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Expanding the scope of electron paramagnetic resonance spectroscopy for structural studies of proteins and peptides

Date
03/12/2019
Author
Shah, Anokhi
Supervisor
Lovett, Janet Eleanor
Funder
Engineering and Physical Sciences Research Council (EPSRC)
Keywords
Electron paramagnetic resonance
EPR
Mass spectrometry
Spin labelling
New labels
Structural biology
Metals
Copper
Gadolinium
Membrane protein
Protein
Peptide
Distances
DEER
Metadata
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Abstract
Electron paramagnetic resonance spectroscopy is a versatile tool for probing structural information about systems with unpaired electrons, in particular, biological systems with metal centres or chemically attached spin labels. This work uses a variety of electron paramagnetic resonance techniques to investigate the ability to measure distances between two spins (radical and metal), as well as characterise the local environment. Distance measurements are used to probe the mechanism of translocation across a membrane, where a change in distance is observed upon ATP cycling, indicating channel movement of the SecYEG:SecA complex. Furthermore, to expand the scope of distance measurements for more complex, cysteine-rich systems, spin labelling regimes are developed and optimised on the test protein myoglobin. Specifically, next generation maleimide spin labels are demonstrated to label and selectively cleave. Myoglobin is further exploited to successfully introduce the unnatural amino acid, dehydroalanine, for selective and orthogonal labelling. The development of the labelling and measurement strategy for the gadolinium-based spin label, [Gd.sTPATCN]-SL, is also shown, where the narrow central transition of the label allows a long phase memory time and increased DEER modulation depth, to give increased measurement sensitivity. In addition, gadolinium(III) distances are used to characterise the binding site of a peptide system for the application of magnetic resonance imaging. The optimisation of measuring inter-gadolinium(III) distances between 2-5 nm at both Q- and W-band is also demonstrated in the corresponding peptide ruler series. The additional benefit of the peptide to act as a metal ruler is further investigated using copper(II), where hyperfine spectroscopy is utilised to successfully confirm the nature of the binding site as all oxygen binding.
Type
Thesis, PhD Doctor of Philosophy
Rights
Embargo Date: 2023-09-13
Embargo Reason: Thesis restricted in accordance with University regulations. Electronic copy restricted until 13th September 2023.
Collections
  • Physics & Astronomy Theses
Description of related resources
Expanding the scope of EPR for structural studies of proteins and peptides (Thesis data) Shah, A., University of St Andrews. DOI: https://doi.org/10.17630/4bc143fb-ff43-4d1c-8c52-60d2e112a860
Related resources
https://doi.org/10.17630/4bc143fb-ff43-4d1c-8c52-60d2e112a860
URI
http://hdl.handle.net/10023/18488

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