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Date of Award

Spring 2023

Document Type

Masters Thesis

Department or Program Affiliation

College of Science and Engineering

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Amacher, Jeanine

Second Advisor

Antos, John M.

Third Advisor

Smirnov, Sergey L.

Abstract

Gram-positive bacteria attach many proteins to their cell walls via sortase enzymes. Sortases are cysteine transpeptidases and are grouped into 6 classes, A-F. Sortase enzymes, particularly sortase A from Staphylococcus aureus, have been used extensively for in vitro protein ligations. Here, we investigate substrate-binding in sortase A from Streptococcus pyogenes. In addition, class B sortases are typically overlooked for research and development due to low in vitro activity and incomplete knowledge of substrate specificity. Here, we investigate the activity of class B sortases from Bacillus anthracis (baSrtB), Clostridioides difficile (cdSrtB), Listeria monocytogenes (lmSrtB), and Staphylococcus aureus (saSrtB). Of these, baSrtB was the most active in our hands and was selected for further study. Mutant enzymes were created to study the impact of a class B N-terminal α-helix and a structurally conserved, but sequentially variable, loop on baSrtB activity. Mutations to the structurally conserved loop were impactful on enzyme activity, with some mutations decreasing activity while others greatly increased it. A substrate-bound enzyme model generated using Alphafold2 (Galaxy) allowed us to explore enzyme-substrate interactions in greater detail. This model was validated through molecular dynamics simulations and mutagenesis. This work shows that baSrtB is a viable tool for protein engineering studies and lends greater insight into the structural features that underpin sortase activity and selectivity.

Type

Text

Keywords

sortases, enzymes, protein engineering, sortase-mediated ligation, target selectivity, structural biology

Publisher

Western Washington University

OCLC Number

1379210894

Subject – LCSH

Enzymes; Protein engineering; Bacillus anthracis; Peptidase

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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