STRUCTURE AND FUNCTION OF BACTERIORHODOPSIN

目的是了解细菌视紫红质的功能 (bR)在化学和原子结构的水平上。 bR是一个 一种跨膜蛋白的原型， 直接参与跨膜的化学过程 运输 一个目的是实现制备性纯化， 从PBR 322 β- 在E.杆菌 的 将掺入发色团视网膜色素以产生 具有与野生型相同性质的细菌视紫红质 bR. bR的许多单个残基将被位点改变- 定向诱变，并通过三个评估对功能的影响 化学计量和效率的定量分析，通过这些分析， 蛋白质对光的反应是输送质子。 每一个的结构 突变的蛋白质将使用电子探针定量评估。 衍射高达2.65 A的分辨率，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， 化学作用在功能中的作用。 第二个目标是在高温下实现三维结构， 分辨率为bR。 自从电子衍射方法证明 分辨率超过3.5埃时， 膜平面，并在较低的分辨率为标题的图像3-D bR晶体是X射线衍射的基础。 bR从 将盐生盐杆菌溶解在Triton X-100中， 在甾醇去污剂中通过尺寸排阻、异质性 在壬基葡糖苷中通过等电聚焦除去，和 在第四洗涤剂中结晶。 连接在一起的 个别成功的步骤旨在超越目前的 晶体尺寸限制的X射线分辨率为5.5埃。 电子显微镜将有助于解决晶体结构。 含重金属标记的结合位点残基已经 定位在结构中的位置将被定位在序列中， 定点诱变和电子衍射。 这将 进一步在结构内限定顺序折叠路径， 膜。 研究结果将有助于深入了解能源的基本过程 转导和跨膜信号传导。 与哺乳动物视紫红质和跨膜视紫红质的相似性 通道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.