ORCID

https://orcid.org/0000-0001-8227-6892

Date of Award

9-12-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Molecular Biophysics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Disordered protein regions play crucial roles in various cellular functions, exhibiting high heterogeneity and sampling an ensemble of conformations distinct from folded domains. However, our understanding of their behavior and contributions to protein-protein and protein-nucleic acid interactions remains limited. This dissertation focuses on investigating the interactions between disordered regions and RNA, as well as folded regions of proteins, utilizing computational modeling and single-molecule fluorescence spectroscopy. The SARS-CoV-2 Nucleocapsid (N) protein serves as a model system to address broader questions concerning disordered protein behavior and N protein-mediated RNA genome packaging. I employed coarse-grained molecular dynamic simulations to characterize the cooperative binding of the first two domains of the N protein (NTD-RBD) to RNA. These simulations align with results from single-molecule experiments and offer insights into the sequence-specific contributions to binding. Notably, the simulations confirm that the disordered N-terminal domain enhances binding by approximately 50-fold through a transient and highly heterogeneous bound state, rather than by acquiring a three-dimensional structure. Interestingly, while the folded RBDs of coronaviruses exhibit structural conservation, the disordered NTDs lack sequence conservation. By simulating six orthologous NTD-RBD constructs and conducting NTD-RBD chimeric swaps, this study suggests that complementary interactions between the NTD and RBD facilitate RNA binding, ensuring functional conservation despite variations in both RBD surface residues and NTD sequences. Furthermore, the research demonstrates that adjacent disordered regions to folded RNA binding domains can either enhance or suppress RNA binding depending on the specific sequence of the disordered region. Overall, this work provides valuable insights into the encoding of behavior within disordered regions and their impact on biological function.

Language

English (en)

Chair and Committee

Andrea Soranno

Committee Members

Alex Holehouse

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