The first step towards the development of an electrophoretic prion detector
Madampage, Claudia Avis
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In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorLee, Jeremy S.
CommitteeMoore, Stan; Geyer, Ron; James, Michael; Roesler, Bill; Howard, Peter
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