title: Developing Novel Methods for High-Field Electron Paramagnetic Resonance Measurements of Biomolecules
Creators: name: Judd, Martyna
Publisher: name: The Australian National University
Year: 2025

description: The scope of Electron Paramagnetic Resonance (EPR) spectroscopy has expanded dramatically in recent decades as a powerful tool for biomolecular structure determination. Advancements in high-frequency microwave instrumentation have motivated the exploration of novel measurement methods, probehead design, and biomolecular spin technologies, which take advantage of the increased sensitivity and resolution that comes with detecting unpaired electrons at high magnetic fields. High-field EPR provides a promising avenue for visualising molecular events in biological cells and addressing key challenges of modern drug discovery. This thesis aims to contribute to ongoing high-field EPR developments and their application to structural biology. The thesis is divided into two parts. Part I focuses on the Electron-Nuclear Double Resonance (ENDOR) experiment at 94 GHz to measure 5-30 A distances between an electron and nuclear spin. This range is notoriously difficult to capture using standard techniques, but can be accessed by ENDOR through exploiting the spectroscopic advantages of 19F and the S = 7/2 paramagnetic Gd3+ ion at 94 GHz. Chapter 3 shows that 19F ENDOR can resolve distances and conformational dynamics in proteins tagged with Gd3+ and 19F spin labels for dipolar interactions up to 16 A. The 19F ENDOR detection limit is pushed to &gt;20 A by using the distance dependence of the ENDOR intensity, overcoming the chase for linewidth resolution. Systematic 19F ENDOR measurements of molecular rulers with well-defined 19F-Gd3+ distances validate the integrated-intensity approach to reliably access distances up to 30 A in spin-labelled protein. This approach is thoroughly benchmarked by lineshape modelling and statistical error analysis. Lastly, by using a novel, complementary set of luorinated aromatic amino acids, the orientation of buried side chains can be accurately constrained in metalloproteins with rigid Gd3+ sites. In proteins labelled with flexible Gd3+ tags, 19F ENDOR lineshape analysis combined with rotamer simulations provides information on the conformational distribution of trifluoromethyl-labelled side chains. In Chapter 4 this approach is expanded to other magnetically active nuclei. 31P Mims ENDOR is used in tandem with EXAFS measurements to validate the binding mode of luminescent lanthanide complexes to different polyphosphates, to understand and optimize these complexes in biological sensing and cell imaging applications. ENDOR provides important insight into binding mode heterogeneity which cannot be easily obtained by other techniques. Part 2 pivots to the development of new resonators for EPR measurements at Q-band (34 GHz) which bridge the requirements of high-sensitivity, narrowband detection for CW EPR, with the need for efficient off-resonance excitation in a broadband resonator for pulse EPR experiments. In Chapter 6 the standard 34 GHz TE011 cavity is modified with a dielectric plate covering the coupling iris to increase the resonator bandwidth while retaining sensitivity. Experiments demonstrate the dependence of the resonator bandwidth on the dielectric thickness, coupling short position, and cavity dimensions and show good agreement with finite elements electromagnetic calculations. This modification was implemented into a movable dielectric coupling short, to easily interchange between the original critically coupled CW mode and the dielectrically overcoupled mode. Finally a new 34 GHz TE01d3 dielectric probe is presented, which outperforms both the TE011 cavity and Bruker EN 5107D2 dielectric resonator sensitivities, and overcomes limitations of standard dielectric resonators, i.e. lack of frequency tunability and sample-size restrictions. The TE01d3 resonator is also compatible with the dielectric coupling mechanism. These developments are well-positioned to allow EPR experiments with different resonator requirements, to be measured in a unified, general-purpose set up.