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Master of Science (MS)
Antos, John M.
Spiegel, P. Clint
Vyvyan, James R.
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.
Sortase, Enzyme, Substrate, Nucleophile, Homolog, Crystallography, Nuclear Magnetic Resonance, Chemoenzymatic, Sorting Motif, Protein Engineering
Western Washington University
Subject – LCSH
Staphylococcal protein A--Research; Streptococcus pneumoniae; Staphylococcal infections--Treatment
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Horvath, Nicholas M., "Progress Towards the Substrate-bound Structure of Streptococcus pneumoniae Sortase A" (2019). WWU Graduate School Collection. 897.