Developing new tools to probe membrane protein-lipid interactions for biomedical applications

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。生物膜的环境是动态的，由丰富的化学物质组成 脂质分子的多样性。除了生物膜的复杂性之外，人们越来越认识到 脂质分子在膜蛋白的折叠、结构和功能中发挥重要作用。事实上，有 通常由 X 晶体学和冷冻电镜确定的结构图中的密度归因于脂质，但 他们的身份很大程度上仍不为人知。尽管存在一些示例可以深入了解 膜蛋白-脂质相互作用，单个脂质分子如何影响膜蛋白-脂质的结构和功能 分子水平上的膜蛋白在很大程度上仍然知之甚少。选择性由什么决定 膜蛋白对脂质的作用？脂质化学有多重要，例如脂质尾长、立体化学 以及蛋白质-脂质相互作用中不饱和双键的位置？膜蛋白是否通过招募 变构，他们自己的微环境？在此，本提案旨在通过以下方式解决这些基本问题： 开发新工具和试剂，利用氨通道探测膜蛋白-脂质相互作用 (AmtB) 来自大肠杆菌，与其调节蛋白 GlnK 形成复合物，作为模型膜蛋白系统。更多 具体来说，本地质谱（MS）技术，通过该技术，非共价相互作用被保留在 质谱仪，将与 Laganowsky 首创的新 MS 方法结合使用 与其他生物物理方法不同，允许单独的脂质结合事件与膜蛋白 需要解决和询问的情结。拟议的研究建立在先前工作的基础上，其中 原生 MS 技术与其他生物物理技术相集成，例如表面等离子共振 (SPR) 和 X 射线晶体学，解决有关膜蛋白-脂质的基本问题 互动。更具体地说，拟议的研究旨在阐明相当多的人的合作性 通过 (i) 使用电荷还原分子和 (ii) 合成，（最多 20）个与 AmtB 的单独脂质结合事件 专为原生 MS 应用而设计的新型去污剂。接下来，拟议的研究突破了技术极限 MS 技术旨在推断 AmtB 异质脂质结合事件中的变构。在这里，这些 研究超越了之前对两种不同脂质头基混合物的研究，转向了更复杂的脂质混合物， 由三到四种不同的脂质种类组成，与 AmtB 结合。提出了一种新方法来推导 结合蛋白质工程和共价键结合脂质对 AmtB 的位置依赖性影响 标签策略。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产生了重大影响，更广泛地说，对我们的理解产生了重大影响 了解膜蛋白生物学，特别是生物膜的变化如何调节 膜蛋白的生理功能。

Integral membrane proteins reside in the biological membrane where they function and intimately interact with lipid molecules. The environment of the biological membrane is dynamic and composed of a rich chemical diversity of lipid molecules. Alongside the complexity of the biological membrane is the growing realization of the important roles of lipid molecules in the folding, structure, and function of membrane proteins. In fact, there is often density in maps of structures determined by X-crystallography and cryoEM that are ascribed to lipids but their identity remains largely unknown. Although a handful of examples exist which provide insight into membrane protein-lipid interactions, how individual lipid molecules influence the structure and function of membrane proteins on the molecular level largely 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? Do membrane proteins recruit, through allostery, their own microenvironment? Here, this proposal seeks to address these fundamental questions by developing new tools and reagents to probe membrane protein-lipid interactions using the ammonia channel (AmtB) from E. coli in complex with its regulatory protein GlnK as a model membrane protein system. More specifically, native Mass Spectrometry (MS) technology, whereby non-covalent interactions are preserved in the mass spectrometer, will be employed in combination with new MS approaches pioneered in the Laganowsky group that, unlike other biophysical methods, allow individual lipid binding events to membrane protein complexes to be resolved and interrogated. The proposed studies build off the foundation of previous work where native MS technology is integrated with other biophysical techniques, such as Surface Plasmon Resonance (SPR) and X-ray crystallography, to address fundamental questions regarding membrane protein-lipid interactions. More specifically, proposed studies are aimed at unravelling cooperativity for a considerable number (up to 20) of individual lipid binding events to AmtB by the (i) use of charge-reducing molecules and (ii) synthesis of new detergents engineered for native MS applications. Next, proposed studies pushing the technological limits of MS technology aimed at deducing allostery within heterogeneous lipid binding events to AmtB. Here, these studies move beyond previous work on mixtures of two different lipid headgroups to more complex lipid mixtures, composed of three to four different lipid species, binding to AmtB. Novel approach is proposed to deduce the position-dependent effects of bound lipids on AmtB by using a combination of protein engineering and covalent labeling strategies. 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, to our understanding of membrane protein biology, especially how changes in the biological membrane may regulate membrane protein physiological function.