NMR Studies of Biological Membranes

生物膜的核磁共振研究

• 批准号: 6873928
• 负责人: JUDITH HERZFELD
• 金额: $ 32.07万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-30 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6873928
• 关键词: Halobacteriaceae; active sites; bacteriorhodopsins; bioimaging /biomedical imaging; cell membrane; chemical bond; chloride ion; chromophore; high energy particle; hydrogen ions; infrared spectrometry; interferometry; intermolecular interaction; ion transport; membrane activity; membrane structure; nuclear magnetic resonance spectroscopy; photochemistry; structural biology

DESCRIPTION (provided by applicant): Ion pumps, which maintain the internal milieu of cells and control the potentials of cell membranes, have demanding requirements with respect to the timing and vectoriality of their action. In the proposed work we will compare the manner in which these requirements are met by three different light-driven ion pumps, a green-driven proton/hydroxyl pump (wild type bacteriorhodopsin), a green-driven halide pump (acid purple bacteriorhodopsin), and a blue-driven proton/hydroxyl pump (D85N bacteriorhodopsin at high pH). Our hypotheses are that (1) chromophore distortion and relaxation are central to the action of bacteriorhodopsin and (2) there is no need for such complications in the other two pumps. The pump mechanisms are to be studied by SSNMR using in situ illumination to accumulate critical photocycle intermediates and low temperature to stabilize them during data acquisition. Samples will be uniformly and selectively labeled with 13C and 15N as needed. Dynamic nuclear polarization (DNP) will be used to enhance signals, thereby (1) greatly expediting measurements of internuclear distances in the active site and (2) extending the range of feasible experiments to include sensitive measurements of torsion angles along the chromophore. The evolution of these variables over the course of each photocycle will provide insight into the mechanism by which vectorial action is enforced in the system. In addition, REDOR and high-field MQMAS will be used to study the interactions of deuterons and chloride with elements lining the transport channels of the proton/hydroxyl and chloride pumps, respectively. The evolution of these interactions during the photocycle will provide a picture of substrate movement through the pump. Interpretations of chemical shifts in terms of local structure will be assisted by ab initio density functional calculations and NMR-derived distances and angles will be compared with crystallographic results. Taken together, the experiments will significantly enhance our understanding of ion pump function and dysfunction.

描述（由申请人提供）：维持细胞内部环境并控制细胞膜的电势的离子泵在其作用的时机和矢量性方面有苛刻的要求。在提议的工作中，我们将比较三种不同的光驱动离子泵，一种绿色驱动的质子/羟基泵（野生型细菌紫红质），一种绿色驱动的卤化物泵（酸性紫色细菌蛋白）和蓝色驱动的Proton/hydroxyl phicoptin（d855n phateropt）。我们的假设是（1）发色团扭曲和放松是细菌紫红素的作用至关重要的，（2）其他两个泵中不需要这种并发症。 SSNMR应使用原位照明来研究泵的机理，以积累关键的光循环中间体和低温，以在数据采集期间稳定它们。样品将根据需要均匀，有选择地用13C和15N标记。动态核极化（DNP）将用于增强信号，因此（1）大大加快了活性位点中核切间距离的测量，以及（2）扩展可行的实验范围，以包括沿着成创伪像的扭转角度的敏感测量。这些变量在每个光循环过程中的演变将提供有关系统在系统中实施矢量作用的机制的洞察力。此外，还将使用重复和高场MQMA来研究迪特龙和氯化物与质子/羟基和氯化物泵的传输通道的相互作用。这些相互作用在光循环过程中的演变将提供通过泵的底物运动的图片。从头算密度的功能计算，将在局部结构方面对化学移位进行解释，而NMR衍生的距离和角度将与晶体学结果进行比较。综上所述，实验将显着增强我们对离子泵功能和功能障碍的理解。