The vast majority of theses in this collection are open access and freely available. There are a small number of theses that have access restricted to the WWU campus. For off-campus access to a thesis labeled "Campus Only Access," please log in here with your WWU universal ID, or talk to your librarian about requesting the restricted thesis through interlibrary loan.
Date Permissions Signed
Date of Award
Master of Science (MS)
Antos, John M.
Spiegel, P. Clint
Vyvyan, James R.
Bacterial sortases have been widely studied for their usefulness in protein modification, however, the variable substrate specificity and activity between homologs of these enzymes is not yet fully characterized. To attempt to further understand sorting signal recognition, we have made advances towards a substrate bound structure of Streptococcus pneumoniae sortase A (SrtApneu). This enzyme displays a wide tolerance for alternate amino acids within the canonical LPXTG sorting motif. Our strategy involves a non-cleavable peptide analog that can be docked into the active site, allowing for elucidation of a structure displaying the key contacts that allow the enzyme to recognize alternate sorting signals. To this end, ketomethylene-linked isosteres were designed and synthesized, one of which was incorporated into a peptide via solid phase synthesis to produce a non-cleavable sorting signal for SrtApneu. Preliminary analysis of the substrate analog LPAG(keto)G for inhibition of SrtApneu activity in a model transpeptidation reaction suggested that this peptide was an effective inhibitor. Work towards understanding the activity of SrtApneu in relation to its oligomeric state was also undertaken, revealing a strong relationship between the extent of oligomerization and relative activity of SrtApneu, where extensive oligomerization resulted in minimally active samples. Purification of SrtApneu samples was optimized to produce pure monomeric samples of the enzyme, which showed improved transpeptidation activity. This work has helped lay the foundation for future efforts in producing a substrate-bound structure of SrtApneu.
Streptococcus pneumoniae--Research, Enzymes--Synthesis--Research
Western Washington University
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 thesis for commercial purposes, or for financial gain, shall not be allowed without the author' written permission
Banks, Orion, "Towards the Substrate-bound Structure of Streptococcus pneumoniae Sortase A" (2017). WWU Graduate School Collection. 609.