Solid State NMR Studies of Membrane Proteins

膜蛋白的固态核磁共振研究

• 批准号: 7082782
• 负责人: ROBERT Guy GRIFFIN
• 金额: $ 50.1万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-05-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7082782
• 关键词: Solid; State; NMR; Studies; Membrane

DESCRIPTION (provided by applicant): We plan research projects in four areas. (1) We propose parallel studies of the photointermediates of bR -- bR568, L550, M412, and N520. (a) Structure of the retinal, the Lys216 sidechain, and the Schiff base 15N: We plan to determine the orientation and structure of the retinal and the Schiff base linkage. The Schiff base experiments will employ angular dependent 15N studies of partially oriented samples of bR to determine the orientation of the SB linkage in the bR568 L, M and N intermediates. The experiments will be performed at low temperatures where it is possible to trap these intermediates and to enhance the signal-to-noise. In other experiments, the structure of the retinal and the Lys sidechain in these intermediates will be determined with 1H13C13C1H and 1H13C15N1H MAS recoupling experiments designed to measure the torsion about C-C and C-N bonds. With 12-13C and e-15N-Lys labeled bR we plan to resolve whether the L state is 13-cis, 14-trans, or 13,14-dicis. (b) Structure of the Retinal Binding Pocket: The SB linkage and the retinal are surrounded by a number of amino residues -- D85, D212, R82, Y185, M118, W182, etc. -- whose positions in bRDA are known with varying precision from the diffraction models. We plan to study the changes in the position of these residues, and thus the structure of the retinal binding pocket, which occur in bR568 L, M and N intermediates with 2D MAS homo and heteronuclear recoupling techniques. (2) H+/OH- pumping pathway and H2O in bR: A third area of interest is the H+ pathway during the pumping cycle and the position and disposition of H2O associated with bR. These aspects of bR will be investigated with 17O NMR experiments designed to establish the proximity of H217O to amino acid sidechains and to resolve shifts of different H217O's with multiquantum MAS experiments. (3) 35Cl- NMR of Low pH bR: We plan to look for Cl- binding with MQMAS experiments. REDOR and REAPDOR will be used to measure distance to adjacent amino acid sidechains. (4) Structural Studies of Membrane Proteins: We propose to determine the structure of bR with multidimensional, MAS NMR experiments spectroscopy

描述（由申请人提供）：我们计划在四个领域进行研究项目。 (1) 我们建议对 bR 的光中间体——bR568、L550、M412 和 N520 进行并行研究。 (a) 视网膜、Lys216 侧链和希夫碱 15N 的结构：我们计划确定视网膜和希夫碱连接的方向和结构。 希夫碱实验将采用对 bR 部分定向样品的角度依赖性 15N 研究来确定 bR568 L、M 和 N 中间体中 SB 连接的方向。 实验将在低温下进行，这样可以捕获这些中间体并增强信噪比。 在其他实验中，这些中间体中的视黄醛和赖氨酸侧链的结构将通过1H13C13C1H和1H13C15N1H MAS重偶联实验来确定，该实验旨在测量C-C和C-N键的扭转。 利用标记为 bR 的 12-13C 和 e-15N-Lys，我们计划解析 L 态是 13-顺式、14-反式还是 13,14-二顺式。 (b) 视网膜结合袋的结构：SB 连接和视网膜被许多氨基残基包围 - D85、D212、R82、Y185、M118、W182 等 - 其在 bRDA 中的位置已知，且不同衍射模型的精度。 我们计划使用 2D MAS 同核和异核重偶联技术研究这些残基位置的变化，从而研究 bR568 L、M 和 N 中间体中发生的视网膜结合袋的结构。 (2) bR 中的 H+/OH- 泵送途径和 H2O：第三个感兴趣的领域是泵送循环期间的 H+ 途径以及与 bR 相关的 H2O 的位置​​和分布。 bR 的这些方面将通过 17O NMR 实验进行研究，这些实验旨在确定 H217O 与氨基酸侧链的接近程度，并通过多量子 MAS 实验解决不同 H217O 的位移。 (3) 低 pH bR 的 35Cl- NMR：我们计划通过 MQMAS 实验寻找 Cl- 结合。 REDOR 和 REAPDOR 将用于测量与相邻氨基酸侧链的距离。 (4) 膜蛋白的结构研究：我们建议通过多维、MAS NMR 实验光谱来确定 bR 的结构