STRUCTURE AND FUNCTION OF BACTERIORHODOPSIN

目的是了解细菌视紫红质的功能。 (Br)在化学和原子结构水平上。BR是一个 跨膜蛋白的原型，其中许多氨基酸 直接参与跨膜的化学过程 运输。一个目标是实现制备性纯化 从PBR322β-产生的细菌螺旋体蛋白- 半乳糖苷酶-细菌螺蛋白融合载体在大肠杆菌中的表达。这个 生色团视网膜将被并入以产生 具有与野生型相同性质的细菌视紫红质 Br.BR的许多个体残留物会因位点而改变- 定向诱变及其对功能的影响 化学计量学和效率的定量分析 蛋白质对光的反应是泵送质子。每种语言的结构 突变的蛋白质将用电子定量评估 衍射率最高可达2.65埃，这将允许清晰的定义 突变效应是化学的，还是仅仅是 扰乱折叠或结构。只有通过这种综合，才能 在功能中的化学角色被分配。 第二个目标是实现高分辨率的三维结构 BR的解决方案。自从电子衍射法证明 在超过3.5埃的分辨率下变得越来越复杂 膜平面，并在较低分辨率的标题图像3-D 溴的晶体是X射线衍射的基础。BR发件人 盐生杆菌将在Triton X-100中增溶， 通过大小排斥、异质性在甾醇洗涤剂中提纯 通过等电聚焦在壬基葡萄糖苷中去除，以及 结晶成了第四种洗涤剂。链接在一起的 个人成功的步骤旨在超越当前的 晶体尺寸--X射线分辨率限制为5.5埃。 电子显微镜将有助于解决晶体结构问题。 含重金属标记物的结合部位残基已经存在 在结构中定位的是在序列中使用 定点诱变和电子衍射。这将是 进一步定义结构内序列的折叠路径 膜。 结果将对能量的基本过程产生洞察力 生物学中的转导和跨膜信号传递。 与哺乳动物视紫红质和跨膜的相似性 通灵受体将照亮视觉过程， 神经化学和细胞-细胞调节。

The objective is to understand the function of bacteriorhodopsin (bR) at the level of chemistry and atomic structure. bR is an archetype of a transmembrane protein where many amino acids are directly involved in the chemical process of transmembrane transport. One aim is to achieve a preparative purification of bacterioospin protein produced from a PBR322 beta- galactosidase-bacterioospin fusion vector in E. coli. The chromophore retinal is to be incorporated to generate bacteriorhodopsin with the same properties as those of wild type bR. Many individual residues of bR will be altered by site- directed mutagenesis, and effects on function assessed by three quantitative assays of stoichiometry and efficiency by which the protein pumps protons in response to light. The structure of each mutated protein will be assessed quantitatively using electron diffraction up to 2.65 A resolution, that will allow clear definition of whether a mutational effect is chemical or is merely one that perturbs the folding or structure. Only through this synthesis can chemical role in the function be assigned. A second aim is to achieve a three-dimensional structure at high resolution for bR. Since electron diffraction methods proved increasingly complex at resolutions beyond 3.5 Angstroms in the membrane plane, and at lower resolutions for titled images 3-D crystals of bR are the basis for x-ray diffraction. bR from Halobacterium halobium will be solubilized in Triton X-100, purified in a sterol detergent by size exclusion, heterogeneity removed by isoelectric focusing in nonyl glucoside, and crystallized in a fourth detergent. Linking together of individually successful steps is aimed at exceeding the current crystal size-limited x-ray resolution of 5.5 Angstroms resolution. Electron microscopy will assist in solving the crystal structure. Binding site residues for heavy metal-containing labels already localized in the structure are to be located in the sequence using site-directed mutagenesis and electron diffraction. This will further define the folding path of sequence within the structure in membranes. Results will yield insights in fundamental processes of energy transduction and transmembrane signalling in biology. Similarities to mammalian rhodopsin and to transmembrane channeling receptors will illuminate processes in vision, neurochemistry and cell-cell regulation.