In-cell Structural Biology using Electron Paramagnetic Resonance Spectroscopy

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The aim of this research is to understand the interactions that occur within the proteome and as a consequence, the interactions required for life. To achieve this, we first need to understand the structures and modes of action of the proteins involved. This project uses electron paramagnetic resonance (EPR) spectroscopy to determine how proteins fit together and in turn how this structure enables their function. EPR allows us to measure the energies of electron spins, and so determine the interactions they have with each other. The interactions change dependent on the distance between spins, and so we can use EPR to infer spin separation. By attaching spins to proteins at specific positions we can determine how proteins physically interact with one another. EPR is best suited to measuring nanometre distances, and so is a tool that is complementary to the more familiar methods of X-ray crystallography, electron microscopy and NMR. To-date most structural studies of proteins using EPR have looked at the proteins outside the cellular environment. This gives us a good indication of the function, but doesn't take into account interactions and changes in protein behaviour as part of a larger, regulated system. There is great scope to use EPR for measurements inside cells and thus obtain structural information in a native environment. The motivation for this research into the biomolecular action of proteins is to determine with quantitative evidence how proteins perform their roles in the cell and how changes to this mode of action can give rise to disease. We will be working with a variety of different cell lines, proteins and spin labels to test the applicability of our methodology as well as the extent to which it can be applied.