The Application of Pulsed Electron Paramagnetic Resonance to the Investigation of Protein Structure
Pulsed electron-electron double resonance (PELDOR) is a pulsed electron paramagnetic resonance (pulsed EPR) technique used for measuring distances and on rare occasions orientations within biomolecular structures. Site directed spin labelling (SDSL) has provided a method for introducing EPR active species into protein structures, the majority of these species are too flexible for the use of orientation measurements unless their introduced into sites that restrict the motion of the label. Additionally the quality of information gathered by PELDOR is dependent on the signals strength and persistence, with deuteration of both the underlying protein structure and surrounding media found to increase both the signal intensity and persistence. Within this thesis the current use of PELDOR for investigating disordered areas of protein structure along with the orientation of homodimeric domains has been demonstrated, followed by investigations into determining the extent of the increase in distance available upon deuteration of the protein backbone and the possibility of using a bifunctional spin label for making orientation measurements within protein structures. Within this thesis chapters 1 and 2 give an introduction into the theory behind EPR. Chapter 1 introduces a brief theoretical background behind EPR and chapter 2 introduces the use of EPR for protein structure determination. Chapter 3 outlines the methods used to produce the data in chapters 4 to 7, and chapter 8 gives the overall conclusions from the thesis. The projects presented here introduce some of the current applications of EPR into structural biology (chapters 4 and 5) along with advancements in sample preparation that allow a greater quantity of data to be collected (chapters 6 and 7).
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