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

项目摘要 完整的膜蛋白驻留在生物膜中，它们在那里发挥作用并与之密切相互作用。 脂类分子。膜环境是动态的，由丰富的脂类化学多样性组成。 随着生物膜的复杂性，人们越来越认识到脂质的重要作用。 调节膜蛋白结构和功能的分子。尽管有一小部分示例 提供了对膜蛋白-脂相互作用的洞察，单个脂分子如何影响 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？脂类化学有多重要，比如脂类尾巴长度， 立体化学和不饱和双键在蛋白质-脂类相互作用中的位置？解决这些问题 基本的问题不仅因为识别与膜蛋白密切结合的脂类而受到阻碍，而且 以及蛋白质-脂质相互作用的生物物理特征。在此，这项建议旨在发展和 应用高度创新和综合的方法，更好地了解脂类如何影响结构和 膜蛋白的功能。我们的第一个目标是开发综合方法，结合 脂类组学与天然质谱学(MS)相结合，从天然提取物中鉴定特定的蛋白质-脂质相互作用 通过使用(I)固定化的膜蛋白的渐进洗涤和(Ii)空的和 膜蛋白负载的纳米盘。在我们的第二个目标中，原生MS技术将用于生物物理 表征单个脂类与膜蛋白的结合事件，提供对亲和力和选择性的洞察。 此外，将使用纳米盘中膜蛋白的MS方法来收集对脂质的洞察 富含在膜蛋白周围。这些新方法将识别与靶标密切相关的脂类。 膜蛋白，为我们的第三个目标提供了路线图，重点是了解脂类结合得有多紧密 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类的情况下，被重组为脂质体和纳米盘。结构性 而功能研究将导致脂质结合部位的可视化以及由此引起的结构和功能变化 通过结合的脂类。来自结构和功能研究的观察结果将被严格地用 突变研究。综上所述，我们拟议研究的结果和结果预计将具有 对我们理解膜蛋白-脂质相互作用的重大影响，更广泛地说，如何 生物膜的变化可能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.