NMR STUDIES OF LIPID EFFECTS ON BACTERIORHODOPSIN

脂质对细菌视紫红质影响的核磁共振研究

• 批准号: 3295008
• 负责人: BARBARA A LEWIS
• 金额: $ 9.61万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295008
• 关键词: Halobacteriaceae; X ray crystallography; acidity /alkalinity; bacteriorhodopsins; circular dichroism; flash photolysis; infrared radiation; ions; lipid structure; membrane lipids; membrane proteins; membrane reconstitution /synthesis; nuclear magnetic resonance spectroscopy; protein structure

Many membrane proteins show altered activity when their lipid environment is modified, and such changes may well be important in the regulation of membrane protein function in vivo. The major focus of the proposed research will be to determine the effects of specific aspects of the lipid environment, particularly bilayer thickness, on both the structure and the function of bacteriorhodopsin (BR), an integral membrane protein which functions as a light-activated proton pump. In addition, lipid effects on the structure of smaller fragments of BR will be investigated. By examining the effects of lipid bilayer thickness, pH, and other ionic conditions on the structure of these proteins and peptides, the relative roles of hydrophobic and electrostatic forces in determining their structures will be elucidated. In addition, structural studies on the lipids themselves under the same conditions will permit the observation of both sides of the protein-lipid interaction simultaneously. Finally, by correlating the effects of environmental perturbations on the BR structure with the effects on its function, a better understanding of the fundamental mechanism of proton pumping will be gained. The experimental approaches will involve mainly nuclear magnetic resonance (NMR) and optical methods. The structural studies of BR and its fragments will use a novel application of solid state NMR methods, which has recently been developed and tested on BR, to obtain information on the orientation of labeled groups in the protein as it executes rotational diffusion in fluid membrane environments. Both 13C and 2H solid-state NMR studies will be carried out on the protein, and 2H NMR studies will also be performed on the lipids. Infared (IR) and circular dichroism (CD) spectroscopic studies will complement the NMR structural data. The functional studies will involve measurement of proton pumping activity and optical measurements of both steady-state and kinetic spectral properties of BR; the latter will be carried out in collaboration with the University of Virginia, using their flash photolysis apparatus. Finally, x-ray scattering will be used to measure the bilayer thickness for lipids not previously studied.

许多膜蛋白在其脂质环境时显示出改变的活性 修改了，这种变化在调节中很重要 体内膜蛋白功能。 提议的主要重点 研究将是确定脂质特定方面的影响 环境，尤其是双层厚度，结构和 细菌淡淡蛋白质（BR）的功能，一种整体膜蛋白，该蛋白质的功能 充当光激活的质子泵。 另外，脂质对 将研究较小的BR碎片的结构。 经过 检查脂质双层厚度，pH和其他离子的影响 这些蛋白质和肽结构的条件，相对 疏水性和静电力在确定它们的作用 结构将被阐明。 此外，关于 在相同条件下脂质本身将允许观察 蛋白质脂质相互作用的两侧同时。 最后，by 将环境扰动对BR结构的影响相关联 对其功能的影响，对基本的更好理解 将获得质子泵送的机理。 实验方法将主要涉及核磁共振 （NMR）和光学方法。 BR及其碎片的结构研究 将使用固态NMR方法的新颖应用，该方法最近 已在BR上开发和测试，以获取有关方向的信息 在蛋白质中执行旋转扩散时的标记组 流体膜环境。 13C和2H固态NMR研究都将 在蛋白质上进行，并且还将在2H NMR研究上进行 脂质。 炎症（IR）和圆形二科主义（CD）光谱研究 将补充NMR结构数据。 功能研究将 涉及质子抽水活性的测量和光学测量 Br的稳态和动力学光谱特性;后者将是 与弗吉尼亚大学合作进行的 闪光光解仪。 最后，X射线散射将用于 测量以前未研究的脂质的双层厚度。