X-ray diffraction analysis of cytochrome P450 2B4 reconstituted into liposomes.
X-ray diffraction analysis of cytochrome P450 2B4 reconstituted into liposomes.
复制标题
重组至脂质体中的细胞色素 P450 2B4 的 X 射线衍射分析。
DOI：
10.1021/bi9514572
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
White,RE
中科院分区：
文献类型：
--
作者：
Miller,JP;Herbette,LG;White,RE
微粒体细胞色素P450的膜拓扑结构有两种一般模型：（1）深浸在膜中，（2）P450 cam样血红素结构域锚定在膜上，具有一个或两个跨膜螺旋。取向膜多层膜的层状X射线衍射被用来区分这些替代品。将细胞色素P450 2B 4重构为单层磷脂蛋白脂质体（蛋白与脂质摩尔比为1：90）。蛋白脂质体的沉积产生了有序的双层堆叠，其一维重复距离（d）垂直于双层平面。将堆叠的多层膜暴露于近掠入射的X射线束（λ = 1.54 λ），并记录层状衍射图案。与蛋白脂质体多层，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adof 63.6 μ m，包括脂质双层、掺入的蛋白质在双层之外的投影和膜间水层。不含P450的脂质体多层膜的包封率为59.6%。这些数据表明，由于P450 2B 4的存在，堆叠中连续双层之间的距离仅增加约4 μ m。该距离远小于膜拓扑结构的“N-末端膜锚”模型的预期距离，其中P450分子大部分延伸超出膜表面（≥35 μ m）。此外，从傅立叶合成推导出的质量分布证实了蛋白质深深地浸入膜中。
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 adof 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 observeddwas 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.