Protein structure and dynamics are essential for understanding the molecular mechanisms and functions of biological macromolecules, especially those involved in viral infection and disease. Viral proteins, such as the nucleocapsid protein (N) and the spike protein (S) of SARS-CoV-2, are potential targets for drug and vaccine development, as they play key roles in viral assembly, genome packaging, host recognition, and entry. However, the structural and conformational diversity and complexity of viral proteins pose significant challenges for their characterization and analysis. Mass spectrometry (MS) is a powerful analytical technique that can provide valuable information on protein structure and dynamics at different levels of resolution and under different conditions. This thesis investigates the relationship between protein primary sequence and conformational dynamics with mass spectrometry, focusing on four research projects that cover different aspects of protein structure and function. The first project uses variable-temperature ion mobility mass spectrometry (VT-IMMS) to study the conformational transition of polyproline-13 (Pro13), a model peptide that can adopt different secondary structures depending on the solvent and temperature conditions. The second project uses ion mobility mass spectrometry (IM-MS) to compare the conformational landscape of nucleocapsid proteins (NCAPs) from SARS-CoV-2 and influenza A viruses. The third project describes a novel method for mapping disulfide bonds in proteins using non-specific proteolysis via HDX-MS. The method is applied to map the disulfide bonds in the spike protein of SARS-CoV-2 delta variant, which is responsible for viral entry and host recognition. The fourth project applied native mass spectrometry (native-MS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural flexibility and RNA binding of SARS-CoV-2 nucleocapsid protein (Ncap). The thesis concludes with a summary of the main findings and contributions of each project, as well as a discussion of the limitations and future directions. The thesis demonstrates how mass spectrometry techniques can be used to investigate the relationship between protein primary sequence and conformational dynamics. The thesis hopes to inspire further research in this field and contribute to the advancement of structural biology and proteomics.
Date of Award | 1 Aug 2024 |
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Original language | English |
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Awarding Institution | - The University of Manchester
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Supervisor | Perdita Barran (Supervisor) & Bruno Bellina (Supervisor) |
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- Polyproline 13
- SARS-CoV-2 Spike
- SARS-CoV-2 nucleocapsid protein
- Proteomics
- IMMS
- HDX-MS
- TWIMS
- Native MS
Investigating the Relationship between Protein Primary Sequence and Conformational Dynamics with Mass Spectrometry
Dai, J. (Author). 1 Aug 2024
Student thesis: Phd