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.

所提出的研究的目的是通过2H-NMR研究 细菌视紫红质的发色团的结构， 这个光驱动的质子泵。质子转移反应的速率为 关键取决于质子供体的距离和键角， 受体。质子化的希夫碱是这一过程中的质子源。 获得的泵和结构信息将是必不可少的， 开发其机制的详细模型。 每一个的角度 连接发色团的五个甲基与 多烯链和β-紫罗兰酮环将相对于 膜正常。这些角度将以高精度从 定向单轴样品~2H-NMR谱的四极分裂 相应选择性再生的紫色膜 氘视网膜实验将在黑暗中进行 态以及关键的光循环中间体M、N和O，其中 所述发色团为13-顺式构型或具有去质子化Schiff 基地因此，角度的变化将提供关于以下的结构信息： 在这些功能上， 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中间体是质子转移的关键中间体 将特别注意其作为一个新的国际组织的潜在作用。 音准开关 补充自旋-晶格弛豫实验 将执行，以了解各部分的流动性， 不同中间体中的发色团。 此信息将 对理解其他医学上重要的离子移位 膜蛋白，对于它的结构知之甚少。 此外，细菌视紫红质是重要的细菌视紫红质家族的原型。 具有七个跨膜α-螺旋的受体蛋白，包括 视色素视紫红质。 视紫红质的激活涉及结构 其M-II中间体的变化，该中间体起源于发色团， 传播到细胞质环。对类比的研究 bR的M中间体中的步骤预计将有助于 对视紫红质在视觉信号中激活机制的认识 转导