Cholera toxin (CT) is a classic A-B type protein toxin that has an A subunit (A1 + A2) and a pentameric B subunit. The catalytic A1 domain is linked to the A2 domain via a disulfide linkage. CTA1 must be dissociated from the rest of the toxin to cause a cytopathic effect. Protein disulfide isomerase (PDI) can reduce the CTA1/CTA2 disulfide bond, but disassembly of the reduced toxin requires the partial unfolding of PDI that occurs when it binds to CTA1. This unfolding event allows PDI to push CTA1 away from the rest of the toxin.
My research question is whether the efficiency of PDI in disassembling CT would be affected by molecular crowding, where a dense internal cell environment is recreated in vitro by the use of chemical agents such as Ficoll. This will give insight on how CT behaves inside a cell. Our hypothesis was that molecular crowding would make CTA1 disassembly more efficient by recreating the tight packing of macromolecules in cells, which provides an extra nudge to enhance toxin disassembly. We then used enzyme-linked immunosorbent assays (ELISAs), a pull-down assay and a biochemical assay to determine how molecular crowders affect the binding, reduction, and disassembly of CT by PDI. Our results will bring about a deeper understanding of the cellular events that may affect the course of a cholera infection.
From the preliminary results, molecular crowders increased PDI's ability to bind to CTA1 and did not prevent PDI from cleaving the CTA1/CTA2 disulfide bond. Based off the disassembly results, molecular crowders reduced PDI's ability to displace CTA1 from the rest of the toxin. This contradicts our original hypothesis. Our new hypothesis is that crowders block PDI unfolding, which is required for CT disassembly. Biophysical experiments using Fourier Transform Infrared Spectroscopy will test this prediction in future work.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:honorstheses-2525 |
| Date | 01 January 2023 |
| Creators | Shah, Niral |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Honors Undergraduate Theses |
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