title: Nanopore analysis of wild-type and mutant prion protein (PrP(C)): single molecule discrimination and PrP(C) kinetics.
abstract: Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP(C)) in the central nervous system into the infectious isoform (PrP(Sc)). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP(Sc). A number of pathogenic PrP(C) mutants exist that are characterized by an increased propensity for conversion into PrP(Sc) and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP(C) conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP(C), (a PrP(C) mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP(C) capture event. Moreover, we present a four-state model to describe wild-type PrP(C) kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP(C). These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.
authors: Department of Physics and Astronomy, University of British Columbia, Vancouver, British Columbia, Canada. nahid@phas.ubc.ca
authors: Nahid N
authors: NN
authors: Jetha
authors: Valentyna
authors: V
authors: Semenchenko
authors: David S
authors: DS
authors: Wishart
authors: Neil R
authors: NR
authors: Cashman
authors: Andre
authors: A
authors: Marziali
mesh_terms: D007700:Kinetics; D009154:Mutation; D058608:Nanopores; D017096:Prion Diseases / Q000378:metabolism*; D011328:Prions / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation
chemical_list: D011328:Prions
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