STRUCTURE/FUNCTION OF ARCHAEAL SENSORY RHODOPSINS

古细菌感觉视紫红质的结构/功能

• 批准号: 2850033
• 负责人: JOHN LEE SPUDICH
• 金额: $ 43.15万
• 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 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2850033
• 关键词: Halobacteriaceae; biological signal transduction; chemotaxis; cryoelectron microscopy; gene mutation; infrared spectrometry; nonvisual photoreceptor; photoactivation; photochemistry; protein kinase; protein structure function; receptor coupling; rhodopsin; site directed mutagenesis

Sensory rhodopsin I (SR-I) is a phototaxis receptor in the archaeon Halobacterium salinarium. The receptor protein is similar in structure to visual pigments, consisting of a single polypeptide which folds into 7- membrane-spanning alpha-helical segments forming an internal pocket where the chromophore retinal is bound. A second integral membrane protein, Htrl, which exhibits sequence homology with eubacterial chemotaxis transducers, is essential for SR-I signaling, and there is compelling but indirect evidence for the presence of SR-I and Htrl in a molecular complex. Our goal is to understand the mechanism of SR-l/Htrl coupling during phototaxis signaling. Experiments are designed to analyze the physical association and structural features of SR-I and Htrl in native membranes by spectrophotometric and protein quantitation to assess SR-I to Htrl stoichiometry; sulfhydryl engineering to probe transmembrane topology of both proteins and their oligomeric states in the dark and after photoactivation; deletion analysis and localized random mutagenesis combined with selection for phototaxis-deficient mutants and intergenic suppressors to define the interacting surfaces of the proteins. Knowledge gained will be applied to in vitro experiments, including interaction of Htrl and active fragments with purified SR-I. High yield purification methods are being developed for these experiments as well as to support crystallization efforts. To provide a comparison to SR-I and Htrl interaction, identification and cloning of related sensory receptors and transducers in the cell will be pursued. Photoconversion of SR-I to its signaling state is accompanied by proton transfer reactions initiated in its photoactive center. In the absence of Htrl, SR-I photoreactions result in light-driven electrogenic proton ejection from the cell. This proton pumping is suppressed by interaction with Htrl which blocks proton release. These results suggest that light- induced proton transfer to Htrl or to a site on the receptor coupled to Htrl is an important step for signal transduction. This hypothesis will be tested by site-specific mutagenesis of protonatable residues in SR-I likely to participate in proton transfers within the molecule and possibly to Htrl. Mutants will be characterized for phototaxis signaling in vivo. The 7-transmembrane helix motif is characteristic of a large family of membrane receptor proteins which sense light, hormones neurotransmitters, and chemotaxis stimuli in humans and analogous photo-and chemo-stimuli in microorganisms. A fundamental question in 7-helix sensors is how the activation of the receptor by photon absorption or ligand binding is communicated to its transducer. Because Htrl modulates SR-I photoreactions we have assays available for the interaction based on kinetic flash spectroscopy. This and the availability of genetic methods provides an exceptional opportunity to understand the chemistry of signal relay from a 7-helix receptor to its transducer. Principles elucidated are likely to be relevant to visual pigments and other 7-helix receptors. Additionally, the SR-I transducer is eubacterial in origin and this novel receptor/transducer combination may reveal a new mechanism of signal transduction.

感觉性视紫红质I(SR-I)是古细菌中的趋光性受体 盐生盐杆菌。受体蛋白在结构上类似于 视觉色素，由单一多肽组成，可折叠成7- 跨膜的阿尔法螺旋段形成内部口袋，其中 生色团视网膜是被束缚的。第二个完整的膜蛋白， Htrl，与真细菌趋化性序列同源 换能器，对于SR-I信令是必不可少的，而且有引人注目的 分子中存在SR-I和Htrl的间接证据 很复杂。我们的目标是了解SR-L/Htrl偶联的机制 在趋光性信号传递过程中。实验的设计是为了分析 天然SR-I和Htrl的物理联系和结构特征 用分光光度和蛋白质定量测定膜的SR-I Htrl化学计量学.探测跨膜拓扑学的硫基工程 这两种蛋白质及其寡聚体在黑暗中和之后的状态 光活化、缺失分析和定位随机诱变 结合趋光性缺失突变体和基因间的选择 抑制子来定义蛋白质的相互作用表面。知识 所获得的将应用于体外实验，包括相互作用 Htrl和与纯化的SR-I的活性片段。高产率提纯 正在为这些实验开发方法，以及支持 结晶的努力。与SR-I和Htrl进行比较 相关感觉神经受体的相互作用、鉴定和克隆 牢房中的换能器将被追查。 SR-I到其信号态的光转换伴随着质子 转移反应在其光活性中心开始。在没有的情况下 Htrl，SR-I光反应产生光驱电生质子 从细胞中排出。这种质子泵浦受到相互作用的抑制 Htrl可以阻止质子释放。这些结果表明，光- 诱导质子转移到Htrl或到受体上偶联的位置 Htrl是信号转导的重要步骤。这一假说将 通过SR-I中可质子化残基的定点突变进行检测 可能参与分子内的质子转移，并可能 敬Htrl。突变体的特征是体内的趋光性信号。 7-跨膜螺旋基序是一个大家族的特征 感受光线的膜受体蛋白，激素神经递质， 以及人类的趋化刺激和类似的光和化学刺激 微生物。七螺旋传感器的一个基本问题是 通过光子吸收或配体结合激活受体是 传送到它的传感器。因为Htrl调节SR-I光反应 我们有基于动态闪光的相互作用的分析方法 光谱学。这一点和遗传方法的可用性提供了一种 了解信号继电器化学的绝佳机会 一个7-螺旋的受体连接到它的转导器。阐明的原则可能会 与视觉色素和其他7-螺旋受体相关。另外， SR-I换能器起源于优生菌，而这一新颖的 受体/转导结合可能揭示一种新的信号机制 转导。