Furthermore, the mass distribution deduced from Fourier synthesis confirms that the protein is deeply immersed in the membrane.
机译：已经提出了微粒体细胞色素P450的膜拓扑结构的两个通用模型：（1）深浸在膜中，和（2）带有一个或两个跨膜螺旋的锚定在膜上的P450（cam）样血红素结构域。定向膜多层的层状X射线衍射用于区分这些替代方案。将细胞色素P450 2B4重构为单层磷脂蛋白脂质体（蛋白与脂质的摩尔比为1:90）。蛋白脂质体的沉淀产生了双层的有序堆叠，其垂直于双层的平面具有一维重复距离（d）。堆叠的多层在接近掠入射的情况下暴露于X射线束（λ= 1.54埃），并记录层状衍射图。使用多层脂质体，可以观察到多达六个衍射级。根据布拉格定律计算，它们的间距对应于63.6埃的d，包括脂质双层，掺入的蛋白质超出双层的投影以及膜间水层。对于不含P450的脂质体多层，观察到的d为59.6埃。这些数据表明，由于P450 2B4的存在，堆栈中连续双层之间的距离增加仅为约4埃。该距离远小于采用膜拓扑结构的“ N末端膜锚”模型所预期的距离，在该模型中，P450分子在很大程度上延伸超出了膜的表面（大于或等于35埃）。此外，从傅里叶合成推导的质量分布证实了蛋白质深深地浸没在膜中。

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