Date of Award

5-2-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Gregory M. K. Poon

Second Advisor

W. David Wilson

Third Advisor

Ivaylo Ivanov

Fourth Advisor

Samer Gozem

Abstract

Specificity for protein/DNA complexes is well-studied in context of DNA sequence requirements, but the precise mechanisms for how segregation of affinity in consensus-bearing DNA has not been well characterized. Nonspecific sites are dictated by the lack of a consensus site, but high/low affinity binding rise from discrepancies in flanking sequences. Low-affinity DNA has been recently implicated as a general mechanism used by cells to regulate gene expression. Ets-1, the progenitor member of the ETS transcription family, offers a useful model to understand how cognate site discrimination occurs. We then performed molecular dynamics simulations of Ets-1 unbound and bound to nonspecific, low- and high-affinity DNA. These indicated two distinct mechanisms at work- nonspecific complexes exhibited reduced protein/DNA contacts, while specific complexes deviated principally at three residues (Q336, E343, R378). In conjunction with binding assays and point mutations, an allosteric mechanism was determined by which Q336 detects low-affinity DNA to trigger the loss of a salt bridge (E343-R378) to expose the hydrophobic core of the protein. Desensitization by mutation of Q336 rendered Ets-1 unable to readily distinguish between high/low-affinity sequences, suggesting a direct response by the protein to subtle variations in the DNA structure. To further probe the atomistic basis for how specificity arises by consequence of shifts in protein dynamics, we sought to parametrize a number of non-canonical bases for use in molecular dynamics in AMBER. However, there is neither a unified scheme for partial charge derivation of unnatural bases, nor has there been attempts to find one that is compatible with the original formulism of ff94. Assessing several quantum mechanically derived structures, and another set prepared from the original minimization procedure with ff86, it was found the latter best recaptured the reference values. To account for the lack of non-canonical bases within ff86, a process for calculating the charges was found that remained congruent with the ff86 derivation philosophy. In summary, we have determined a model for how proteins use internal dynamics to differentiate high- and low-affinity cognate sites, and a workflow for deploying unnatural bases with molecular dynamics simulations to probe the atomistic basis of specificity discrimination.

DOI

https://doi.org/10.57709/28901209

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