Biological NMR: probing biomolecular condensates, exploration of a disordered virus capsid protein and reducing molecular size limitations

Thesis

Solution-state nuclear magnetic resonance (NMR) spectroscopy is capable of characterising atom-specific structural and dynamical information of biomolecules. NMR excels in studying dynamical and liquid-state systems; however, its use in slowly tumbling biomolecules is restricted, due to signal intensity and resolution limitations. In this thesis, NMR was used to characterise biomolecular condensates and a disordered virus capsid protein. This was followed by an exploration of methods to enhance NMR spectral quality for slowly tumbling biomolecules.

Biomolecular condensates are formed through liquid-liquid phase separation and provide cellular compartmentalisation without a phospholipid membrane. The ability of condensates to partition metabolites was determined using an NMR-based method, which showed trends similar to “like-dissolves-like”, used extensively to predict partitioning into organic solvents. Additionally, NMR revealed a significant change in the dynamical and chemical properties of water inside condensates. These measurements support the growing evidence that biomolecular condensates act as distinct solvents.

Secondly, the properties of an adenovirus capsid protein, N-terminal truncated protein VI (∆54-pVI), were explored using NMR. Adenoviruses can cause common respiratory illnesses and are exploited as viral vectors for vaccination. Protein VI is essential for membrane disruption and endosomal escape into a host cell, crucial for adenoviral infectivity. Diffusion NMR showed the highly disordered nature of ∆54-pVI; however, the assignment of chemical shifts revealed regions with amphipathic α-helical propensity, which is thought to enhance interactions with membranes.

Finally, a review of methodologies to combat size limitations in NMR is provided through simulations. The benefits of ¹³CF₃ labelling on slowly tumbling biomolecules were also assessed. Experimental measurements on a 395 kilodalton protein showed the ¹³CF₃ spin system had favourable relaxation properties. Simulations predicted that the relaxation rate of the slowest relaxing coherence would be largely independent of molecular tumbling. Therefore, ¹³CF₃ labelling schemes could extend the applications of NMR in slower-tumbling biomolecules.

Authors: Eaton, J

• DOI: 10.5287/ora-8j2rz7ybd
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: adenovirus capsid proteins; nuclear magnetic resonance (NMR); phase-separation; biomolecular condensates; disordered proteins; relaxation rates
• Subjects: Biochemistry; Physics; Chemistry, Physical and theoretical