STRUCTURE AND FUNCTION OF BACTERIAL SENSORY RHODOPSIN

细菌感觉视紫红质的结构和功能

• 批准号: 3274996
• 负责人: JOHN LEE SPUDICH
• 金额: $ 23.59万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3274996
• 关键词: Halobacteriaceae; active transport; atomic absorption spectrometry; bacteriorhodopsins; bioenergetics; biological signal transduction; cell motility; chemotaxis; conformation; hydrogen transport; immunochemistry; laboratory rabbit; membrane proteins; nonvisual photoreceptor; nucleic acid probes; photochemistry; protein structure function; radiotracer; retina; rhodopsin; site directed mutagenesis

Three proteins in the Halobacterium halobium phototaxis system have been identified: The ~25 kDa retinal-containing receptors sensory rhodopsins I and II (SR-I and SR-II), and a carboxylmethylated protein of 94 kDa Mr (MPP-I, methyl-accepting photoaxis protein I) involved in SR-I signalling. SR-I exists in two photoactive forms, one serving as an attractant receptor for orange-red light (lambdamax 587 nm), and the second as a repellent receptor for UV-blue light (lambdamax 373 nm). SR-II provides additional repellent sensitivity in the blue-green (lambda 487 nm). Photoisomerization of retinal in the SR-I or SR-II chromoprotein triggers a sequence of thermal steps returning the molecule to its original state. This cyclic process (or photocycle) produces receptor conformations (signalling states) which activate post-receptor components to modulate the flagellar motors. The signalling states of SR-I and SR-II are being identified by incorporating retinal analogs which alter lifetimes of photocycle intermediates, and measuring the photoaxis signalling efficiency of the analog receptors by computer-assisted motion analysis of swimming cells. SR-I, the better characterized of the two receptors, is similar in structure and in its retinal/apoprotein interactions to the light-driven proton pump bacteriorhodopsin (BR), yet unlike BR, SR-I is not active in ion translocation. To identify molecular processes during the SR-I photocycle which are essential for signalling, a synthetic SR-I chromoprotein gene (sopI) designed for casette mutagenesis will be site- specifically mutated and expressed in H. halobium by plasmid transformation. Initially a set of 12 residues implicated in photosignal transduction will be modified. The mutant receptors will be analyzed for spectroscopic properties in native membranes and for phototaxis signalling by analysis of cell swimming behavior. Our working hypothesis is that activated SR-I interacts with MPP-I, which relays the signal to the flagellar motor. SR-I/MPP-I protein/protein interaction will be tested and the stoichiometric relationship of MPP-I to SR-I determined. Sequences of proteolytic fragments of MPP-I will be used to design oligonucleotides probes for cloning. The gene-derived sequence will aid in the characterization of the MPP-I protein for the presence of membrane spanning segments, intracellular and extracellular domains, and the number and location of methylation sites.

盐杆菌趋光系统中的三种蛋白质已被 鉴定：约 25 kDa 的视网膜受体感觉视紫红质 I 和 II（SR-I 和 SR-II），以及 94 kDa Mr 的羧甲基化蛋白 （MPP-I，甲基接受光轴蛋白 I）参与 SR-I 信号传导。 SR-I 以两种光敏形式存在，其中一种作为引诱受体 用于橙红光（lambdamax 587 nm），第二个作为驱虫剂 UV-蓝光受体（λmax 373 nm）。 SR-II 提供额外的 蓝绿色（λ 487 nm）的驱避灵敏度。 SR-I 或 SR-II 色素蛋白中视网膜的光异构化触发 将分子恢复到原始状态的一系列热步骤。 这种循环过程（或光循环）产生受体构象 （信号状态）激活受体后成分来调节 鞭毛马达。 SR-I 和 SR-II 的信号状态正在 通过合并改变寿命的视网膜类似物来识别 光循环中间体，并测量光轴信号效率 通过计算机辅助游泳运动分析模拟受体 细胞。 SR-I 是两种受体中特征较好的，其相似之处在于 结构及其视网膜/脱辅基蛋白与光驱动的相互作用 质子泵细菌视紫红质 (BR)，但与 BR 不同，SR-I 在 离子易位。 识别 SR-I 期间的分子过程 光循环对于信号传导至关重要，是一种合成的 SR-I 设计用于盒式诱变的色蛋白基因（sopI）将被定点 通过质粒在 H. halobium 中特异性突变和表达 转变。 最初涉及光信号的一组 12 个残基 转导将被修改。 将分析突变受体 天然膜的光谱特性和趋光性信号传导 通过分析细胞游泳行为。 我们的工作假设是激活的 SR-I 与 MPP-I 相互作用，这 将信号传递给鞭毛马达。 SR-I/MPP-I蛋白/蛋白 将测试相互作用以及 MPP-I 与 SR-I 已确定。 将使用 MPP-I 的蛋白水解片段的序列 设计用于克隆的寡核苷酸探针。 基因衍生序列 将有助于表征 MPP-I 蛋白是否存在 跨膜片段、细胞内和细胞外结构域，以及 甲基化位点的数量和位置。