Understanding how membrane composition directs membrane protein structure and function

了解膜成分如何指导膜蛋白结构和功能

基本信息

批准号：
10630518
负责人：
Joel Adam Butterwick
金额：
$ 207.21万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10630518
关键词：
Address Affect Area Binding Biochemical Biochemistry Biological Assay Biophysics Butyric Acids Cells Cellular biology Chemistry Clinic Complex Coupled Cryoelectron Microscopy Dehydration Deuterium Development Drug Targeting Drug usage Environment Faculty Family G-Protein-Coupled Receptors Goals Hydrogen Insecta Integral Membrane Protein Ion Channel Ion Channel Gating Libraries Ligands Lipid Binding Lipids Lipopolysaccharides Mass Spectrum Analysis Membrane Membrane Proteins Methods Molecular Molecular Conformation Outcome Pharmaceutical Preparations Pharmacology Property Protein Dynamics Proteins Proteolysis Proteome Proteomics Receptor Protein-Tyrosine Kinases Regulation Research Role Science Signal Transduction Smell Perception Structure Technology Thinking Water copolymer imaging approach innovation interdisciplinary approach interest lipidome lipidomics member mimetics multiple omics nanodisk new technology novel strategies olfactory receptor optical imaging particle pharmacologic protein function protein oligomer protein structure receptor reconstitution recruit screening sensor single molecule skills small molecule structural biology

项目摘要

The goal of this interdisciplinary team science proposal is to extend and inform biochemical and structural biology approaches for studying membrane proteins by understanding how their native environments define structure and function. To date, the majority of mechanistic studies of integral membrane proteins (IMPs) have not captured the properties and functional contributions of the membranes in which the IMPs are embedded. The central goal of our collaborative team is to develop new technologies and approaches that will allow us to: i.) Define lipid components and protein co-receptor components of functional complexes; ii.) Evaluate the role of the local membrane environment in function and regulation of the IMPs; and iii.) Determine the structures of these assemblies. Driven – and made possible – by the recruitment of six key junior faculty to the Departments of Pharmacology and Cell Biology over the past 5 years, we have assembled an interdisciplinary team with shared interest in transmembrane protein structure and function. Our team members bring complementary expertise to the project with skills in cryo electron microscopy (cryoEM), top-down and bottom-up mass spectrometry (MS), multi-omic analysis, optical imaging, biochemistry, and cellular signaling. We are very well placed to make unique advances in understanding membrane proteins involved in regulation of bacterial lipopolysaccharide synthesis, insect olfaction, mammalian ion channels, and mammalian receptors in the G protein-couple receptor (GPCR), Frizzled, and receptor tyrosine kinase families. Our Specific Aims are: 1: Identify native environments of integral membrane proteins To achieve this, we will identify new membrane-active copolymers that efficiently extract IMPs of interest, and use state-of-the-art lipidomics, proteomics, and native mass spectrometry to elucidate the molecular components of the protein’s membrane environment. 2: Understand how the native membrane environment modulates or determines membrane protein function Using a wide variety of assays – tailored to each IMP – we will ask how the specific membrane environment identified in Aim 1 influences IMP activity and oligomerization. We will also use limited proteolysis and H/D exchange mass spectrometry approaches to assess the influence of the membrane composition on conformation and structural dynamics 3: Determine structures of membrane proteins and complexes in native membrane environments We will determine structures of the target IMPs in defined membrane environments using cryoEM, to ask how known specific IMP-associated lipids and other components interact with and modulate IMP structure.

这个跨学科团队科学提案的目标是扩展生物化学和结构并为其提供信息通过了解膜蛋白的原生环境如何定义来研究膜蛋白的生物学方法迄今为止，大多数整合膜蛋白（IMP）的机制研究已经完成。没有捕获嵌入 IMP 的膜的特性和功能贡献。我们协作团队的中心目标是开发新技术和方法，使我们能够： i.) 定义功能复合物的脂质成分和蛋白质辅助受体成分 ii.) 评估作用；局部膜环境对 IMP 的功能和调节的影响；以及 iii.) 确定 IMP 的结构；这些集会的推动和实现是通过向各系招募了六名重要的初级教员。在过去的5年里，我们组建了一支跨学科团队，我们的团队成员对跨膜蛋白结构和功能有着共同的兴趣，具有互补性。具有冷冻电子显微镜 (cryoEM)、自上而下和自下而上质量方面技能的项目专业知识我们在光谱分析 (MS)、多组学分析、光学成像、生物化学和细胞信号传导方面非常擅长。在理解参与细菌调节的膜蛋白方面取得独特进展脂多糖合成、昆虫嗅觉、哺乳动物离子通道和 G 中的哺乳动物受体蛋白偶联受体 (GPCR)、Frizzled 和受体酪氨酸激酶家族我们的具体目标是： 1：识别整合膜蛋白的天然环境为了实现这一目标，我们将鉴定能够有效提取感兴趣的 IMP 的新型膜活性共聚物，以及使用最先进的脂质组学、蛋白质组学和天然质谱来阐明分子蛋白质膜环境的组成部分。 2：了解天然膜环境如何调节或决定膜蛋白功能使用针对每种 IMP 量身定制的多种检测方法，我们将询问特定的膜环境如何目标 1 中确定的影响 IMP 活性和寡聚化的方法我们还将使用有限的蛋白水解和 H/D。交换质谱法来评估膜成分对构象和结构动力学 3：确定天然膜环境中膜蛋白和复合物的结构我们将使用冷冻电镜确定特定膜环境中目标 IMP 的结构，以了解如何已知的特定 IMP 相关脂质和其他成分与 IMP 结构相互作用并调节 IMP 结构。