Dynamic Mass Photometry: A new method for studying membrane protein dynamics and interactions

膜蛋白使细胞与其环境之间的通信。虽然对健康至关重要，并且是治疗一系列疾病的最有针对性的蛋白质类，但它们非常难以研究。在这个项目中，我们将开发一种新技术，使人们能够更好地了解膜蛋白的功能，使人们能够更深入地了解依赖于其活性的许多生命过程，并为药物的开发提供动力。一种类型的膜蛋白是整合膜蛋白，其具有至少一次（通常是几次）穿过细胞外脂膜的区域。它们占我们体内蛋白质的近四分之一。另一种类型是膜相关蛋白，当它们与膜相互作用时，它们执行重要的细胞功能，例如信号传导和运输。膜蛋白难以研究的一个原因是它们需要疏水环境，即脂质双层，以实现稳定性和功能，但脂质双层难以在细胞外重建。现有的模拟细胞膜的系统在形状和组成上与细胞不同，因此不能依赖它们来诱导蛋白质在细胞中显示的行为。数据收集方面的挑战构成了另一个难题。现有的技术只能提供时间上的快照，而不是跟踪动态，或者它们需要荧光标记，这可能会破坏蛋白质功能并产生难以量化的数据。我们的目标是通过开发，优化和应用动态质量光度法（Dynamic Mass Photometry）来克服这些挑战，动态质量光度法是一种用于脂质膜中单个膜蛋白及其复合物的成像，跟踪和质量测量的无标记方法。该方法建立在质量光度法的基础上，这是我们开发的一种技术，可以对蛋白质及其在溶液中的相互作用进行单分子质量成像。我们的第一个目标将是开发实验装置和数据分析方法，这将使我们能够将现有的质量光度学平台转化为测量膜蛋白。我们将使用支持的脂质双层，在那里蛋白质可以自由扩散和相互作用，我们可以选择性地修改脂质组成，以适应个别膜蛋白和模拟不同的组织细胞类型。接下来，我们将进一步研究我们在溶液中广泛研究的两种不同类型的膜蛋白，使我们能够评估在溶液中发现的行为在多大程度上代表双层膜上或双层膜中的行为。首先，我们将量化发动蛋白的组装和相互作用动力学，发动蛋白是一种在细胞运输中起核心作用的膜相关蛋白。其次，我们将研究血管紧张素转换酶-2（ACE2）受体，由于其与刺突糖蛋白的结合，该受体在人类SARS-CoV-2感染中起关键作用。这项工作将由牛津大学新成立的Kavli纳米科学发现研究所的生物制药学家团队进行。这将使人们有可能建立一个详细的定量图片的相互作用和组装的膜蛋白在其自然设置-带来前所未有的洞察力的关键类别的蛋白质。同时，开发的分析方法将广泛适用于任何系统，其中了解生物分子的质量，相互作用，动力学和扩散是有价值的，但目前还不可能。将其应用于不同类型的膜蛋白将展示如何使用它，同时阐明生物功能的机制，以及关键的膜在这些过程中的作用。以SARS-CoV-2 spike和ACE2的研究为例，将生物信息学应用于病原体-宿主-细胞界面，将为病毒如何参与人类细胞提供前所未有的理解，并为开发阻断这些相互作用的药物开辟新途径。

Membrane proteins enable communication between a cell and its environment. Though critically important for health and the most targeted class of proteins for treating a range of illnesses, they are notoriously difficult to study. In this project, we will develop a new technology that will make it possible to better understand how membrane proteins function, enabling a deeper understanding of the many life processes that rely on their activity and empowering the development of drugs. One type of membrane protein is the integral membrane proteins, which have regions that cross the outer lipid membrane of the cell at least once (and often several times). They represent nearly one quarter of the proteins in our bodies. Another type is the membrane-associated proteins, which perform their vital cellular functions, such as signalling and transport, when they interact with the membrane. One reason why membrane proteins are difficult to study is that they require a hydrophobic environment, i.e. the lipid bilayer, for stability and function, but lipid bilayers are difficult to recreate outside the cell. Existing systems that mimic cellular membranes differ from cells in shape and composition, so they cannot be relied on to induce the behaviour that proteins display in cells. Data collection challenges pose another difficulty. Existing techniques provide only snapshots in time, rather than tracking dynamics, or they require fluorescent labels, which may disrupt protein function and produce data that can be difficult to quantify. We aim to overcome these challenges by developing, optimising and applying Dynamic Mass Photometry (DMP), a label-free approach for the imaging, tracking and mass measurement of individual membrane proteins and their complexes in lipid membranes. The approach builds on mass photometry, a technology we developed that enables single-molecule mass imaging of proteins and their interactions in solution. Our first objective will be to develop the experimental setup and data analysis approach for DMP, which will enable us to translate the existing mass photometry platform to measuring membrane proteins. We will use supported lipid bilayers, where proteins can freely diffuse and interact, and we can selectively modify the lipid composition to suit individual membrane proteins and mimic different tissue cell types. Next, we will advance DMP to study two different types of membrane proteins that we have studied extensively in solution, enabling us to assess the degree to which the behaviour found in solution is representative of that on or in a bilayer membrane. First, we will quantify the assembly and interaction dynamics of dynamin, a membrane-associated protein that plays a central role in cellular trafficking. Second, we will investigate the angiotensin converting enzyme-2 (ACE2) receptor, which is central to SARS-CoV-2 infection in humans due to its binding to the spike glycoprotein. This work will be carried out by our team of biophysicists at the University of Oxford's newly established Kavli Institute for NanoScience Discovery. DMP will make it possible to establish a detailed quantitative picture of the interactions and assembly of membrane proteins in their natural setting - bringing unprecedented insight to a critical class of proteins. Meanwhile, the analysis approach developed will be broadly applicable to any system where understanding the mass, interaction, dynamics and diffusion of biomolecules is valuable, but currently not possible. Applying DMP to different types of membrane proteins will demonstrate how it can be used, while shedding light on mechanisms of biological function, and, critically, the role of the membrane in those processes. Applying DMP to pathogen-host-cell interfaces, exemplified with the study of SARS-CoV-2 spike and ACE2, will provide unprecedented understanding of how viruses engage human cells and open a new route for developing drugs that block these interactions.