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

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。随着生物膜的复杂性，人们越来越认识到， 脂质分子在膜蛋白的折叠、结构和功能中的重要作用。其实有 通常在由X-晶体学和cryoEM确定的结构图中密度被归因于脂质， 他们的身份仍然基本上不为人知。虽然有一些例子提供了深入了解 膜蛋白-脂质相互作用，单个脂质分子如何影响膜蛋白的结构和功能， 在分子水平上的膜蛋白在很大程度上仍然知之甚少。是什么决定了 膜蛋白转化为脂质？脂质化学有多重要，如脂质尾长、立体化学 和不饱和双键的位置，在蛋白质-脂质相互作用？膜蛋白是否通过 变构作用，它们自己的微环境？在此，本提案寻求通过以下方式解决这些基本问题： 开发新的工具和试剂，利用氨通道探测膜蛋白-脂质相互作用 （AmtB）从E.大肠杆菌与其调节蛋白GlnK复合作为模型膜蛋白系统。更 具体地，天然质谱（MS）技术，由此非共价相互作用被保留在 质谱仪，将与Laganowsky开创的新MS方法结合使用 与其他生物物理方法不同，该方法允许单个脂质结合事件与膜蛋白结合 复杂的问题需要解决和询问。拟议的研究建立在以前工作的基础上， 天然MS技术与其他生物物理技术相结合，例如表面等离子体共振 (SPR)和X射线晶体学，以解决有关膜蛋白-脂质的基本问题 交互.更具体地说，拟议的研究旨在解开相当多的合作性， (up通过（i）使用电荷减少分子和（ii）合成 为天然MS应用而设计的新型洗涤剂。接下来，提议的研究推动了技术极限 MS技术旨在推断AmtB异质脂质结合事件中的变构。这里，这些 研究超越了先前关于两种不同脂质头基的混合物的工作而进入更复杂的脂质混合物， 由三到四种不同的脂质物质组成，与AmtB结合。提出了一种新的方法来推导 结合脂质对AmtB的位置依赖性作用，通过使用蛋白质工程和共价结合的组合， 标签策略。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-脂质相互作用产生了重大影响，更广泛地说， 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.