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

基本信息

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
项目类别：
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&apos 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

项目摘要

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发挥着至关重要的作用。去垢剂影响溶解膜蛋白的脂质组成、功能和稳定性的方式是膜蛋白结构生物学的一个鲜为人知的方面。在过去的几十年里，用于膜蛋白溶解和/或结晶的去污剂的选择一直是随机实验。我们在本申请中要解决的主要问题是是否可以开发一种针对特定膜蛋白系统（非随机）选择去污剂的综合方法。尽管烟碱乙酰胆碱受体 (nAChR) 是研究最广泛的离子通道之一，但它是多聚体膜复合物的一个主要例子，其高分辨率结构仍然难以捉摸。我们最近对溶解在不同去污剂中的 nAChR 进行了脂质组学/活性研究（Asmar-Rovira 等，2008），表明专注于去污剂结构和溶解条件的综合方法可以为功能活性复合膜蛋白的制备提供重要信息。。我们建议显着扩展基于脂质的方法来评估 nAChR-去污剂复合物 (nAChR-DC) 的功能和稳定性，主要目标是确定去污剂结构影响脂质组成、离子通道功能、激动剂结合的方式、 nAChR-DC 的聚集状态，以及最终形成膜蛋白晶体的能力。中心假设是某些去垢剂可以改变溶解膜蛋白的天然脂质组成，从而导致结构改变，从而导致功能丧失、不可逆变性、聚集和无法形成晶体。我们提出了四个具体目标：(1) 确定去污剂的化学性质如何影响从加州鱼雷组织中去污剂提取的 nAChR 的脂质组成，(2) 确定去污剂的化学性质如何影响配体结合和离子通道功能nAChR，（3）检查不同去污剂中 nAChR 的聚集程度，（4）估计脂质成分对 nAChR-DC 在去污剂中的移动分数和扩散系数的影响脂质立方相（LCP）。我们对 nAChR-DC 的脂质分析揭示了制备高质量 nAChR 晶体的创新视角。我们首次提议对 LCP 中的 nAChR 进行生物物理学研究。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。该项目在技术上具有重要意义，因为它将同时系统地评估 nAChR-DC 的脂质成分，并且在 LCP 中使用光漂白后荧光恢复 (FRAP) 方法来估计 nAChR 移动分数和扩散系数以与受体稳定性和/或聚集。这种基于脂质的方法的隐含假设是，关于 nAChR-DC 在脂质组成、功能、稳定性和 nAChR 聚集状态方面发现的原理将与其他重要的膜受体系统、离子通道和膜相关蛋白质。