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: 8158503
• 负责人: JOSE Antonio LASALDE-DOMINICCI
• 金额: $ 27.79万
• 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 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8158503
• 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; Lipid A; 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; burden of illness; chemical property; design; functional loss; innovation; liquid chromatography mass spectrometry; novel; peripheral membrane protein 43K; prevent; protein function; protein structure; public health relevance; receptor; reconstitution; research study; screening; structural biology; 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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because a high-resolution structure of the nAChR is of crucial importance for the design of novel agents that target defined nervous system pathologies such as Alzheimer's disease, schizophrenia, depression, attention deficit hyperactivity disorder and tobacco addiction. Thus, the proposed research is relevant to NIH's mission in that it will increase current knowledge about an important ligand-gated ion channel which could help extend healthy life and reduce the burdens of illness.

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知识。在过去的几十年中，选择用于膜蛋白溶解和/或结晶的洗涤剂是一个随机的实验。我们将在此应用程序中解决的主要问题是，是否可以开发用于系统（非随机）选择特定膜蛋白清洁剂的综合方法。尽管研究最广泛的离子通道之一，但烟碱乙酰胆碱受体（NACHR）是多聚体膜络合物的一个主要例子，其高分辨率结构仍然难以捉摸。最近，我们在不同的洗涤剂中溶解了NACHR（Asmar-Rovira等，2008），表明综合方法侧重于洗涤剂结构和溶解条件，可以提供有关功能上功能活跃的复杂膜蛋白的准备的重要信息。我们建议在评估NACHR污染物复合物（NACHR-DC）的功能和稳定性方面显着扩展基于脂质的方法，其主要目标是确定洗涤剂结构影响脂质组成，离子通道功能，激动剂粘合状态，NACHR-DC聚集状态的方式，NACHR-DC的聚集状态以及最终的表型晶状体蛋白蛋白蛋白蛋白蛋白蛋白蛋白蛋白质蛋白蛋白质蛋白质蛋白。中心假设是某些洗涤剂可以以一种导致结构性改变的方式改变溶解化膜蛋白的天然脂质组成，从而导致功能损失，不可逆的变性，聚集和无法形成晶体。我们提出了四个具体目的：（1）确定清洁剂的化学性质如何影响从加利福尼亚州索雷雷斯科组织中提取的清洁剂提取的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如何影响NACHR的NACHR，（（3）nachr的nachr frientation anachr in anachr in Detripation in Detription in Detripation in lip and（4）（4）（4）（4）（4）（4）（4）（4） NACHR-DC在脂质立方相（LCP）中的扩散系数。我们对NACHR-DC的基于脂质的分析揭示了关于制备高质量NACHR晶体的创新观点。我们第一次提议对LCP中的NACHR进行生物物理研究。我们的初步数据表明，LCP与基于脂质的方法结合使用可能导致高质量的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，并且在LCP中使用光漂白（FRAP）方法的荧光恢复具有创新性，以估算NACHR移动分数和扩散系数与受体稳定性和/或聚集。这种基于脂质方法的隐含假设是，关于NACHR-DC在脂质组成，功能，稳定性和NACHR聚集状态的原理将与其他重要的膜受体系统，离子通道和膜蛋白有关。 公共卫生相关性：拟议的研究与公共卫生有关，因为NACHR的高分辨率结构对于针对定义的神经系统病理（例如阿尔茨海默氏病，精神分裂症，抑郁症，注意力缺陷多动障碍和tobaccococococococococococococococo otion）的新型药物的设计至关重要。因此，拟议的研究与NIH的使命相关，即它将增加有关重要配体门控离子渠道的当前知识，该渠道可以帮助延长健康的生活并减轻疾病的负担。