Nanopore analysis of the interaction of metal ions with prion proteins and peptidesThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

Abstract

Nanopore analysis can be used to study conformational changes in individual peptide or protein molecules. Under an applied voltage there is a change in the event parameters of blockade current or time when a molecule bumps into or translocates through the pore. If a molecule undergoes a conformational change upon binding a ligand or metal ion the event parameters will be altered. The objective of this research was to demonstrate that the conformation of the prion protein (PrP) and prion peptides can be modulated by binding divalent metal ions. Peptides from the octarepeat region (Octa2, (PHGGGWGQ)2 and Octa 4, (PHGGGWGQ)4), residues 106-126 (PrP106-126), and the full-length Bovine recombinant prion (BrecPrP) were studied with an alpha-hemolysin pore. Octa2 readily translocated the pore but significant bumping events occurred on addition of Cu(II) and to a lesser extent Zn(II), demonstrating that complex formation was occurring with concomitant conformational changes. The binding of Cu(II) to Octa4 was more pronounced and at high concentrations only a small proportion of the complex could translocate. Addition of Zn(II) also caused significant changes to the event parameters but Mg(II) and Mn(II) were inert. Addition of Cu(II) to PrP106-126 caused the formation of a very tight complex, which could not translocate the pore. Small changes were observed with Zn(II), but not with Mg(II) or Mn(II). Analysis of BrecPrP showed that about 37% were translocation events, but on addition of Cu(II) or Zn(II) these disappeared and only bumping events were recorded. Suprisingly, addition of Mn(II) caused an increase in translocation events to about 64%. Thus, conformational changes to prions upon binding metal ions are readily observed by nanopore analysis.

Nanopore analysis

“…For example, in the absence of metal ions Zn-finger peptides, prion peptides from the octarepeat region and myelin basic protein will readily translocate; but in the presence of Zn(II) or Cu(II) the peptides fold into a compact structure and only bumping events are observed. [38][39][40] These results highlight the capacity of the pore to interrogate the conformation of the peptide at the instant at which it interacts with the pore.…”

Binding of bovine T194A PrPCby PrPSc-specific antibodies

“…As shown previously, the proportion of Type-I events are about 60%. 37 Upon addition of antibody SN6b there was no significant change in the percentage of type-I events as would be expected if the antibody did not bind to the native protein (Fig. 1B).…”

“…35 At present these possibilities cannot be distinguished. Previously, our group has used nanopore analysis to probe various aspects of PrP C behavior, including metal binding 37 and the interaction with PrP C specific-antibodies. 35 As well, it was demonstrated that the event profiles after pre-incubation in 1.5 M guanidinium hydrochloride (Gdn-HCl) of wild-type bPrP and mutant bPrP(T194A) were different as were those of full length human PrP(23-231) and truncated PrP(90-231).…”

“…19,20,45 Previously it was shown that wild-type bPrP generated about 35%−40% of type-II events. 35,37 This decrease in the number of type-II events in PrP C as compared with T194A may be due to the orientation of the molecule that may only thread into the pore through the three negatively charged amino acids (DYEDRYYR) located on the helix α1. 37 The interaction of the T194A mutant of PrP C with SN6b by nanopore analysis was validated through ELISAs and immunopreciptation of the mutant but not the wild-type proteins expressed in bacteria or mammalian cells.…”

Nanopore analysis reveals differences in structural stability of ovine PrPCproteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

“…38,39 Previously nanopore analysis has allowed detailed investigation of various aspects of the PrP C structure-activity relationship including the interaction of PrP C with metal ions and characterization of the consequences of disease-associated single amino acid mutations on interaction with a therapeutic PrP Sc -antibody. [42][43][44] Accordingly nanopore analysis was a logical approach to investigate genetic polymorphisms of ovine PrP C that are associated with differential susceptibilities to scrapie infection.…”