NMR Studies of Biological Membranes

描述（由申请人提供）：与可溶性蛋白质一样，膜蛋白具有催化、结构和信号传导功能。然而，它们还具有其在边界位置所特有的其他重要功能，这些功能通常被证明难以通过常规生物物理方法进行探测。在这里，我们建议解决一个长期存在的和令人信服的问题，膜生物物理学使用强大的国家的最先进的固态NMR实验能够探测化学和空间变量具有高灵敏度。特别是，我们试图了解视网膜为基础的膜蛋白创建跨细胞膜的离子梯度的手段。虽然微生物的这种广泛的光捕获过程具有生态重要性，但它也更普遍地重要，因为光激发的发色团可以被认为是高能代谢物的有用类似物，当光不可用时，高能代谢物为其他膜蛋白的离子转运提供动力。然而，即使在细菌视紫红质中，最容易获得和最彻底研究的光驱动泵，迄今为止积累的大量信息还没有加起来成为矢量作用的机制，因为大量的能量存储在非常小但显著的结构变化中。最先进的固态NMR具有很大的优势，能够清楚地区分在光循环期间发生的混合物中的细微不同的中间体，同时还为每个中间体提供原子水平的细节，直到临界质子。在拟议的实验中，我们将遵循的路径中的能量耗散的活性位点在关键步骤，直接离子运动。来自内部离子和水的信号，以及来自发色团和周围蛋白质残基的信号，将为我们提供一个全面的化学和空间变化的图像，这些变化有助于每个早期光循环中间体中的泵机制。这些变化的解释将通过与细菌视紫红质泵的变体进行比较来了解。具体来说，我们将进行平行的研究的阴离子泵的盐视紫红质，蓝光pdriven泵的D85 N突变体的细菌视紫红质，和运输无能的13-顺式，15-顺式异构体的细菌视紫红质的光循环。 公共卫生相关性：除了更一般的蛋白质功能，膜蛋白进行重要的功能，是特定于它们的位置在边界上，往往抵制传统的生物物理方法的调查。在这里，我们建议使用强大的固态核磁共振实验来了解微妙的能量交易，影响强大的主动运输的离子穿过细胞膜。这种转运直接负责神经和肌肉细胞的电兴奋性，并间接负责所有细胞中的营养摄取和渗透平衡。

DESCRIPTION (provided by applicant): Like soluble proteins, membrane proteins have catalytic, structural and signaling functions. However, they also have other vital functions peculiar to their location at boundaries, functions that have often proven difficult to probe by conventional biophysical methods. Here we propose to address a long-standing and compelling problem of membrane biophysics by using powerful state-of-the-art solid state NMR experiments capable of probing both chemical and spatial variables with high sensitivity. In particular, we seek to understand the means by which retinal-based membrane proteins create ion gradients across cell membranes. While this widespread light harvesting process of micro-organisms is of ecological importance, it is also more generally significant because the photo-excited chromophore may be considered a useful analog of the high energy metabolites that power ion transport by other membrane proteins when light is not available. However, even in bacteriorhodopsin, the most accessible and thoroughly studied light- driven pump, the considerable information that has accumulated so far has not added up to a mechanism for vectorial action because large amounts of energy are stored in very small, but significant, structural changes. State-of-the-art solid state NMR has the great advantage of being able to clearly distinguish between the subtly different intermediates in the mixtures that occur during the photocycle, while also providing atomic level detail, down to the critical protons, for each intermediate. In the proposed experiments, we will follow the path of energy dissipation in the active site during the critical steps that direct ion movement. Signals from internal ions and water, as well as from the chromophore and surrounding protein residues, will give us a thorough picture of chemical and spatial changes that contribute to the pump mechanism in each of the early photocycle intermediates. Interpretation of these changes will be informed by comparison with variants of the bacteriorhodopsin pump. Specifically we will carry out parallel studies of the photocycles of the anion pump of halorhodopsin, the blue light pdriven pump of the D85N mutant of bacteriorhodopsin, and the transport incompetent 13-cis,15-syn isomer of bacteriorhodopsin. PUBLIC HEALTH RELEVANCE: In addition to more general protein functions, membrane proteins carry out vital functions that are specific to their locations at boundaries and often resist investigation by conventional biophysical approaches. Here we propose to use powerful solid state NMR experiments to understand the subtle energy transactions that effect robust active transport of ions across cell membranes. Such transport is directly responsible for the electrically excitability of nerve and muscle cells, and indirectly responsible for nutrient uptake and osmotic balance in all cells.