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

描述（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，选择用于膜蛋白增溶和/或结晶的洗涤剂一直是一种随机实验。我们将在本应用中解决的主要问题是，是否可以开发一种系统（非随机）选择特定膜蛋白洗涤剂的综合方法。虽然是研究最广泛的离子通道之一，但烟碱乙酰胆碱受体（nAChR）是多聚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洗涤剂中的nAChR进行了脂质组学/活性研究（Asmar-Rovira et al., 2008），表明专注于洗涤剂结构和溶解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。我们建议在评估nAChR-洗涤剂复合物（nAChR- dc）的功能和稳定性方面显著扩展我们基于脂质的方法，主要目标是确定洗涤剂结构影响脂质组成、离子通道功能、激动剂结合、nAChR- dc聚集状态的方式，并最终形成膜蛋白晶体的能力。核心假设是，某些洗涤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。我们提出四个具体目标：(1)确定洗涤剂的化学性质如何影响加利福尼亚鱼雷组织中洗涤剂提取的nAChR的脂质组成；(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)检测不同洗涤剂中nAChR的聚集程度；(4)估计脂质组成对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。我们首次提出对LCP中的nAChR进行生物物理研究。我们的初步数据表明，LCP结合基于脂质的方法可以产生高质量的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- dcs的脂质组成，并且在LCP中使用光漂白后荧光恢复（FRAP）方法来估计与受体稳定性和/或聚集相关的nAChR移动分数和扩散系数是创新的。这种基于脂质方法的隐含假设是，关于nAChR- dc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。

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.