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，在天然的双层环境中表征两种固有的膜蛋白的结构和动力学：KCSA，变铅青链霉菌的典型K+通道，以及来自大肠杆菌的ATP合成酶的c亚基。固态核磁共振将提供原子级的结构和动力学细节，而不需要晶体或单分散溶液。KCSA是与医学相关的哺乳动物K+通道的同源模型，也是阐明高效和选择性离子传递以及通道门控原理的最佳系统。X射线结晶学在通道闭合状态下的结构工作是膜蛋白结构生物学最好的成就之一，但由于截断的蛋白质是在非功能条件下研究的，提供的动态柔性信息很少或根本没有提供信息，因此研究工作是有限的。双层环境和脂类的组成对内源性膜蛋白的结构、功能和动力学是至关重要的，包括KCSA的功能和折叠。我们建议研究双层环境中蛋白质的全长、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pH态和低pH态、开放态和闭合态、高K+态和低K+态之间的结构差异，以及这些态在双层中的动态相互转化，以及它们与脂质的相互作用。在ATP合成酶中，c亚基在跨双分子层的质子转移中起核心作用，人们认为该亚基的构象变化驱动了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低聚物的整体旋转，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。溶液核磁共振研究表明，有机溶剂中的c亚基单体是螺旋状发夹，其螺旋间环结构是pH的函数。到目前为止，还没有关于c亚基在双层和FO中组装的高分辨率研究。我们将分配这个低聚组件(C10和FO)的光谱，在关键的泵残基D61的pKA上方和下方。我们将研究亚基c和相邻亚基之间的四元联系。对于这两个系统，我们将应用最近开发的核磁共振方法来确定结构，包括用于确定距离的选择性重新耦合技术，用于约束扭角的基于偶极张量的矢量角相关方法，以及化学位移分析。初步数据包括两个体系在双层中的部分序列特异性指定，以及pH相关构象的核磁共振证据。与公共卫生相关：膜蛋白是至关重要的医学靶点中最重要的，然而大多数的结构和机制仍然不能用传统的方法来描述。我们计划应用固体核磁共振来阐明两个重要的案例：(1)KCSA，一个典型的K+通道，也是哺乳动物医学上相关K+通道的一个重要同源模型，以及(2)ATP合成酶亚单位c，一个质子泵，驱动合成ATP的旋转机制，一直被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.