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Date of Award

Spring 2024

Document Type

Masters Thesis

Department or Program Affiliation

Chemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

McCarty, Jay

Second Advisor

Amacher, Jeanine

Third Advisor

Antos, John M.

Abstract

In vitro experiments are critical to understanding the biochemistry of molecular systems but it can be a challenge to obtain atomistic resolution. Computational chemistry gives insight to these results with molecular dynamics (MD) simulations using Newton’s equations of motion to analyze the motion of atoms and molecules. However, due to insufficient sampling and long-lived metastable states separated by high energy intermediates, enhanced sampling methods must be applied. Metadynamics (MetaD) enhances the exploration of MD by adding time-dependent bias. Here, we applied MetaD to two enzymes to study their substrate binding/unbinding pathways with their respective substrates. Yeast cytosine deaminase (yCD) deaminates cytosine to uracil and is a potential enzyme-prodrug cancer treatment, converting prodrug 5-flurocytosine (5-FC) to the anti-cancer drug 5-flurouracil (5-FU). However, product release is the rate-limiting step, hindering its drug effectiveness. MD simulations with a volume-based metadynamics approach were used to compare the (un)binding pathways of uracil and 5-FU from yCD, while infrequent metadynamics simulations were performed for their kinetics. The binding free energies showed similar thermodynamics and kinetics showed 5-FU binding tighter to yCD. MD simulations also helped explain in vitro substrate-enzyme interactions of the class B sortase from Bacillus anthracis (baSrtB) for our protein engineering studies. Finally, we successfully applied funnel metadynamics to predict peptide binding conformations comparable to the peptide-bound X-ray crystallography structure of the Streptococcus pyogenes class A sortase (spySrtA). As a result, we used it to sample binding conformation of peptides with other sortase A/B enzymes that have yet to be structurally characterized; baSrtB and sortase A from Staphylococcus aureus (saSrtA).

Type

Text

Keywords

Computational Chemistry, Molecular Dynamics, Metadynamics, GROMACS, PLUMED, Yeast Cytosine Deaminase, Sortases, Enzymes

Publisher

Western Washington University

OCLC Number

1439049076

Subject – LCSH

Computational chemistry--Research; Molecular dynamics--Simulation methods; Sampling; Enzymes--Analysis; Enzymes--Mechanism of action

Format

application/pdf

Genre/Form

masters theses

Language

English

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

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