NMR Studies of Biological Membranes

描述（由申请人提供）：离子泵维持细胞的内部环境并控制细胞膜的电位，其对动作的时间和矢量性有严格的要求。在拟议的工作中，我们将比较的方式，其中这些要求是由三种不同的光驱动的离子泵，绿色驱动的质子/羟基泵（野生型细菌视紫红质），绿色驱动的卤化物泵（酸性紫色细菌视紫红质），和蓝色驱动的质子/羟基泵（D85 N细菌视紫红质在高pH值）满足。我们的假设是：（1）发色团的扭曲和松弛是细菌视紫红质作用的核心，（2）在其他两个泵中不需要这样的复杂性。泵浦机制进行了研究，SSNMR使用原位照明积累关键的光循环中间体和低温稳定它们在数据采集过程中。根据需要，使用13 C和15 N对样本进行均匀和选择性标记。动态核极化（DNP）将被用来增强信号，从而（1）大大加快了活性部位核间距的测量，（2）扩大了可行实验的范围，包括沿发色团沿着扭转角的灵敏测量。这些变量在每个光循环过程中的演变将提供深入了解的机制，其中矢量行动是强制执行的系统。此外，REDOR和高场MQMAS将分别用于研究氘核和氯离子与质子/羟基和氯离子泵的传输通道内衬元素的相互作用。这些相互作用在光循环过程中的演变将提供通过泵的基板运动的图片。解释化学位移的局部结构将有助于从头计算密度泛函计算和NMR推导的距离和角度将与晶体学结果进行比较。总之，这些实验将显著增强我们对离子泵功能和功能障碍的理解。

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.