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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.

Original publication

DOI

10.1073/pnas.1312739110

Type

Journal article

Journal

Proc Natl Acad Sci U S A

Publication Date

19/11/2013

Volume

110

Pages

E4417 - E4426

Keywords

Coulombic interaction, phosphorylation, single-molecule sensor, substrate binding kinetics, Bacterial Toxins, Hemolysin Proteins, Kinetics, Peptides, Protein Binding, Protein Engineering, Proteolysis, Proto-Oncogene Proteins c-pim-1, Signal Transduction, Static Electricity, Stochastic Processes