Mapping membrane protein dynamics in time and space with mass spectrometry

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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.