Understanding how membrane composition directs membrane protein structure and function

这个跨学科团队科学提案的目标是扩展和告知生化和结构 通过了解膜蛋白的天然环境来研究膜蛋白的生物学方法 结构和功能。到目前为止，对完整膜蛋白(Imps)的大部分机制研究都有 没有捕捉到IMP嵌入其中的膜的性质和功能贡献。 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.)定义功能复合体的脂类成分和蛋白质共受体成分；二.评估角色 局部膜环境在IMPS功能和调节中的作用；确定的结构 这些组件。推动--并使之成为可能--为各系招聘了六名主要初级教员 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感兴趣。我们的团队成员带来了互补 项目专业知识，掌握低温电子显微镜、自上而下和自下而上的质量 光谱(MS)、多组体分析、光学成像、生物化学和细胞信号。我们都很好 在理解参与细菌调控的膜蛋白方面取得了独特的进展 G细胞内毒素合成、昆虫嗅觉、哺乳动物离子通道和哺乳动物受体 蛋白偶联受体(GPCR)、Frizzled型和受体酪氨酸激酶家族。我们的具体目标是： 1：鉴定完整膜蛋白的天然环境 为了实现这一点，我们将确定新的膜活性共聚物，有效地提取感兴趣的IMP，以及 使用最先进的脂质组学、蛋白质组学和自然质谱学来阐明分子 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 使用针对每个IMP量身定做的各种化验方法，我们将询问特定的膜环境如何 在目标1中确定的影响IMP活性和寡聚化。我们还将使用有限的蛋白质分解和H/D 用交换质谱法评价膜成分对 构象与结构动力学 3：测定天然膜环境中膜蛋白和复合体的结构 我们将在定义的膜环境中使用CryoEM来确定目标IMP的结构，以询问如何 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。

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.