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

通过固态核磁共振研究通道和泵的结构和功能

• 批准号: 8325732
• 负责人: ANN E MCDERMOTT
• 金额: $ 27.49万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8325732
• 关键词: ATP Synthesis Pathway; ATP phosphohydrolase; Address; Binding; Chemicals; Comparative Study; Crystallography; Data; Dependence; Detergents; Disease; Energy Supply; Environment; Escherichia coli; Event; Exhibits; Four-dimensional; Goals; Health; Helix-Turn-Helix Motifs; Homology Modeling; Integral Membrane Protein; Ions; Label; Lead; Learning; Length; Lipid Bilayers; Lipids; Mammals; Measurement; Measures; Medical; Membrane; Membrane Proteins; Methods; Molecular Conformation; Mono-S; Organic solvent product; Organism; Phospholipids; Play; Positioning Attribute; Potassium Channel; Proteins; Proton Pump; Protons; Pump; Resolution; Roentgen Rays; Role; Rotation; Sampling; Scheme; Site; Solutions; Structure; System; Systems Analysis; Techniques; Torsion; Tuberculosis; Validation; Work; X-Ray Crystallography; base; flexibility; in vivo; interest; ionization; magnetic field; monomer; protein structure; protonation; solid state nuclear magnetic resonance; structural biology; transmission process; vector

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.

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，变青链球菌的典型 K+ 通道，以及来自大肠杆菌的 ATP 合酶的 c 亚基。固态核磁共振将提供有关结构和动力学的原子级详细信息，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，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，但其局限性是因为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关于动态灵活性的信息。众所周知，双层环境和脂质组成对于内膜蛋白的结构、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，使用许多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状态和低pH状态、开放状态和封闭状态、高K+状态和低K+状态之间的结构差异，以及双层中这些状态之间的动态相互转换，以及它们与脂质的相互作用。在 ATP 合酶中，c 亚基在质子跨双层转移中起着核心作用，人们认为该亚基的构象变化驱动 F1 的构象变化，从而实现 ATP 合成。残基 D61 的质子化被认为驱动寡聚物的整体旋转，以及 c 亚基的构象变化，涉及与 F1 直接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亚基单体是螺旋发夹，其螺旋间环结构是pH的函数。迄今为止，还没有对双层或 FO 中的 c 亚基组装进行高分辨率研究。我们将分配该寡聚组装体（c10 和 FO）的光谱高于和低于关键泵残基 D61 的 pKa。我们将研究亚基 c 和邻近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核磁共振方法来确定结构，包括用于确定距离的选择性重耦合技术、用于约束扭转角的基于偶极张量的矢量角相关方法以及化学位移分析。初步数据包括双层中两个系统的部分序列特异性分配，以及 pH 依赖性构象的 NMR 证据。公共健康相关性：膜蛋白是极其重要的医学靶点中最重要的，但大多数膜蛋白的结构和机制仍然很难通过传统方法来表征。我们计划应用固态核磁共振来阐明两个重要案例：(1) KcsA，一种典型的 K+ 通道，也是哺乳动物医学相关 K+ 通道的重要同源模型；(2) ATP 合酶亚基 c，一种质子泵，驱动 ATP 合成的旋转机制，并一直被视为与结核病相关的生物体特异性抑制靶点。