STRUCTURE/FUNCTION OF ARCHAEAL SENSORY RHODOPSINS

古细菌感觉视紫红质的结构/功能

• 批准号: 6385382
• 负责人: JOHN LEE SPUDICH
• 金额: $ 48.94万
• 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/6385382
• 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.

感觉视紫红质I（SR-I）是古细菌的一种趋光性受体 盐生盐杆菌 受体蛋白质在结构上类似于 视色素，由一种多肽组成，折叠成7- 形成内袋的跨膜α-螺旋片段，其中 发色团视黄醛被结合。第二种膜蛋白， 与真细菌趋化性具有序列同源性的HSP 70 传感器，是必不可少的SR-I信号，有引人注目的，但 间接证据表明，SR-I和HSP 70存在于一个分子中， 复杂.我们的目标是了解SR-1/HSP 70偶联的机制 在趋光性信号传导过程中。设计实验来分析 SR-I和HSP 70在天然植物中的物理结合和结构特征 膜通过分光光度计和蛋白质定量来评估SR-I， 受阻化学计量;巯基工程化以探测跨膜拓扑结构 这两种蛋白质和它们的寡聚状态在黑暗中和之后， 光活化、缺失分析和局部随机诱变 结合选择趋光性缺陷突变体和基因间 抑制子来定义蛋白质的相互作用表面。知识 将获得的应用于体外实验，包括相互作用 用纯化的SR-I抑制和活性片段。 高产纯化 正在为这些实验开发方法， 结晶的努力。 提供与SR-I和阻滞剂的比较 相关感觉受体的相互作用、鉴定和克隆， 将追踪单元中的换能器。 SR-I到其信号状态的光转换伴随着质子的释放。 转移反应在其光活性中心开始。在没有 受阻，SR-I光反应导致光驱动生电质子 从细胞中排出。 这种质子泵被相互作用抑制 与阻止质子释放的Hexylamine一起使用。这些结果表明，光- 诱导质子转移到受体或受体上与受体偶联的位点， 阻断是信号转导的重要步骤。 这一假设将 通过SR-I中可质子化残基的位点特异性诱变进行检测 可能参与分子内的质子转移， 到哈登。将在体内表征突变体的趋光性信号传导。 7-跨膜螺旋基序是一个大家族的特征， 感受光的膜受体蛋白，激素神经递质， 和趋化性刺激，以及类似的光和化学刺激， 微生物的 7-螺旋传感器中的一个基本问题是 通过光子吸收或配体结合激活受体， 传送到它的传感器。因为Hisperin调节SR-I光反应， 我们有基于动力学闪光的相互作用的检测方法 谱这一点和遗传方法的可用性提供了一个 一个特殊的机会来了解信号中继的化学反应， 一个7螺旋受体连接到它的传感器上。所阐明的原则很可能 与视色素和其他7-螺旋受体有关。此外，本发明还 SR-I转换器起源于真细菌， 受体/传感器组合可能揭示一种新的信号机制 转导