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

描述（由申请人提供）：膜蛋白在许多细胞和生理过程中起关键作用。洗涤剂影响溶解的膜蛋白的脂质组成、功能和稳定性的方式是膜蛋白的结构生物学的一个知之甚少的方面。在过去的几十年中，选择用于膜蛋白溶解和/或结晶的洗涤剂一直是随机实验。在本申请中我们将解决的主要问题是是否可以开发用于系统（非随机）选择特定膜蛋白的去污剂的综合方法。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。我们最近用溶解在不同去污剂中的nAChR进行了脂质组学/活性研究（Asmar-Rovira等人，2008），表明集中于去污剂结构和增溶条件的综合方法可以提供关于功能活性复合膜蛋白的制备的重要信息。我们建议显着扩大我们的脂质为基础的方法，在评估的功能和稳定性的nAChR-去污剂复合物（nAChR-DC）的主要目标是定义的方式，其中去污剂结构影响的脂质组成，离子通道功能，激动剂结合，聚集的nAChR-DC的状态，并最终形成膜蛋白晶体的能力。中心假设是某些洗涤剂可以改变溶解的膜蛋白的天然脂质组成，其方式导致结构改变，从而导致功能丧失、不可逆变性、聚集和不能形成晶体。我们提出四个具体目标：（1）确定去污剂的化学性质如何影响来自加利福尼亚电鳐组织的去污剂提取的nAChR的脂质组成，（2）确定去污剂的化学性质如何影响nAChR的配体结合和离子通道功能，（3）检查nAChR在不同去污剂中的聚集程度，（4）研究脂质成分对nAChR-DC在脂立方相中移动的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制备高质量nAChR晶体的创新观点。对于第一次，我们建议进行生物物理研究的nAChR在LCP。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，因为它将同时和系统地评估脂质组成nAChR-DCs，并且它在LCP中使用光漂白后荧光恢复（FRAP）方法来估计nAChR移动的分数和扩散系数以与受体稳定性和/或聚集相关方面具有创新性。这种基于脂质的方法的隐含假设是，关于nAChR-DC发现的有关nAChR的脂质组成、功能性、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.