Advances in electron paramagnetic resonance through synthetic chemistry
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Date
10/06/2024Author
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Grant ID
EP/L015110/1
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Electron paramagnetic resonance spectroscopy (EPR) is one of the most powerful and versatile tools for investigating the local structure and dynamics of paramagnetic substances. In this work, dipolar spectroscopy (PDS) and hyper fine spectroscopy (HFS) are used to investigate the binding and coordination of metal ions such as gadolinium (Gd(III)) and copper (Cu(II)) to de novo coiled-coil (Cc) peptides to characterise their local environment, and probe the nature of the binding site. This work highlights the versatility of designer miniature protein frameworks, which give access to unique and functional binding sites.
This flexibility is seen through investigations of the miniature protein Cc MB1-2, presenting the characterisation of a novel oxophilic Cu(II) binding site, within a protein scaffold and a series of Cc's based on modi cation of this binding site. Mutation and translation of this site shows how the EPR parameters of the Cu(II) ion can be tuned depending on the nature of the available amino acids.
High-spin EPR spin labels such as Gd(III) are of interest owing to their stability within cellular environments. However, PDS measurements using Gd(III) are known to be inaccurate at short distances (<4 nm) when excitation of the central transition (CT) often leads to unwanted excitation of flip-flop transitions causing spectral distortions. New PDS techniques are detailed that can provide high-sensitivity, artefact free data in high-spin systems by avoiding the CT at W-band. RIDME measurements show when measuring short-distances, RIDME is preferred over DEER and provides higher quality data. Amplitude and phase modulated chirp pulses are also compared against standard rectangular pulses in a variety of PDS experiments. Finally, translation of a double Gd(III) binding Cc with known distances, shows promise as a ruler system for PDS measurements, moving away from purely synthetic model rulers whose flexibility do not mirror those of native biological systems.
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Thesis, PhD Doctor of Philosophy
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Embargo Date: 2026-05-14
Embargo Reason: Thesis restricted in accordance with University regulations. Restricted until 14 May 2026
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Data underpinning Michael James Taylor's thesis Taylor, M. J. (Creator), University of St Andrews, 14 May 2026. DOI: https://doi.org/10.17630/48cec54e-ef27-40d6-b5bf-03ca102c85b2Shah, A., Taylor, M. J., Molinaro, G., Anbu, S., Verdu, M., Jennings, L., Mikulska, I., Diaz-Moreno, S., El Mkami, H., Smith, G. M., Britton, M. M., Lovett, J. E., & Peacock, A. F. A. (2023). Design of the elusive proteinaceous oxygen donor copper site suggests a promising future for copper for MRI contrast agents. Proceedings of the National Academy of Sciences of the United States of America, 120(27), Article e2219036120. https://doi.org/10.1073/pnas.2219036120
Starck, M., Fradgley, J., De Rosa, D. F., Batsanov, A., Papa, M., Taylor, M., Lovett, J., Lutter, J., Allen, M., & Parker, D. (2021). Versatile para-substituted pyridine lanthanide coordination complexes allow late stage tailoring of complex function. Chemistry - A European Journal, Early View. Advance online publication. https://doi.org/10.1002/chem.202103243
Keeley, J., Choudhury, T., Galazzo, L., Bordignon, E., Feintuch, A., Goldfarb, D., Russell, H., Taylor, M. J., Lovett, J. E., Eggeling, A., Fabregas Ibanez, L., Keller, K., Yulikov, M., Jeschke, G., & Kuprov, I. (2022). Neural networks in pulsed dipolar spectroscopy: a practical guide. Journal of Magnetic Resonance, 338, Article 107186. https://doi.org/10.1016/j.jmr.2022.107186
El Mkami, H., Hunter, R. I., Cruickshank, P. A. S., Taylor, M. J., Lovett, J. E., Feintuch, A., Qi, M., Godt, A., & Smith, G. M. (2020). High-sensitivity Gd3+-Gd3+ EPR distance measurements that eliminate artefacts seen at short distances. Magnetic Resonance, 1(2), 301-313. https://doi.org/10.5194/mr-1-301-2020
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