Structural and Functional Studies of Channels and Pumps by Solid State NMR

描述（由申请人提供）：我们将在与其功能一致的条件下表征两种内在膜蛋白在其天然双层环境中的结构和动力学：KcsA，S的原型K+通道。和E.杆菌固态NMR将提供结构和动力学的原子级细节，而不需要晶体或单分散溶液。KcsA是哺乳动物医学相关K+通道的同源模型，并且是阐明高效和选择性离子传输以及通道门控原理的最佳表征系统。通过X射线晶体学对通道的闭合状态进行的结构研究是膜蛋白结构生物学的最佳成就之一，但由于在非功能条件下研究了截短的蛋白质，提供了很少或根本没有动态灵活性的信息，因此受到限制。已知双层环境和脂质的组成对内在膜蛋白的结构、功能和动力学（包括KcsA的功能和折叠）至关重要。我们建议在双层环境中研究蛋白质的全长，活性形式，将其与晶体中的蛋白质进行对比，使用一些最近开发的方法来稳定双层中的开放状态。我们将阐明高和低pH值状态，开放和封闭状态，高和低K+状态之间的结构差异，以及这些状态在双层之间的动态相互转换，以及它们与脂质的相互作用。在ATP合成酶中，c亚基在质子跨双分子层转移中起着核心作用，并且据信该亚基中的构象变化驱动F1的构象变化，从而使ATP合成成为可能。残基D61的质子化被认为驱动寡聚体的整体旋转，以及c亚基的构象变化，涉及直接与F1相互作用的螺旋间环。溶液NMR研究表明，在有机溶剂中的c亚基单体是一个螺旋发夹，其螺旋间的环结构是pH值的函数。迄今为止，没有高分辨率的研究的c亚基组装在双层，也没有FO。我们将分配光谱的低聚物组装（c10和FO）以上和以下的关键泵残基，D61的pKa。我们将研究亚基c和相邻亚基之间的四级接触。对于这两个系统，我们将应用最近开发的NMR方法来确定结构，包括选择性再耦合技术确定距离，偶极张量为基础的矢量角相关方法约束扭转角，和化学位移分析。初步数据包括部分序列特定的分配两个系统的双层，和NMR的pH依赖性构象的证据。公共卫生相关性：膜蛋白是至关重要的医学靶点之一，但传统方法对大多数膜蛋白的结构和机制仍知之甚少。我们计划应用固态NMR来阐明两个重要的情况：（1）KcsA，一个原型K+通道，以及哺乳动物医学相关K+通道的重要同源模型，和（2）ATP合酶亚基c，一个质子泵，驱动ATP合成的旋转机制，并已被追求作为与结核病相关的生物体特异性抑制靶点。

DESCRIPTION (provided by applicant): We will characterize the structure and dynamics of two intrinsic membrane proteins in their native bilayer environments, under conditions consistent with their functions: KcsA, the prototypical K+ channel of S. lividans, and the c subunit of ATP synthase from E. coli. Solid State NMR will provide atomic level details on structure and dynamics, without any requirement for crystals or mono-dispersed solutions. KcsA is a homology model for medically relevant K+ channels of mammals, and is the best characterized system for clarifying the highly efficient and selective ion transmission, and the principles underlying channel gating. Structural work by X-ray crystallography on the closed state of the channel stands among the best accomplishments of membrane protein structural biology, and yet is limited because a truncated protein was studied under nonfunctional conditions, providing little or no information on dynamical flexibility. The bilayer environment and the composition of lipids are known to be crucial for structure, function, and dynamics of intrinsic membrane proteins, including the function and folding of KcsA. We propose to study the full length, active form of the protein in a bilayer environment, contrasting it to the protein in the crystal, using a number of recently developed approaches to stabilize the open state in the bilayer. We will clarify structural differences between the high and low pH states, the open and closed states, and between the high and low K+ states, and the dynamic interconversion between these states in the bilayer, and their interactions with lipids. In ATP synthase, the c subunit plays the central role in proton transfer across the bilayer, and it is believed that conformation changes in this subunit drive the conformation changes of F1, enabling ATP synthesis. Protonation of residue D61 is believed to drive overall rotation of the oligomer, as well as a conformation change in the c subunit, involving an interhelix loop that interacts directly with F1. Solution NMR studies have shown that the c subunit monomer in organic solvents is a helical hairpin whose interhelical loop structure is a function of pH. To date, there is no high-resolution study of the c subunit assembly in the bilayer nor in FO. We will assign spectra of this oligomeric assembly (c10 and FO) above and below the pKa of the crucial pump residue, D61. We will study quaternary contacts between subunit c and neighboring subunits. For both systems, we will apply recently developed NMR methods for determining structure, including selective recoupling techniques for determining distances, dipolar tensor-based vector angle correlation methods for constraining torsion angles, and chemical shift analysis. Preliminary data include partial sequence-specific assignments for both systems in bilayers, and evidence for NMR for pH-dependent conformations. PUBLIC HEALTH RELEVANCE: Membrane proteins are foremost among crucially important medical targets, and yet the structures and mechanisms of most remain poorly characterized by traditional methods. We plan to apply a solid state NMR to elucidate two important cases: (1) KcsA, a prototypical K+ channel, and an important homology model for the medically relevant K+ channels of mammals, and (2) ATP synthase subunit c, a proton pump that drives a rotary mechanism for the synthesis of ATP and has been pursued as an organism specific target for inhibition in connection with tuberculosis.