A lipid-based approach towards a high-resolution structure of a functional nAChR

基于脂质的方法获得功能性 nAChR 的高分辨率结构

• 批准号: 9068277
• 负责人: JOSE Antonio LASALDE-DOMINICCI
• 金额: $ 28.31万
• 依托单位: UNIVERSITY OF PUERTO RICO RIO PIEDRAS
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-30 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9068277
• 关键词: ATP phosphohydrolase; Address; Affect; Affinity; Agonist; Alzheimer' s Disease; Attention deficit hyperactivity disorder; Binding; Biological Assay; Bungarotoxins; Cell membrane; Cell physiology; Chemicals; Cholesterol; Complex; Coupled; Coupling; Crystal Formation; Crystallization; Data; Defect; Detergents; Diffusion; Disease; Electron Microscopy; Exclusion; Gated Ion Channel; Goals; Head; Health; High Pressure Liquid Chromatography; Ion Channel; Knowledge; Lead; Life; Ligand Binding; Ligands; Lipids; Mass Fragmentography; Membrane; Membrane Proteins; Mental Depression; Micelles; Mission; Modeling; Molecular Sieve Chromatography; Nature; Nervous system structure; Nicotinic Receptors; Pathology; Pattern; Phase; Phospholipids; Photobleaching; Physiological Processes; Play; Preparation; Proteins; Public Health; Recovery; Research; Resolution; Role; Sampling; Schizophrenia; Shapes; Structure; System; Tail; Tertiary Protein Structure; Testing; Time; Tissues; Tobacco Dependence; Torpedo; acyl group; base; biophysical analysis; burden of illness; chemical property; design; functional loss; innovation; liquid chromatography mass spectrometry; novel; peripheral membrane protein 43K; prevent; protein function; protein structure; receptor; reconstitution; research study; screening; structural biology; targeted agent; vapor

DESCRIPTION (provided by applicant): Membrane proteins play crucial roles in many cellular and physiological processes. The manner by which detergents affect lipid composition, functionality, and stability of solubilized membrane proteins is a poorly understood aspect of the structural biology of membrane proteins. For the past several decades, the selection of detergents for membrane protein solubilization and/or crystallization has been a random experiment. The main question that we will address in this application is whether a comprehensive approach for a systematic (non-random) selection of a detergent for a particular membrane protein can be developed. Although one of the most widely studied ion channels, the nicotinic acetylcholine receptor (nAChR) is a prime example of a multimeric membrane complex whose high-resolution structure has remained elusive. We have recently carried out lipidomics/activity studies with nAChR solubilized in different detergents (Asmar-Rovira et al., 2008) indicating that a comprehensive approach focused on detergent structure and solubilization conditions could provide important information on the preparation of functionally active complex membrane proteins. We propose to significantly expand our lipid-based approach in assessing the function and stability of the nAChR- detergent complex (nAChR-DC) with the primary goal of defining the manner in which detergent structure affects the lipid composition, ion channel function, agonist binding, state of aggregation of the nAChR-DC, and ultimately the ability to form membrane protein crystals. The central hypothesis is that certain detergents can alter the native lipid composition of solubilized membrane proteins in a manner that results in structural alterations that lead to functional loss, irreversible denaturation, aggregation, and inability to form crystals. We propose four Specific Aims: (1) Determine how the chemical nature of detergents affects the lipid composition of detergent-extracted nAChR from Torpedo californica tissue, (2) Determine how the chemical nature of detergents affects the ligand binding and ion channel function of the nAChR, (3) Examine the degree of aggregation of the nAChR in different detergents, and (4) Estimate the effect of lipid composition on mobile fraction and diffusion coefficient of nAChR-DC in the lipidic cubic phase (LCP). Our lipid-based analysis of the nAChR-DC has revealed an innovative perspective on the preparation of high-quality nAChR crystals. For the first time, we propose to perform biophysical studies of the nAChR in the LCP. Our preliminary data demonstrated that the LCP in combination with the lipid-based approach could lead to high-quality nAChR crystals. This project is technically significant because it will concurrently and systematically assess the lipid composition nAChR-DCs, and it is innovative in its use of the fluorescent recovery after photobleaching (FRAP) approach in the LCP to estimate nAChR mobile fraction and diffusion coefficient to correlate with receptor stability and/or aggregation. An implicit assumption of this lipid-based approach is that principles discovered about the nAChR-DC with respect to lipid composition, functionality, stability, and state of aggregation of the nAChR will be relevant to other important membrane receptor systems, ion channels, and membrane proteins.

描述(由申请人提供)：膜蛋白在许多细胞和生理过程中起着至关重要的作用。洗涤剂影响溶解的膜蛋白的脂组成、功能和稳定性的方式是膜蛋白结构生物学中一个鲜为人知的方面。在过去的几十年里，膜蛋白增溶和/或结晶的去污剂的选择一直是一个随机的实验。我们在这一应用中要解决的主要问题是，是否可以开发一种全面的方法，为特定的膜蛋白系统(非随机)选择洗涤剂。尽管烟碱型乙酰胆碱受体(NAChR)是研究最广泛的离子通道之一，但它是多聚体膜复合体的一个典型例子，其高分辨结构仍然难以捉摸。我们最近对nAChR在不同洗涤剂中的增溶进行了脂质组学/活性研究(Asmar-Rovira等人，2008年)，表明专注于洗涤剂结构和增溶条件的综合方法可以为制备具有功能活性的复合膜蛋白提供重要信息。我们建议显著扩展我们基于脂质的方法来评估nAChR-洗涤剂复合体(nAChR-DC)的功能和稳定性，主要目标是定义洗涤剂结构对nAChR-DC的脂质组成、离子通道功能、激动剂结合、聚集状态以及最终形成膜蛋白晶体的能力的影响方式。中心假设是，某些洗涤剂可以改变溶解的膜蛋白的天然脂组成，导致结构改变，导致功能丧失、不可逆转的变性、聚集和不能形成晶体。我们提出了四个具体目标：(1)确定洗涤剂的化学性质如何影响从鱼雷加州组织中提取的nAChR的脂组成；(2)确定洗涤剂的化学性质如何影响nAChR的配体结合和离子通道功能；(3)检测nAChR在不同洗涤剂中的聚集程度；以及(4)估计脂组成对nAChR-DC在脂质立方相(LCP)中的流动分数和扩散系数的影响。我们对nAChR-DC的基于脂质的分析揭示了高质量nAChR晶体制备的创新前景。我们首次提出对LCP中的nAChR进行生物物理研究。我们的初步数据表明，LCP与基于脂质的方法相结合可以产生高质量的nAChR晶体。这个项目具有重要的技术意义，因为它将同时和系统地评估nAChR-DC的脂质成分，并且它在LCP中使用光漂白后荧光恢复(FRAP)方法来估计nAChR的移动分数和扩散系数以与受体的稳定性和/或聚集相关，这一点是创新的。这种基于脂质的方法的一个隐含的假设是，关于nAChR-DC关于nAChR的脂质组成、功能、稳定性和聚集状态的原理将与其他重要的膜受体系统、离子通道和膜蛋白相关。