LIGHT-DRIVEN ION TRANSPORT IN BACTERIAL RHODOPSINS

细菌视紫红质中的光驱动离子传输

• 批准号: 3277140
• 负责人: JANOS K LANYI
• 金额: $ 21.74万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277140
• 关键词: Halobacteriaceae; Raman spectrometry; X ray crystallography; bacteriorhodopsins; chloride channels; conformation; fluorescent dye /probe; gene expression; ion transport; laser spectrometry; lighting; membrane channels; membrane transport proteins; mutant; photobiology; photochemistry; protein sequence; protein structure function; protonation; retina; rhodopsin; site directed mutagenesis; spectrometry; visual photosensitivity

The fundamental unsolved questions of membrane ion pumps can be studied to greatest advantage with bacteriorhodopsin and halorhodopsin, the light-driven electrogenic ion pumps in, Halobacterium halobium, the best characterized active transport proteins. Using the models we developed in the last few years for the photoreaction sequences and the energetics of ion transport in these proteins, we will describe the mechanism of specific steps of the transport cycles and the coupling between the chromophore reactions and the ion gradients created. By comparing two closely similar transport systems which transport different ions we expect to gain mechanistic and conceptual insights, and expect that these will be relevant to ionic pumps in general. A variety of approaches will be used to evaluate the role of specific amino acid residues in the structure and function of these small retinal proteins, most importantly by site-specific mutagenesis. Bacteriorhodopsins and balorhodopsins with single and multiple residue changes will be generated, using a recently developed shuttle vector and transformation system for Halobacterium halobium to introduce and express the cloned bop and hop genes. These homologously expressed proteins have different phenotypes from those expressed earlier in E. coli. The mutagenesis approach will be complemented by replacement of the retinal with retinal analogues with altered fit into the binding pocket. We will analyze the altered proteins with time-resolved optical multichannel and single wavelength spectroscopy, low temperature spectroscopy, and transport studies in cell envelope vesicles. Through collaborations FTIR and resonance Raman spectroscopy, photoelectric and electro-optical measurements, retinal analogues, and x-ray diffraction will be available to us when needed.

膜离子泵的基本未解决的问题可以研究， 细菌视紫红质和盐视紫红质的最大优点是， 光驱动的产电离子泵，盐生盐杆菌，最好的 活性转运蛋白。 使用我们在1999年开发的模型 过去几年的光反应序列和能量学 离子转运在这些蛋白质，我们将描述的机制，具体 传递循环的步骤和发色团之间的耦合 反应和产生的离子梯度。 通过比较两个非常相似的 我们期望获得的不同离子的传输系统 机械和概念的见解，并期望这些将是相关的 离子泵。 将采用各种方法， 评估特定氨基酸残基在结构中的作用， 这些小的视网膜蛋白的功能，最重要的是通过位点特异性 诱变 细菌视紫红质和balorhodopsins与单一和 将使用最近开发的 用于盐生盐杆菌穿梭载体和转化系统， 导入并表达克隆的bop和hop基因。 这些同源 表达的蛋白质具有与先前表达的不同的表型 在大肠杆菌 诱变方法将通过替换来补充 视网膜类似物与视网膜的结合， 口袋 我们将用时间分辨光学技术分析改变的蛋白质， 多通道和单波长光谱，低温 光谱学和细胞包膜囊泡中的运输研究。 通过 合作FTIR和共振拉曼光谱，光电和 电光测量、视网膜类似物和X射线衍射将 在我们需要的时候提供。