Date of Graduation

7-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Joshua Sakon

Committee Member

Paul Adams

Second Committee Member

Francis Millet

Third Committee Member

Roger Koeppe

Fourth Committee Member

Mack Ivey

Keywords

Bacterial Collagenase, Collagen, Hathewaya histolytica, Histidine Kinase, PKD domains

Abstract

In this research the mechanisms by which Hathewaya (Clostridia) histolytica collagenases are secreted and work together to degrade collagens are investigated. While H. histolytica collagenases Col G and Col H have similar multi-domain structures the difference in number of and orientation of the domains hint that the two target different regions in collagen. Study small angle x-ray scattering (SAXS) was used to give a low-resolution envelope of full-length Col G and Col H and Col G/Col H non-catalytic domains bound to a collagen-like peptide (mini-collagen). SAXS derived envelopes along with structural information was used to tease out the mechanisms by which Col G and Col H degrade collagen. Structures of collagenase:mini-collagen complexes along with acid solubilized collagen hydrolysis studies was used to hypothesis a mechanism of synergy between Col G and Col H. Using all information from this research Col G to Col H ratio of 3:1 was proposed for optimal isolation pancreatic islets.

For V. alginolyticus, the VarS/VarA two-component systems is responsible for regulating the expression of collagenase. All two-component systems consist of a extracellular sensing domain, a catalytic and ATP binding (CA) domain, a dimerization and histidine phosphor-transfer (dHp) domain and a response regulator. For REC domain, itasser homology model and limited proteolysis revealed a two-domain structure connected by a flexible linker. Based on B-factor of linker truncation may be necessary for crystallization of the domain. In this study we initially sought to gain structural information on VarS, a sensor histidine kinase from VarS/VarA two-component system in V. aglinolyticus. It was discovered that the protein forms soluble aggregates in the absence of reducing agent. Trypsin digestion coupled with mass spectrometry revealed the existence of Cys883/Cys892 disulfide bridge. Homology modeling and DALI server was used to show that Cys892 is fairly conserved in HPT domains across species. qRT-PCR showed that in the presence of menadione VarS induced transcription of sRNA2 was slightly downregulated. This study speculates that Cys883/Cys892 disulfide bond stops collagenase expression in the presence of oxidative stress allowing V. alginoltyicus to turn on necessary repair pathways.

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