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Date of Award

Summer 2025

Document Type

Masters Thesis

Department or Program Affiliation

Department of Chemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Amacher, Jeanine

Second Advisor

Antos, John M.

Third Advisor

Smirnov, Sergey L.

Abstract

Many different proteins decorate the surface of bacterial cells. A cysteine-transpeptidase, sortase, binds, cleaves, and ligates target proteins to the bacterial surface by acknowledging a specific motif within surface protein precursors. Our previous work showed that a structurally conserved ß7-ß8 loop near the active site of Staphylococcus aureus sortase A (referred to as saSrtA in this document) enzyme plays an important role in activity and specificity, however, its exact contribution to the saSrtA catalytic mechanism is not fully understood.1 saSrtA undergoes an allosteric activation upon calcium binding at the C-terminus of the ß6-ß7 loop.2 We predicted that the conformational shift of the ß7-ß8 loop from a closed to an open conformation, upon allosteric activation by calcium, directly impacts catalytic efficiency. We found an important hydrophobic interaction between Y187 in the ß7-ß8 loop and P94 in an adjacent alpha helix. We predicted that interrupting this interaction by negative-negative charge repulsion would push the loop into an open conformation by introducing the P94D mutation, increasing enzymatic activity. Here we use several saSrtA variants (WT, pentamutant (5M), and P94D) to show that the P94 position strongly influences the ß7-ß8 loop conformation and flexibility and that introducing mutations at P94 elevates the substrate P2 position to an affinity modulator. By heteronuclear solution NMR15N-HSQC experiments, we calculate the Kd for these 3 variants. These constructs were also applied to the development of a protein-based FRET reporter assay. Holistically, this research improves the tools available for sortase-mediated ligation technology.

Type

Text

Keywords

sortase, FRET, protein, structure, activity

Publisher

Western Washington University

OCLC Number

1533965020

Subject – LCSH

Staphylococcus aureus--Analysis; Bacterial proteins--Analysis; Enzymes--Analysis; Enzymes--Structure; Protein engineering--Research; Allosteric regulation

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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