High sensitivity electron diffraction analysis. A study of divalent cation binding to purple membrane.
High sensitivity electron diffraction analysis. A study of divalent cation binding to purple membrane.
复制标题
高灵敏度电子衍射分析。
DOI：
10.1016/s0006-3495(90)82532-7
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发表时间：
1990
影响因子：
3.4
通讯作者：
Stroud,RM
中科院分区：
文献类型：
--
作者：
Mitra,AK;Stroud,RM
描述了一种灵敏的高分辨率电子衍射分析结构变化的方法，并利用该方法分析了二价阳离子与盐杆菌紫色膜（PM）的结合。低剂量电子衍射图服从于匹配的滤波算法（Spencer， S. a .和a . a . Kossiakoff, 1980）。j:。Crystallogr 13:563 - 571)。提取准确的反射强度值。这种方法，再加上在显微镜下解释孪生和标本倾斜的方案，产生的结果足够敏感，可以快速量化由位点定向诱变引起的PM中的任何结构变化，直至小于两个碳原子的水平。从PM中去除紧密结合的二价阳离子（主要是Ca2+和Mg2+）会导致颜色变为蓝色，并伴随着蛋白质细菌紫红质（bR）的光循环严重改变，这是一种光驱动质子泵。我们描述了在3:1二价阳离子与PM结合时发生的结构变化，而不是通过添加过量的Na+而呈现紫色的膜。从室温下Pb2+和Na+重组PM的葡萄糖包埋样品中获得的高分辨率、低剂量电子衍射数据确定了几个总占用量为2.01 +/- 0.05 Pb2+当量的位点。Ca2+或Mg2+和Pb2+的颜色过渡作为离子浓度的函数是严格可比的。投影中的（Pb2(+)-Na+） PM傅立叶差分图是在5a处合成的，使用了几个名义上未倾斜的补丁的平均数据，校正了孪生和标本倾斜。我们发现6个主要位点位于螺旋7,5,4,3,2 （Engelman et al. 1980的命名法）。Proc。国家的。学会科学。美国。77:2023-2027)与bR密切相关。这些部分占据的位点（0.55-0.24 Pb2+当量）代表了二价阳离子的优先结合位点，并补充了我们早先通过x射线衍射得到的结果（Katre et al. 1986）。Biophys。j . 50:277 - 284)。
A sensitive high-resolution electron diffraction assay for change in structure is described and harnessed to analyze the binding of divalent cations to the purple membrane (PM) of Halobacterium halobium. Low-dose electron diffraction patterns are subject to a matched filter algorithm (Spencer, S. A., and A. A. Kossiakoff. 1980. J. Appl. Crystallogr. 13:563–571). to extract accurate values of reflection intensities. This, coupled with a scheme to account for twinning and specimen tilt in the microscope, yields results that are sensitive enough to rapidly quantitate any structure change in PM brought about by site-directed mutagenesis to the level of less than two carbon atoms. Removal of tightly bound divalent cations (mainly Ca2+ and Mg2+) from PM causes a color change to blue and is accompanied by a severely altered photocycle of the protein bacteriohodopsin (bR), a light-driven proton pump. We characterize the structural changes that occur upon association of 3:1 divalent cation to PM, versus membranes rendered purple by addition of excess Na+. High resolution, low dose electron diffraction data obtained from glucose-embedded samples of Pb2+ and Na+ reconstituted PM preparations at room temperature identify several sites with total occupancy of 2.01 +/- 0.05 Pb2+ equivalents. The color transition as a function of ion concentration for Ca2+ or Mg2+ and Pb2+ are strictly comparable. A (Pb2(+)-Na+) PM Fourier difference map in projection was synthesized at 5 A using the averaged data from several nominally untilted patches corrected for twinning and specimen tilt. We find six major sites located on helices 7, 5, 4, 3, 2 (nomenclature of Engelman et al. 1980. Proc. Natl. Acad. Sci. USA. 77:2023–2027) in close association with bR. These partially occupied sites (0.55–0.24 Pb2+ equivalents) represent preferential sites of binding for divalent cations and complements our earlier result by x-ray diffraction (Katre et al. 1986. Biophys. J. 50:277–284).