BACTERIORHODOPSIN CHROMOPHORE STRUCTURE BY DEUTERIUM NMR

通过氘核磁共振测定细菌视紫红质发色团结构

• 批准号: 2655000
• 负责人: MAARTEN P HEYN
• 金额: $ 13.7万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2655000
• 关键词: Halobacteriaceae; Schiff bases; acidity /alkalinity; bacteriorhodopsins; chemical structure function; chromophore; deuterium; hydrogen transport; ionic bond; ionic strengths; isomer; methyl group; molecular dynamics; mutant; nuclear magnetic resonance spectroscopy; photochemistry; polyenes; protonation; retinaldehyde; stereochemistry; structural biology; temperature

The objective of the proposed research is to investigate by 2H-NMR the structure of the chromophore of bacteriorhodopsin, the key component of this light-driven proton pump. The rate of proton transfer reactions is critically dependent on the distances and bond angles of proton donors and acceptors. The protonated Schiff base is the source of protons in this pump and the structural information obtained will be essential in developing detailed models of its mechanism. The angles of each of the bonds connecting the five methyl groups of the chromophore with the polyene chain and beta-ionone ring will be determined with respect to the membrane normal. These angles will be obtained with high accuracy from the quadrupole splittings of the 2H-NMR spectra of oriented uniaxial samples of purple membranes regenerated with the corresponding selectively deuterated retinals. Experiments will be carried out in the dark initial state as well as in the key photocycle intermediates M, N and O, in which the chromophore is in a 13-cis configuration or has a deprotonated Schiff base. The changes in angle will thus provide structural information about curvature, twists and tilt of the chromophore in these functionally important intermediates. External parameters (pH, ionic strength and temperature) and mutants will allow trapping of the various intermediates. The M intermediate is the key intermediate in the proton translocation cycle, and particular attention will be paid to its potential role as a reprotonation switch. Complementary spin-lattice relaxation experiments will be performed to learn about the mobility of various parts of the chromophore in the different intermediates. This information will be valuable in understanding other medically important ion translocating membrane proteins, for which much less is known about the structure. Bacteriorhodopsin is moreover the prototype of the important family of receptor proteins with seven transmembrane alpha-helices, which includes the visual pigment rhodopsin. Activation of rhodopsin involves structural changes in its M-II intermediate which originates in the chromophore and propagate to the cytoplasmic loops. An investigation of the analogous steps in the M intermediate of bR is expected to contribute to an understanding of the mechanism of activation of rhodopsin in visual signal transduction.

拟议研究的目的是通过2H-NMR进行研究 细菌紫红素发色团的结构，这是关键成分 这个轻驱动的质子泵。质子转移反应的速率为 严格依赖质子供体的距离和键角和 受体。质子化的Schiff基础是质子的来源 泵和获得的结构信息至关重要 开发其机制的详细模型。 每个角度的角度 连接发色团五个甲基的键与 将根据 膜正常。这些角度将以高精度从 定向单轴样品的2H-NMR光谱的四极分裂 与相应的选择性再生的紫色膜 氘化的视网膜。实验将在黑暗的初始 状态以及关键光循环中间体m，n和o，其中 发色团以13盘配置或具有去质子化的Schiff 根据。因此，角度的变化将提供有关的结构信息 曲率，发色团的曲折和倾斜 重要的中间体。 外部参数（pH，离子强度和 温度）和突变体将允许捕获各种中间体。 M中间体是质子易位的关键中间体 周期，并特别关注其作为一个潜在作用 反应开关。 互补的自旋放松实验 将执行以了解该地区各个部分的移动性 不同中间体的发色团。 这些信息将是 在理解其他重要的离子易位的情况下有价值 膜蛋白，对结构知之甚少。 细菌视紫红质是重要家族的原型 具有七个跨膜α-螺旋的受体蛋白，其中包括 视觉色素视紫红质。 视紫红质的激活涉及结构 其M-II中间体的变化起源于发色团和 传播到细胞质环。对类似的调查 预计BR中级的步骤有助于 了解视觉信号中视紫红质激活的机理 转导。