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Date Permissions Signed

8-8-2019

Date of Award

Summer 2019

Document Type

Masters Thesis

Department or Program Affiliation

Biochemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Antos, John M.

Second Advisor

Spiegel, P. Clint

Third Advisor

Vyvyan, James R.

Abstract

Sortases are cysteine transpeptidases found primarily on the cell surface of Gram-positive bacteria. Sortase-mediated ligations have become an attractive option for protein modification chemistry, enabling the synthesis of a wide range of non-natural polypeptide derivatives. Attempts at understanding how these enzymes recognize and bind substrates are integral to furthering their usefulness in protein engineering and, potentially, treatment of bacterial diseases. However, the variable substrate specificity and activity between homologs of these enzymes is not yet fully understood. Of specific interest to us is sortase A from Streptococcus pneumoniae (SrtApneu), as it demonstrates a broad substrate tolerance not observed in other sortase A homologs. Correspondingly, we have made advances towards characterizing a substrate bound structure of SrtApneu in an effort to further understand its unique substrate promiscuity, deviating from the canonical LPXTG sorting signal. Our strategy initially involved generating a non-cleavable peptide analog capable of docking into the active site, however, synthesis of a ketomethylene-linked dipeptide isostere and its insertion into a peptide via solid phase peptide synthesis proved to be more challenging than we anticipated. We revised our approach by designing a substrate harboring an LPACG sorting motif. Peptide preparations with a thiopyridine leaving group favorably facilitated disulfide bridging between the active site and sorting motif cysteines, allowing for elucidation of a SrtApneu structure displaying key interactions that allow the enzyme to recognize a wide-variety of substrates. To this end, we have utilized x-ray crystallography and solution NMR in an attempt to characterize SrtApneu with a bound substrate analog. Although we were unsuccessful, this work has established a foundation for future efforts toward determining the substrate-bound structure of SrtApneu.

Type

Text

Keywords

Sortase, Enzyme, Substrate, Nucleophile, Homolog, Crystallography, Nuclear Magnetic Resonance, Chemoenzymatic, Sorting Motif, Protein Engineering

Publisher

Western Washington University

OCLC Number

1112125876

Subject – LCSH

Staphylococcal protein A--Research; Streptococcus pneumoniae; Staphylococcal infections--Treatment

Format

application/pdf

Genre/Form

masters theses

Language

English

Rights

Copying of this thesis 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.

Included in

Chemistry Commons

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