Structural and Functional Insights into the F Plasmid Type IV Secretion System proteins TrbI and TrbB

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dc.contributor.advisorAudette, Gerald F.
dc.contributor.authorApostol, Arnold Jan
dc.date.accessioned2024-03-18T17:59:36Z
dc.date.available2024-03-18T17:59:36Z
dc.date.issued2024-03-16
dc.date.updated2024-03-16T10:48:39Z
dc.degree.disciplineChemistry
dc.degree.levelMaster's
dc.degree.nameMSc - Master of Science
dc.description.abstractBacteria have evolved elaborate mechanisms to thrive in stressful environments. One mechanism that bacteria utilize are secretion systems that can traverse protective lipid cell membranes and serve as mediators for a diverse set of goals, including the secretion of toxins implicated with target host pathogenesis. F-like plasmids in gram-negative bacteria encode for the multi-protein Type IV Secretion System (T4SSF) that is functional for bacterial proliferation and adaptation through the process of conjugation. The periplasmic protein TrbB is believed to have a stabilizing chaperone role in the T4SSF assembly, with TrbB exhibiting disulfide isomerase (DI) activity. In the current report, we demonstrate that residues W57-K181, which include the active thioredoxin motif, are sufficient for DI activity. Moreover, a structural model of GST-TrbBWT based on ColabFold-AlphaFold2 and Small Angle X-Ray Scattering data indicate that TrbBWT’s N-terminus is disordered, and this disordered nature likely contributes to the protein’s dynamicity and recalcitrance to crystallization. A truncation construct, TrbB57-181, was designed and found to exhibit higher physicochemical stability using 1H-15N Heteronuclear Single Quantum Correlation spectroscopy and Circular Dichroism spectroscopy. Binding studies of TrbB and other T4SSF proteins TrbI and TraW were performed, and results do not support the inference of a stable complex forming in vitro. Comparative studies of TrbB, TraF, and TrbI also provide insights into the structure of these T4SSF component proteins. Lastly, crystallization trials of GST-TrbBWT and GST-TrbI provide leads for future crystallization campaigns.
dc.identifier.urihttps://hdl.handle.net/10315/41882
dc.languageen
dc.rightsAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.
dc.subjectBiochemistry
dc.subjectMicrobiology
dc.subjectMolecular biology
dc.subject.keywordsF-like type IV secretion system
dc.subject.keywordsDisulfide isomerase
dc.subject.keywordsSmall angle X-ray scattering
dc.subject.keywordsCircular dichroism spectroscopy
dc.subject.keywords1H-15N Heteronuclear single quantum coherence spectroscopy
dc.subject.keywordsColabFold-AlphaFold2
dc.subject.keywordsProtein design
dc.titleStructural and Functional Insights into the F Plasmid Type IV Secretion System proteins TrbI and TrbB
dc.typeElectronic Thesis or Dissertation

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