Understanding the role of lipids in structure and function of membrane proteins

了解脂质在膜蛋白结构和功能中的作用

基本信息

批准号：
10703408
负责人：
Erin S Baker
金额：
$ 147.78万
依托单位：
TEXAS A&M UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-15 至 2027-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10703408
关键词：
Address Affect Affinity Binding Binding Sites Biological Biology Biophysics Brain Cardiolipins Carrier Proteins Cellular Membrane Chemicals Complex Complex Mixtures Cryoelectron Microscopy Data Detergents Development Discipline Environment Event Exhibits Experimental Designs Glean Heart Immobilization Impairment Individual Integral Membrane Protein Ion Channel Knowledge Length Lipid Binding Lipid Chemistry Lipids Liposomes Mass Spectrum Analysis Measurement Membrane Membrane Lipids Membrane Proteins Membrane Structure and Function Methods Molecular Mutation Natural Extract Outcome Physiological Physiology Positioning Attribute Protein Biochemistry Proteins Protocols documentation Research Role Structure Tail Technology Testing Visualization Work X-Ray Crystallography biophysical properties biophysical techniques cofactor density innovation insight lipidomics medical specialties member method development nanodisk novel novel strategies pi bond preference protein function protein reconstitution protein structure reconstitution stereochemistry stoichiometry structural biology

项目摘要

Project Summary Integral membrane proteins reside in the biological membrane where they function and intimately interact with lipid molecules. The membrane environment is dynamic and composed of a rich chemical diversity of lipids. Alongside the complexity of the biological membrane is the growing realization of the important roles of lipid molecules in modulating the structure and function of membrane proteins. Although a small subset of examples exist that provide insight into membrane protein-lipid interactions, how individual lipid molecules influence the structure and function of membrane proteins on the molecular level remains poorly understood. What determines the selectivity of membrane proteins towards lipids? How important is the lipid chemistry such as lipid tail length, stereochemistry, and position of unsaturated double bonds in protein-lipid interactions? Addressing these fundamental questions is hindered not only by identifying the lipids that bind avidly to membrane proteins but also the biophysical characterization of protein-lipid interactions. Herein, this proposal seeks to develop and apply a highly innovative and integrative approaches to better understand how lipids impact the structure and function of membrane proteins. Our first objective is the development of integrative methods, combining lipidomics with native mass spectrometry (MS), to identify specific protein-lipid interactions from natural extracts by using (i) progressive washes of the immobilized membrane proteins and (ii) lipid exchange within empty and membrane protein loaded nanodiscs. In our second objective, native MS technology will be used to biophysically characterize individual lipid binding events to membrane proteins, providing insight into affinity and selectivity. Moreover, MS approaches of membrane proteins in nanodiscs will be employed to glean insight into lipids enriched around membrane proteins. These new methods will identify lipids that avidly associate with the target membrane protein, providing a roadmap for our third objective focused on understanding how tightly bound lipids affect function and structure of membrane proteins. Membrane proteins devoid of any contaminating lipids will be reconstituted into liposomes and nanodiscs in the presence and absence of a tightly bound lipid. Structural and functional studies will lead to visualization of lipid binding sites and structural and functional changes induced by the bound lipids. Observations from structural and functional studies will then be rigorously examined with mutational studies. Taken together, the results and outcomes from our proposed studies are anticipated to have a significant impact in our understanding of membrane protein-lipid interactions and, more generally, how changes in the biological membrane may regulate membrane protein physiological function.

项目概要整合膜蛋白驻留在生物膜中，在那里它们发挥作用并与脂质分子。膜环境是动态的，由丰富的化学多样性的脂质组成。随着生物膜的复杂性，人们越来越认识到脂质的重要作用调节膜蛋白结构和功能的分子。虽然例子的一小部分存在提供了对膜蛋白-脂质相互作用的深入了解，单个脂质分子如何影响膜蛋白在分子水平上的结构和功能仍然知之甚少。是什么决定了膜蛋白对脂质的选择性？脂质化学（例如脂质尾长）有多重要，立体化学以及蛋白质-脂质相互作用中不饱和双键的位置？解决这些问题基本问题不仅通过识别与膜蛋白紧密结合的脂质而受到阻碍，而且还有蛋白质-脂质相互作用的生物物理特征。在此，本提案旨在发展和应用高度创新和综合的方法来更好地了解脂质如何影响结构和膜蛋白的功能。我们的首要目标是开发综合方法，结合使用天然质谱 (MS) 进行脂质组学，以识别天然提取物中的特定蛋白质-脂质相互作用通过使用（i）固定膜蛋白的渐进洗涤和（ii）空和内的脂质交换负载膜蛋白的纳米圆盘。在我们的第二个目标中，原生 MS 技术将用于生物物理学表征单个脂质与膜蛋白的结合事件，提供对亲和力和选择性的深入了解。此外，纳米圆盘中膜蛋白的质谱方法将用于深入了解脂质富集在膜蛋白周围。这些新方法将识别与目标密切相关的脂质膜蛋白，为我们的第三个目标提供了路线图，重点是了解脂质如何紧密结合影响膜蛋白的功能和结构。不含任何污染脂质的膜蛋白将在存在和不存在紧密结合的脂质的情况下可被重构为脂质体和纳米盘。结构性和功能研究将导致脂质结合位点以及诱导的结构和功能变化的可视化通过结合的脂质。然后，将严格检查结构和功能研究的观察结果突变研究。总而言之，我们拟议的研究的结果和成果预计将对我们对膜蛋白-脂质相互作用的理解以及更一般地说，如何理解膜蛋白-脂质相互作用产生了重大影响生物膜的变化可能调节膜蛋白的生理功能。