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ORCID

https://orcid.org/0000-0001-6935-2010

Access Type

Open Access Thesis

Document Type

thesis

Degree Program

Molecular & Cellular Biology

Degree Type

Master of Science (M.S.)

Year Degree Awarded

2023

Month Degree Awarded

September

Abstract

p53 is a transcription factor and an important tumor suppressor protein that becomes activated due to DNA damage. Because of its role as a tumor suppressor, mutations in the gene that encodes it are found in over 50% of human cancers. The N-terminal transactivation domain (NTAD) of p53 is intrinsically disordered and modulates the function and interactions of p53 in the cell. Its disordered structure allows it to be controlled closely by post-translation modifications that regulate p53’s ability to bind DNA and interact with regulatory binding partners. p53 is an attractive target for developing cancer therapeutics, but its intrinsically disordered region makes it difficult for traditional experimental techniques to resolve its heterogeneous conformational ensemble. This challenge necessitates the use of techniques that can capture the transient structural features and interactions of p53 to aid in designing effective drugs that can modulate and stabilize its activity. Hybrid-resolution (HyRes) II is a coarse-grained molecular dynamics force field that was parameterized specifically to capture the dynamics of IDPs and can give insight into secondary structure propensity and how post-translational modifications affect the structural ensemble of the protein. Nanopore experiments allow for real-time, single-molecule studies of protein dynamics and interactions with binding partners through characteristic changes in the current that passes through the nanopore. Pairing nanopore experiments with simulations can give insight into the molecular detail of IDP ensembles and interactions, revealing a fuller picture of how p53 is controlled in stressed cell conditions and how its structure is affected due to various modifications and small molecules with therapeutic implications. Herein, we show the HyRes II force field can capture the complex, long-range dynamics of the p53 tetramer and provide molecular-level detail of the p53 autoinhibition mechanism, which is enhanced by the phosphorylation of the NTAD. Secondly, we use the MspA nanopore to capture the differences in events of the wild-type NTAD and a cancer-associated NTAD mutant. Lastly, we detect a small molecule binding to the WT NTAD using nanopore sensing. This approach of integrating MD simulations and nanopore experiments can be applied to the study of other IDPs which are prevalent in biology and integral to human health and disease.

DOI

https://doi.org/10.7275/35904425.0

First Advisor

Jianhan Chen

Second Advisor

Min Chen

Third Advisor

Scott Garman

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