Nanopore Analysis of the Folding of Zinc Fingers

Abstract

In the absence of Zn(II) a single Zn‐finger module such as Zif268 is unfolded and can translocate an α‐hemolysin pore (see image). Upon addition of Zn(II), the protein folds and is too large to translocate so that only bumping events are observed. In contrast, upon addition of Co(II) or Mg(II), there are only small changes to the event profiles.

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.…”

Biological Nanopores for Single‐Molecule Biophysics

“…[45] Upon addition of an equimolar concentration of Zn 2 + , the ratio of translocation events to bumping events decreased. Further work examined the unfolding of an 86-residue protein as it translocated the aHL nanopore, and established the generality of the approach for studying the conformations of peptides and small proteins.…”

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

“…Experiments were conducted with an applied potential of -100 mV at a bandwidth of 10 KHz. [36][37][38][39]43 A perfusion unit made up of a black perfusion chamber containing a white perfusion cup that divided two chambers (cis and trans) by a 150 µm aperture was used for nanopore analysis. A lipid bilayer was formed by applying a synthetic lipid, (1, 2-diphytanoyl-sn-glycero-3-phosphocholine [Avanti Polar Lipids]) on the 150 µm aperture (Fig.…”

“…[33][34][35] Nanopore analysis also allows investigation of structural characteristics of individual peptides or proteins via characterization of their interactions with a biological pore. [36][37][38][39] In nanopore analysis, single molecules are interrogated with the pore to give rise to distinct event parameters, blockade current (I) and blockade time (T).…”

“…It is also performed at low concentrations which mitigates against aggregation. 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.…”