Nanopore Analysis of a Small 86‐Residue Protein

Abstract

HPr is a small protein that must unfold to translocate the α‐hemolysin pore (see image). Single amino acid substitutions can cause large changes to the translocation parameters. A conservative mutation is sufficient to alter the event profile; either the mutant must unfold differently or it must interact with the pore differently. A negatively charged mutant is driven through the pore, which facilitates unfolding.

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.…”

“…A single exponential function was used to fit each blockade duration distribution. 36,38,43 Most experiments were done more than once and gave reproducible results. A detailed statistical analysis is not possible, as is frequently the case with nanopore analysis.…”

“…[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.…”

Binding of bovine T194A PrPCby PrPSc-specific antibodies

“…Thus, as previously described for other proteins, such as the 86 amino acid HPr or the 140 amino acid α-synuclein, single amino acid substitutions cause subtle conformational changes which are readily detected by the pore. 40,42 Upon addition of SN6b antibody, however, the proportion of type-II events decreases to 65% demonstrating that some of the native protein has a conformation that exposes the SN6b epitope (Fig. 2B).…”