Electrostatically Enhanced Association of a Pim Kinase Substrate Revealed by Stochastic Detection

Abstract

A wide variety of analytes may be detected stochastically by observing the modulation of ionic current flowing through a single engineered protein pore. Rate and equilibrium constants for the interaction of an analyte with a binding site are obtained without a need for labels, such as fluorescent tags. We engineered alpha-hemolysin pores containing peptide sensor elements within a single trans-side beta-barrel loop. Site-specific proteolysis produced pores bearing peptides tethered by a single N-terminal peptide bond. These pores were used for the single-molecule detection and study of kinase-peptide interactions. Kinetics and affinities of the Pim kinases (Pim-1, Pim-2, Pim-3) for their consensus substrate Pimtide, and of cAMP-dependent protein kinase for an inhibitory peptide, were found to be in good agreement with previously reported values. Distinct current noise behaviors were observed between kinases while bound, which form an additional basis for analyte discrimination. Pim kinases exhibited unusually high association rate constants for their consensus substrate (∼107-108 M−1 s−1), extrapolated to zero applied membrane potential. We found this to be due to electrostatic enhancement, which in vivo may constitute an additional layer of control between cell signaling pathways.