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：
10413702
负责人：
Erin S Baker
金额：
$ 150.28万
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/10413702
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 Glean Heart Immobilization Impairment Individual Integral Membrane Protein Ion Channel Knowledge Length Lipid Binding Lipid Chemistry Lipids Liposomes Mass Spectrum Analysis Measurement Membrane Membrane Proteins Membrane Structure and Function Methods Molecular Mutation Outcome Physiological Physiology Positioning Attribute Protein Biochemistry Proteins Protocols documentation Research Role Structure Tail Technology Testing Visualization Work X-Ray Crystallography base biophysical properties biophysical techniques cofactor density design experimental study innovation insight lipidomics medical specialties member method development nanodisk novel novel strategies pi bond preference protein function protein structure function 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.

项目摘要整合膜蛋白驻留在它们发挥作用并与之紧密相互作用的生物膜中脂质分子。膜环境是动态的，由脂质的丰富化学多样性组成。生物膜的复杂性以及脂质重要作用的实现不断增长分子调节膜蛋白的结构和功能。虽然一小部分示例存在对膜蛋白脂质相互作用的见解，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鲜为人知。什么决定膜蛋白对脂质的选择性？脂质化学（例如脂质尾巴长度）有多重要，立体化学和蛋白质脂质相互作用中不饱和双键的位置？解决这些基本问题不仅通过识别与膜蛋白相关的脂质来阻碍也是蛋白质脂质相互作用的生物物理表征。在此，该建议旨在发展和应用高度创新和综合的方法，以更好地了解脂质如何影响结构和膜蛋白的功能。我们的第一个目标是结合综合方法的发展具有天然质谱法（MS）的脂质组学，以鉴定自然提取物的特定蛋白质脂质相互作用通过使用（i）进行固定的膜蛋白的进行性洗涤和（ii）空中的脂质交换膜蛋白负载纳米盘。在我们的第二个目标中，本机MS技术将用于生物物理表征单个脂质结合事件与膜蛋白，从而洞悉亲和力和选择性。此外，将采用纳米盘中的膜蛋白的MS方法来了解脂质的洞察力富含膜蛋白。这些新方法将识别与目标急切关联的脂质膜蛋白，为我们的第三个目标提供了路线图，重点是了解脂质的紧密结合影响膜蛋白的功能和结构。没有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，将其重构为脂质体和纳米盘。结构功能研究将导致可视化脂质结合位点以及诱导的结构和功能变化通过绑定的脂质。然后，将对结构和功能研究的观察进行严格检查突变研究。综上所述，预计我们提出的研究的结果和结果将有对我们对膜蛋白脂质相互作用的理解的重大影响，更普遍地是如何生物膜的变化可能调节膜蛋白生理功能。