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）：我们将表征其天然双层环境中两种固有膜蛋白的结构和动力学，在与其功能一致的条件下：KCSA，S。lividans的原型K+通道以及来自E. coli的ATP合酶的c subunit。固态NMR将提供有关结构和动力学的原子水平详细信息，而无需任何晶体或单分散溶液。 KCSA是哺乳动物医学相关的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基础通道门控的原理的最佳特征系统。 X射线晶体学在通道的封闭状态下进行的结构性工作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，因此受到限制，几乎没有或根本没有有关动力灵活性的信息。已知双层环境和脂质的组成对于内在膜蛋白的结构，功能和动力学至关重要，包括KCSA的功能和折叠。我们建议在双层环境中研究蛋白质的全长，活性形式，并使用许多最近开发的方法与晶体中的蛋白质进行对比，以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互连以及它们与脂质的相互作用。在ATP合酶中，C亚基在质子跨双层中的核心作用起着核心作用，据信，该亚基中的构象变化驱动F1的构象变化，从而实现了ATP合成。据信，残基D61的质子化可以驱动低聚物的总体旋转，以及C亚基的构象变化，涉及直接与F1相互作用的螺旋环路。溶液NMR研究表明，有机溶剂中的C亚基单体是一种螺旋发夹，其间旋转环结构是pH的函数。迄今为止，尚无对双层中的C亚基组装或FO中的高分辨率研究。我们将在关键的泵残基PKA上方和下方分配此寡聚组件（C10和FO）的光谱，D61。我们将研究亚基C和相邻亚基之间的第四纪接触。对于这两种系统，我们将应用最近开发的NMR方法来确定结构，包括用于确定距离的选择性重耦技术，基于偶极张量的矢量角相关方法来约束扭转角度以及化学移位分析。初步数据包括双层系统中两个系统的部分序列分配，以及pH依赖性构象的NMR证据。公共卫生相关性：膜蛋白在至关重要的医学靶标之间是最重要的，但是大多数人的结构和机制仍然以传统方法为特征。我们计划应用固态NMR来阐明两种重要情况：（1）KCSA，原型K+通道和哺乳动物医学相关的K+通道的重要同源模型，（2）ATP合酶亚基C，一种proton泵，一种驱动ATP和Ongers Connection的旋转机制，可以针对ATP和Ongansism of tu的旋转机制。