Under pressure : measuring nanometre distance changes in biomolecules

Abstract

Pulse dipolar electron paramagnetic resonance (PD EPR) spectroscopy is a technique with the ability to measure dipole-dipole interactions between unpaired electrons. Through careful analysis, distances between specifically selected protein residues can be extracted, and with it, protein structures and changes can be observed. In this thesis, the utility of two methods of PD EPR, double electron electron resonance (DEER) and relaxation induced dipolar modulation enhancement (RIDME), are tested, and their advantages and disadvantages elucidated, alongside a direct and detailed comparison of the numerous analysis softwares available to process their respective data.

After establishing the experimental methods, a foray into a young technique in the field is introduced. Many conformations within a protein sample are often invisible to spectroscopic measurement due to being sparsely populated. However, by applying high pressures to the proteins, we can reversibly shift populations of conformational equilibria and thereby make these states visible and ready for measurement. Within, the details of building and testing a high pressure system capable of working in combination with DEER spectroscopy are discussed, alongside two research projects centring two different and distinct protein samples that prove high pressure EPR to be an exciting and promising experimental technique.