Atomic-level visualization of signalling protein activity in living mammalian cells using In-Cell NMR spectroscopy

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Every living cell contains genetic information encoding a set of instructions to produce their defining component proteins. Generated from a set of just 20 amino acids, cellular proteins come in many shapes (or folds') and sizes. The distinct 3D shape of a protein, and even singular amino acids exposed on the surface of a given protein, bestow specific capabilities that allow proteins to govern all cellular process, including growth, differentiation, metabolism, motility, homeostasis and transport. Of course, proteins do not act independently but instead as part of a vast network of constantly changing, integrated cogs inside of cells. Thus, to understand how cells move or grow, we must understand how proteins behave while part of their normal network. This is an extremely challenging endeavor, as we lack the technology to observe proteins at an atomic level while they reside in cells. Researchers have therefore, by necessity, studied individual proteins in detail by isolating them from their native environment into pure buffers or even water. While this has provided tremendous insight into how proteins function at an atomic level and even their relationships within networks, a major task for the future is to acquire these same data inside a living cell. My research team uses an advanced technique called NMR spectroscopy to observe' the atomic 3D shape and dynamic properties of proteins. This approach requires proteins to be labelled with isotopes such as 15-Nitrogen. While this labelling has no effect on a protein's function, it provides us a massive advantage in that anything we add to our protein that is unlabelled remains invisible' by NMR. Thus, we can observe how proteins behave in complex mixtures i.e. with other proteins, metabolites, drugs, or even entire, broken up cells. This has provided exciting new data detailing how essential cellular proteins function, and we here propose to take this one step further and begin to study our proteins inside of living cells. This technique is called In-Cell NMR spectroscopy, or IC-NMR, and offers tremendous potential to truly understand how proteins integrate and control cell fate as components of their natural networks. We are improving our ability to get labelled proteins into live cells and conduct NMR experiments, and now propose to observe the activity of some very key cellular proteins called GTPases and kinases. These types of data are crucial to advance our understanding of cellular proteins, networks, and ultimately the biology of cells.