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.

Pim Kinase Inhibitors Evaluated with a Single‐Molecule Engineered Nanopore Sensor

“…Then ew heteroheptameric,p seudo-substrate-functionalized pore,( aHL-D127N-PSLM-TEV-D8) 1 (aHL-D127N) 6 (hereafter aHL-D127N-PSLM-TEV), exhibited similar twostate gating behavior to the previously described aHL-D127N-PLM-TEV [7] when inserted into planar lipid bilayers (Figure 1c-g).…”

“…[6] We have previously developed an ovel engineering strategy for producing heptameric alpha-hemolysin (aHL) pores containing as ingle subunit bearing ap eptide sensor element fused to the trans mouth loop. [7] Site-specific proteolysis liberates one end of the sensor-element so that it is attached by as ingle peptide bond ( Figure 1a). Analyte binding is readily observed by monitoring the modulation of ionic current flow through as ingle pore in an artificial membrane under an applied potential.…”

“…In the present work, we applied this strategy to engineer ap seudosubstrate analogue of the Pim consensus substrate sequence "Pimtide" as the sensor element (Figure 1a,b). [7,8] This allowed us to measure kinase binding to the sensor in the presence of MgATP without subsequent phosphorylation. We discovered synergistic binding to Pim-1 by MgATP and the sensor peptide.I nhibition constants of ATP-competitive inhibitors could be determined by measuring their modulation of the synergistic binding.…”

“…Rarely,m uch longer events were seen, presumably due to contaminating ATPf rom ADP dispro- Figure 1c,with the addition of O 3 ,which corresponds to the open pore where kinase-MgATPbinds to the sensor peptide to form aternary complex. Note:anadditional blocked state B 2 connected to O 3 (not shown) was included to properly account for "noise" spikes [7] during long events corresponding to the ternary complex (SI). The ATPconcentration-dependence of the pseudo-first-order association rate constantsf or formation of the c) binary (k' +2 )a nd d) ternary (k' +3 )c omplex are plotted together with their fitted curves accordingtoEquations (1) .…”

Tuning the Selectivity and Sensitivity of an OmpG Nanopore Sensor by Adjusting Ligand Tether Length

“…Typically, an analyte binds to a high affinity site located on the exterior of the nanopore. The binding site can be (i) a peptide sequence introduced into a region of the protein nanopore, 35-38 or (ii) a covalently attached ligand in the lumen 39 or on the rim of the nanopore. 7, 40, 41 Using this approach, a single nanopore can detect proteins of any size, eliminating the need to search for pores of suitable dimensions.…”