title: Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate.
abstract: In stochastic sensing, the association and dissociation of analyte molecules is observed as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free determination of rate and equilibrium constants with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-molecule detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (~10(7) to 10(8) M(-1) · s(-1)), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.
authors.affiliation: Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom.
authors.forename: Leon
authors.initials: L
authors.lastname: Harrington
authors.forename: Stephen
authors.initials: S
authors.lastname: Cheley
authors.forename: Leila T
authors.initials: LT
authors.lastname: Alexander
authors.forename: Stefan
authors.initials: S
authors.lastname: Knapp
authors.forename: Hagan
authors.initials: H
authors.lastname: Bayley
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D059748:Proteolysis; D051573:Proto-Oncogene Proteins c-pim-1 / Q000302:isolation & purification* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D055672:Static Electricity; D013269:Stochastic Processes
chemical_list: D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D010455:Peptides; C021091:staphylococcal alpha-toxin; D051573:Proto-Oncogene Proteins c-pim-1; C406031:proto-oncogene proteins pim
keywords: Coulombic interaction; phosphorylation; single-molecule sensor; substrate binding kinetics