MODEL FOR THE STRUCTURE OF BACTERIORHODOPSIN BASED ON HIGH-RESOLUTION ELECTRON CRYOMICROSCOPY
MODEL FOR THE STRUCTURE OF BACTERIORHODOPSIN BASED ON HIGH-RESOLUTION ELECTRON CRYOMICROSCOPY
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DOI：
10.1016/s0022-2836(05)80271-2
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发表时间：
1990-06-20
影响因子：
5.6
通讯作者：
DOWNING, KH
中科院分区：
文献类型：
--
作者：
HENDERSON, R;BALDWIN, JM;DOWNING, KH
光驱动的质子泵细菌视紫红质天然地以二维晶体的形式存在。通过使用电子冷冻显微镜研究晶体以获得电子衍射图案和高分辨率显微照片，获得了接近原子分辨率的三维结构密度图。开发了用于分析倾斜样本显微照片的新方法，结合了先前为倾斜样本开发的方法，可以分析大面积并校正扭曲。对来自倾斜和未倾斜样本的 72 幅图像的数据进行了分析，以产生该结构的 2700 个独立傅立叶分量的相位。这些成分的振幅是通过 150 个衍射图案精确测量的。这些数据加在一起代表了 3.5°ANG 完整三维变换的大约一半。结构图的分辨率为 3.5°ANG。在平行于膜平面的方向上但低于在垂直方向上的膜平面。它显示了从七个α-螺旋的主密度解析出的密度的许多特征。我们将这些特征解释为苯丙氨酸、酪氨酸和色氨酸残基的庞大芳香族侧链。还有一个非常致密的特征，就是视网膜发色团的β-紫罗兰酮环。使用这些庞大的侧链作为引导点，并考虑到螺旋中的凸起表明较小的侧链（例如亮氨酸），建立了氨基酸残基 8 和 225 之间的细菌视紫红质的完整原子模型。视网膜周围有 21 个氨基酸残基，由所有 7 个螺旋贡献，还有 26 个残基，由 5 个螺旋贡献，形成质子途径或通道。质子通道中部的十个氨基酸残基也是视网膜结合位点的一部分。该模型还为考虑质子泵浦机制提供了有用的基础，并允许对大量其他实验数据进行一致的解释。特别是，该结构表明席夫碱中的 pK 变化必须充当光能在通道中转化为质子泵浦压力的手段。 Asp96 位于从细胞质到希夫碱的途径上，Asp85 位于从希夫碱到细胞外表面的途径上。
The light-driven proton pump bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analyzing micrographs for tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analyzed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analyzed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full three-dimensional transform to 3.5 .ANG.. The map of the structure has a resolution of 3.5 .ANG. in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven .alpha.-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine, and tryptophan residues. There is also a very dense feature, which is the .beta.-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built. There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues, contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site. The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel. Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.