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

• 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
• 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

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和告知生物化学和结构 通过了解其天然环境如何定义膜蛋白来研究膜蛋白的生物学方法 结构和功能。迄今为止，大多数关于膜整合蛋白（IMP）的机制研究都 没有捕捉到嵌入IMP的膜的性质和功能贡献。 我们合作团队的核心目标是开发新技术和方法，使我们能够： （一）定义功能复合物的脂质组分和蛋白质共受体组分; ii.）评估角色 局部膜环境在IMP的功能和调节中的作用;和iii.）确定的结构 这些集会。驱动-并成为可能-通过招聘六个关键的初级教师的部门 药理学和细胞生物学在过去的5年里，我们组建了一个跨学科的团队， 对跨膜蛋白的结构和功能有共同的兴趣。我们的团队成员带来了互补性 具有冷冻电子显微镜（cryoEM）、自上而下和自下而上质量技术的项目专业知识 质谱（MS）、多组学分析、光学成像、生物化学和细胞信号传导。我们非常 在理解参与细菌生长调节的膜蛋白方面取得了独特的进展。 脂多糖合成，昆虫嗅觉，哺乳动物离子通道和哺乳动物受体在G 蛋白偶联受体（GPCR）、卷曲蛋白（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，我们将确定新的膜活性共聚物，有效地提取目标IMP， 使用最先进的脂质组学、蛋白质组学和天然质谱法来阐明 蛋白质的膜环境的组成部分。 2：了解天然膜环境如何调节或决定膜蛋白功能 使用各种各样的测定-为每种IMP量身定制-我们将询问特定的膜环境是如何影响细胞的生长的。 目的1中鉴定的影响IMP活性和寡聚化。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组成的影响， 构象与结构动力学 3：确定天然膜环境中膜蛋白和复合物的结构 我们将使用cryoEM确定在限定的膜环境中目标IMP的结构，以询问如何 已知特异性IMP相关脂质和其它组分与IMP结构相互作用并调节IMP结构。