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

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。生物膜的环境是动态的，由丰富的化学物质组成 脂类分子的多样性。除了生物膜的复杂性之外，人们还日益认识到 脂分子在膜蛋白的折叠、结构和功能中的重要作用。事实上，有 通常由X-结晶学和低温电子显微镜确定的结构图中的密度归因于脂类，但 他们的身份在很大程度上仍然不为人知。尽管有几个例子提供了对 膜蛋白-脂质相互作用，单个脂质分子如何影响细胞的结构和功能 膜蛋白在分子水平上很大程度上仍然知之甚少。是什么决定了 膜蛋白向脂类转化？脂类化学有多重要，如脂类尾长、立体化学 以及不饱和双键在蛋白质-脂类相互作用中的位置？膜蛋白是否通过 变构，他们自己的微环境？在这里，这项提议试图通过以下方式解决这些根本问题 开发利用氨通道探测膜蛋白-脂质相互作用的新工具和试剂 (AmtB)与其调控蛋白GlnK形成复合体，作为膜蛋白系统的模型。更多 具体地说，原生质谱学(MS)技术，其中非共价相互作用被保存在 质谱仪，将与拉加诺夫斯基开创的新的MS方法相结合 与其他生物物理方法不同的是，允许个别脂结合事件与膜蛋白结合的组 复杂的问题有待解决和审问。拟议的研究建立在以前工作的基础上， Native MS技术与其他生物物理技术相结合，如表面等离子体共振 (SPR)和X射线结晶学，以解决与膜蛋白-脂有关的基本问题 互动。更具体地说，拟议的研究旨在解开相当数量的协作性。 通过(I)使用电荷还原分子和(Ii)合成与AmtB的单个脂结合事件(最多20个) 专为本地MS应用而设计的新型洗涤剂。接下来，提出了推动技术极限的研究 MS技术的目的是推断AmtB的异质脂结合事件中的变构。这里，这些 研究超越了以前关于两个不同脂头基团混合物的工作，转向更复杂的脂类混合物， 由三到四种不同的脂类组成，与AmtB结合。提出了一种新的方法来推导 结合蛋白质工程和共价结合的结合脂对AmtB的位置依赖效应 标签策略。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，对我们的 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.