Mapping membrane protein dynamics in time and space with mass spectrometry

新的分析工具和方法的进步是应对生命科学最大挑战的核心。一个这样的未满足的挑战是了解膜嵌入蛋白质的结构动力学支持功能。膜蛋白控制着细胞的进出。因此，它们构成了一半以上已知药物的主要目标。尽管它们至关重要，但现有的方法往往无法揭示这类重要生物分子的结构信息，从而阻碍了与治疗干预和药物发现相关的进展。更重要的是，我们目前缺乏工具来捕获膜蛋白在其移动和操作的天然环境中的动态。这主要是由于这些系统的复杂性，因为它们嵌入到脂质的异质层中，这保护了它们的膜蛋白的疏水核心。因此，迫切需要新的工具来揭示膜蛋白的动态运动，并允许对解决当前和未来与人类健康和疾病相关的挑战至关重要的机制见解。在这里，我们将建立一种新的方法来捕获膜蛋白的分子电影。为此，我们将开发时间分辨氢氘交换质谱（tHDX-MS）。HDX-MS是一种灵敏的分析工具，可以准确监测溶液中氢原子与较重氘的交换，从而提供有关蛋白质动力学的信息。通过将新兴的HDX-MS技术与微流控技术相结合，我们将能够在从微秒到小时的时间内以可调分辨率拍摄膜蛋白状态的快照。为了使我们的策略在膜蛋白发挥功能的天然脂质环境中的适用性，我们将利用膜仿生学的受控补丁，即所谓的纳米盘。纳米圆盘技术将使我们能够微调膜蛋白周围的脂质成分，并评估特定脂质对膜蛋白结构和动力学的个体影响。我们将证明我们的方法在一系列重要系统上的适用性，这些系统的规模和复杂性不断增加，包括具有挑战性的G蛋白偶联受体（GPCR），这些受体是关键的药物靶点。为了使我们的方法适用于大型动态复合物，并规避当前在灵敏度和分辨率方面的挑战，我们将与我们的工业合作伙伴（沃茨公司）合作。利用目前非商业化的原型仪器（循环HDX-MS）。这与我们的方法进步一起将使我们成为第一个在脂质背景下实现膜蛋白的人，从而成为英国和全球这一快速发展的研究领域的领导者。总的来说，这项奖学金不仅将建立一个新的工具，用于解决在破译膜蛋白功能的动态机制的关键挑战，但它也将使我能够领导这个令人兴奋的和新兴的研究领域，目前在英国代表不足。

The advancement of new analytical tools and methods are at the centre of tackling the biggest challenges in the life sciences. One such unmet challenge is understanding the structural dynamics underpinning function in membrane-embedded proteins. Membrane proteins control what comes in and what goes out of the cell. As a consequence, they constitute the main targets of more than half of known drugs. Despite their critical importance, existing methods often fail to uncover structural information about this important class of biomolecules, thus precluding progresses related to therapeutic intervention and drug discovery. More importantly, we currently lack the tools to capture the dynamics of membrane proteins within their native environment wherein they move and operate. This is primarily due to the complexity of such systems as they are embedded into a heterogeneous layer of lipids, which protect their hydrophobic core of membrane proteins. New tools are therefore urgently needed to unveil the dynamic motions of membrane proteins and allow mechanistic insights important for addressing current and future challenges related to human health and disease.Here, we will built a new method to capture molecular movies of membrane proteins in action. To do this, we will develop time-resolved hydrogen deuterium exchange mass spectrometry (tHDX-MS). HDX-MS is a sensitive analytical tool that can accurately monitor the exchange of hydrogen atoms with the heavier deuterium in solution, thus offering information about protein dynamics. By combining the emerging HDX-MS technology with microfluidic techniques, we will enable snapshots of membrane protein states in times ranging from microseconds to hours and with adjustable resolution. To enable applicability of our strategy within the native lipid environment wherein membrane proteins function, we will utilise the controlled patches of membrane biomimetics, the so called nanodiscs. The nanodisc technology will allow us to fine-tune the lipid composition surrounding membrane proteins and assess the individual effect of specific lipids on membrane protein structure and dynamics. We will demonstrate applicability of our approach on a range of important systems of increasing size and complexity including the challenging G protein-coupled receptors (GPCRs) that are the key drug targets. To make our approach amenable to large and dynamic complexes and circumvent current challenges with respect to sensitivity and resolution, we will work with our industrial partner (Waters Corp.) to utilise a currently non-commercial, prototypical instrumentation (Cyclic HDX-MS). This together with our methodological advancements will allow us to be the first to achieve this for membrane proteins in lipid context and thus become the leaders in this rapidly evolving field of research in the UK and worldwide. Overall, this fellowship will not only establish a new tool for tackling key challenges in deciphering the dynamic mechanisms underpinning membrane protein function but it will also allow me to lead this exciting and emerging field of research, currently under-represented in the UK.