Native mass spectrometry (nMS) and ion mobility mass spectrometry (IM-MS) are well suited for investigating biomacromolecular complexes, and their application is particularly valuable to complexes which are challenging to study with traditional structural techniques. IM-MS and nMS can be used to investigate structures, dynamics and binding stoichiometries of molecules and their complexes. In this thesis, nMS and IM-MS were employed to study a wide range of complexes involving nucleic acids. nMS methods were developed for the screening and characterisation of new and existing Strathclyde minor groove binders (S-MGBs). They were used to determine the binding stoichiometries of S-MGBs in complex with DNA and have become an established part of S-MGB analysis. nMS experiments also provided insight into the binding mechanism of S-MGBs. Collision induced dissociation was employed to measure complex stability in the gas phase, which was then compared to solution phase stability data to investigate complementarity.
MS methods were used to investigate the disordered nucleocapsid protein (N-protein) of SARS-CoV-2 and study its binding interactions with RNA. nMS revealed high levels of disorder in both monomer and dimer N-protein and showed RNA binding to both
monomers and dimers. IM-MS reported conformational changes in N-protein upon RNA binding, as well as under changing salt concentrations, which provided insights into the behaviour of N-protein during liquid-liquid phase separation (LLPS).
Finally, the complexes formed by Cre DNA recombinase were probed, with nMS revealing the stoichiometries of Cre’s complexes with DNA. nMS was also able to report on the activity of Cre through detection of recombined DNA. These results enabled the development of an MS workflow for characterising DNA recombinase enzymes, including their binding complexes and recombination activity.
The work conducted for this thesis shows the versatility and wide applicability of mass spectrometry to study nucleic acid and protein systems, with results contributing to the understanding of viral replication and LLPS mechanisms.
| Date of Award | 14 Apr 2025 |
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| Original language | English |
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| Awarding Institution | - University Of Strathclyde
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| Sponsors | EPSRC (Engineering and Physical Sciences Research Council) |
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| Supervisor | Rebecca Beveridge (Supervisor) & Glenn Burley (Supervisor) |
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