Jasmine Cubuk

ORCID

http://orcid.org/0000-0001-6915-8242

Date of Award

Spring 5-15-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Biochemistry)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The COVID pandemic has affected over 760,000,000 individuals worldwide since late 2019. Understanding how SARS-CoV-2, the virus responsible for the disease, functions at a mechanistic level is essential to develop therapeutics and vaccines. SARS-CoV-2 utilizes four structural proteins that work together during the viral life cycle to ensure the spread of infections: spike (S), envelope (E), membrane (M) and nucleocapsid (N). Though much work has focused on the S protein for the purpose of vaccines, the N protein plays a key function in the viral life cycle as well. Nucleocapsid is responsible for packaging the viral genome and incorporating the viral ribonucleocapsid into the virion. In SARS-CoV-2, the packaged viral genome adopts a more “beads on a string” organization than the helical configuration previously observed in other coronaviruses. Little is known about how N protein controls the packaging of the viral genome. N protein is composed of five domains: a folded RNA binding domain, a folded dimerization domain, and three flanking intrinsically disordered regions that were proposed to modulate interaction with RNA. Despite their potential role in modulating genome compaction, properties of corresponding disordered regions in nucleocapsid proteins from other coronaviruses remains largely understudied. At the beginning of the COVID pandemic, there was no insight on whether predicted disordered regions in SARS-CoV-2 remain disordered in the context of the full-length protein and how they modulated protein-RNA interactions. In my thesis work, I made use of single-molecule confocal fluorescence spectroscopy, and in particular, single-molecule Förster Resonance Energy Transfer (FRET) to close this knowledge

gap and investigate conformations, dynamics, and interactions of the disordered regions within the SARS-CoV-2 nucleocapsid protein. I first determined that the three predicted disordered regions of N protein are disordered in the context of full-length protein. The combination of single-molecule FRET experiments and all-atom Monte Carlo simulations revealed that the monomeric full-length protein is flexible and dynamic. In addition, we observed that the protein undergoes phase separation when mixed with RNA. Having characterized the monomeric form of the protein, I next investigated the protein-protein interactions that lead to dimerization. I found that the dimerization domain is partially disordered and flexible when N protein is monomeric. I further determined the concentration under which dimerization occurs (KD = 11 ± 3 nM) to be in good agreement with previous AUC experiments and found that even in the dimeric state, N protein retains some of the dynamic nature of the monomer. I also quantified that dimer formation does not alter the conformations of the disordered NTD and folded RBD, but causes an expansion of the disordered linker and CTD. These observations were consistent with my previous determination of interactions of the linker and CTD with the dimerization domain. As a next step, I started to investigate the interactions of the N protein with RNA. To understand the role of a disordered region in aiding the recruitment of RNA, I started to investigate whether the NTD enhances the affinity of RNA to the RBD. For this, I focused on using truncations of the NTD-RBD and RBD in isolation. My experiments showed that the presence of the NTD enhances the affinity by over 50-fold compared to the RBD in isolation. Furthermore, when in complex with RNA, the NTD forms a dynamic fuzzy complex, as seen also in coarse-grained simulations. Comparison of single- and double-stranded RNA provided evidence that the NTD-RBD preferentially binds to single-stranded RNA. Finally, I examined how a crowded environment (mimicked by polyethylene glycol molecules) can modify binding properties of the NTD-RBD to RNA and found that the NTD binding is sensitive to the solution environment. Comparison of Omicron and wildtype (Wuhan-Hu-1) variants revealed that significant differences in binding affinity observed in absence of crowding are equalized in presence of the crowders. In conclusion, single-molecule fluorescence spectroscopy has offered a powerful toolbox for investigating protein conformations and interactions of disordered regions. The work has

provided new insights on the molecular interactions encoded in the SARS-CoV-2 N protein and paves the way to quantitative studies of interactions with other binding partners, viral genome RNA, and small molecules.

Language

English (en)

Chair and Committee

Andrea Soranno

Committee Members

Timothy Lohman, Kathleen Hall, Janice Robertson, Yuna Ayala,

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