Hayward S B, Grano D A, Glaeser R M, Fisher K A
Proc Natl Acad Sci U S A. 1978 Sep;75(9):4320-4. doi: 10.1073/pnas.75.9.4320.
The direction of orientation of the protein bacteriorhodopsin within the purple membrane of Halobacterium halobium has been determined by selected-area electron diffraction of membranes preferentially oriented by adsorption to polylysine. Purple membrane is known to adsorb preferentially to polylysine by its cytoplasmic surface at neutral pH and by its extracellular surface at low pH. To maintain the adsorbed membranes in a well-ordered state in the electron microscope, an improved technique of preparing frozen specimens was developed. Large areas of frozen-hydrated specimens, devoid of bulk water, were obtainable after the specimen was passed through a Ca stearate film at an air-water interface. High-resolution microscopy was used to relate the orientation observed in the electron diffraction patterns to the orientation of the projected structure that is obtained from images. We have found that the three-dimensional structure determined by Henderson and and Unwin [Henderson, R. & Unwin, P.N.T. (1975) Nature 257, 28--32] is oriented with the cytoplasmic side uppermost--i.e., the helices fan outward on the cytoplasmic side of the membrane.
嗜盐菌紫膜中细菌视紫红质蛋白的取向方向，已通过对优先吸附于聚赖氨酸的膜进行选区电子衍射来确定。已知在中性pH值下，紫膜通过其胞质面优先吸附于聚赖氨酸；在低pH值下，则通过其细胞外表面优先吸附。为了在电子显微镜下使吸附的膜保持有序状态，开发了一种改进的制备冷冻标本的技术。在标本通过气-水界面的硬脂酸钙膜后，可获得大面积不含大量水分的冷冻水合标本。利用高分辨率显微镜将电子衍射图中观察到的取向与从图像获得的投影结构的取向联系起来。我们发现，亨德森和昂温[亨德森，R. & 昂温，P.N.T.（1975年）《自然》257, 28 - 32]确定的三维结构，其取向是胞质侧在最上方——即螺旋在膜的胞质侧向外展开。
