IONIC INTERACTIONS OF BACTERIORHODOPSIN'S ACTIVE SITE DURING THE PHOTOCYCLE

光循环期间细菌视紫红质活性位点的离子相互作用

• 批准号: 6240018
• 负责人: LAURA L SWEETMAN
• 金额: $ 13.94万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240018
• 关键词: Raman spectrometry; active sites; atomic absorption spectrometry; bacteriorhodopsins; calcium flux; cations; cell membrane; chemical binding; hydrogen channel; membrane proteins; photobiology; protein structure; ultraviolet spectrometry

The long term goal of this project is to understand how ions affect the molecular structure of bacteriorhodopsin (bR). This is a membrane protein found in the halophilic Halobacterium halobium, contains the chromophore retinal which undergoes a photocycle of reversible structural and environmental changes upon absorption of light. The protein's similarity to visual rhodopsin which also contains a retinal chromophore makes an excellent model for the visual process. Thus, a better understanding of the molecular processes of cation association with bR will provide an important step in determining if a similar mechanism applies to the visual rhodopsin cycle. Similarly, bR can also be used as a model for Gi regulatory protein involved in signal transduction pathways affecting intracellular calcium levels. The bR system to be used in these experiments is a straightforward and inexpensive model that can be used to gain a fundamental understanding of the molecular mechanisms involved in mammalian systems. The immediate focus of this proposal is to better understand the function of cations in the proton pump mechanism during the photocycle and specifically, to test the hypothesis that cations are displaced from the active site during this mechanism. The three specific aims designed to test this hypothesis are: (1) To determine the steady state intracellular and surface concentration of cations. Initial work will focus on measuring steady state cytosolic calcium ion by using a calcium ion sensitive probe which is loaded into the cells as a methyl ester. Once inside of the cells it is de-esterified by esterases near the cell surface and is able to bind cytosolic calcium. This binding cause the dye to fluoresce, and the fluorescence can be used to measure the concentration of calcium in the cytosol. (2) To determine the changes in intracellular concentration of cations and correlate these changes to the formation of photocycle intermediates. This will allow us to test the present theories regarding the environment at the active site during the photocycle. (3) To determine the changes in the surface concentration of cations and correlate these changes to the formation of photocycle intermediates. By using new lipophilic fluorescent probes, which measure surface cation concentrations, we expect to determine whether cation displacement at the active site results in a movement of the cation to the surface of the cell membrane where it can be taken up again by the protein at a later stage in the photocycle. An integral aim of this research is the training of minority students in biomedically related scientific techniques. This project is particularly suitable for this type of training because it involves an integrated multi-disciplinary approach, and the research can be divided into specific projects which would provide information that is not already known about this bacteria's photocycle and proton pump mechanism. This is an area of bR research which is not actively being studied; nevertheless, it would provide very important information relevant to various biological fields. Each project is focused, result oriented, and can be divided into individual, small or large, subprojects based on each student's background and available time commitments (e.g. undergraduate vs. graduate students.

该项目的长期目标是了解离子如何影响 细菌紫红质（BR）的分子结构。这是一种膜蛋白 在卤素盐杆菌中发现，含有发色团 视网膜经历可逆结构和 吸收光后环境变化。蛋白质的相似性 对于视觉视紫红质，还包含视网膜发色团使 视觉过程的出色模型。因此，更好地理解 阳离子关联与BR的分子过程将提供 确定是否适用于视觉的重要步骤 视紫红质周期。同样，BR也可以用作GI的模型 参与信号转导途径的调节蛋白影响 细胞内钙水平。这些在这些中使用的BR系统 实验是一种直接且廉价的模型，可用于 对涉及的分子机制获得基本理解 哺乳动物系统。 该提案的直接重点是更好地了解功能 质子泵机构中阳离子的阳离子和 具体而言，要检验阳离子从 在此机制中活跃部位。旨在的三个特定目标 检验此假设是： （1）确定稳态细胞内和表面浓度 阳离子。最初的工作将集中于测量稳态胞质 钙离子通过使用钙离子敏感探针加载到 细胞作为甲酯。一旦在细胞内部，它就会被拆除 通过细胞表面附近的酯酶，能够结合胞质钙。 这种结合导致染料荧光，并且可以使用荧光 测量细胞质中钙的浓度。 （2）确定阳离子内浓度的变化和 将这些变化与光循环中间体的形成相关。这 将使我们能够测试有关环境的当前理论 光圈期间的活性位点。 （3）确定阳离子表面浓度的变化和 将这些变化与光循环中间体的形成相关。经过 使用新的亲脂荧光探针，该探针测量表面阳离子 浓度，我们希望确定阳离子位移是否在 主动部位导致阳离子向细胞表面移动 膜可以在以后的蛋白质中再次吸收蛋白 光圈。 这项研究不可或缺的目的是培训少数族裔学生 与生物医学相关的科学技术。这个项目特别是 适合这种类型的培训，因为它涉及集成的 多学科方法，研究可以分为特定 将提供尚未知道的信息的项目 该细菌的光循环和质子泵机制。这是一个领域 不积极研究的BR研究；然而，会 提供与各种生物学领域相关的非常重要的信息。 每个项目都集中于此，以结果为导向，可以分为 基于每个学生的背景 和可用的时间承诺（例如，本科生与研究生。