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

项目摘要 整合膜蛋白存在于生物膜中，在那里它们起作用并与生物膜密切相互作用。 脂质分子膜环境是动态的，由丰富的脂质化学多样性组成。 随着生物膜的复杂性，人们越来越认识到脂质的重要作用 分子调节膜蛋白的结构和功能。虽然有一小部分例子 存在可以深入了解膜蛋白-脂质相互作用、单个脂质分子如何影响膜蛋白-脂质相互作用的方法。 膜蛋白在分子水平上的结构和功能仍然知之甚少。什么决定 膜蛋白对脂质的选择性？脂质化学如脂质尾长， 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 基本问题的解决不仅受到了识别与膜蛋白紧密结合的脂质的阻碍， 也是蛋白质-脂质相互作用的生物物理表征。在此，该提案旨在发展和 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， 膜蛋白的功能。我们的第一个目标是发展综合方法， 脂质组学与天然质谱（MS），以确定特定的蛋白质-脂质相互作用的天然提取物 通过使用（i）固定化膜蛋白的渐进洗涤和（ii）在空的和 膜蛋白负载的纳米盘。在我们的第二个目标中，天然MS技术将用于生物制药， 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，纳米盘中膜蛋白的MS方法将用于收集脂质的洞察力 在膜蛋白周围富集。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，重点是了解如何紧密结合脂质 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 和功能的研究将导致可视化的脂质结合位点和结构和功能的变化诱导 被结合的脂质所束缚。结构和功能研究的观察结果将被严格检查， 突变研究总的来说，我们提出的研究的结果和成果预计将 这对我们理解膜蛋白-脂质相互作用以及更普遍地理解 生物膜的变化可调节膜蛋白的生理功能。

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.