Stochastic detection of Pim protein kinases revealselectrostatically enhanced association ofa peptide substrateLeon Harringtona, Stephen Cheleya,1, Leila T. Alexanderb, Stefan Knappb, and Hagan Bayleya,2

aDepartment of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom; and bNuffield Department of Clinical Medicine, Structural GenomicsConsortium and Target Discovery Institute, University of Oxford, Oxford OX3 7DQ, United Kingdom

Edited by John Kuriyan, University of California, Berkeley, CA, and approved October 1, 2013 (received for review July 5, 2013)

In stochastic sensing, the association and dissociation of analytemolecules is observed as the modulation of an ionic currentflowing through a single engineered protein pore, enabling thelabel-free determination of rate and equilibrium constants withrespect to a specific binding site. We engineered sensors based onthe staphylococcal α-hemolysin pore to allow the single-moleculedetection and characterization of protein kinase–peptide interac-tions. We enhanced this approach by using site-specific proteolysisto generate pores bearing a single peptide sensor element at-tached by an N-terminal peptide bond to the trans mouth of thepore. Kinetics and affinities for the Pim protein kinases (Pim-1,Pim-2, and Pim-3) and cAMP-dependent protein kinase were mea-sured and found to be independent of membrane potential and ingood agreement with previously reported data. Kinase bindingexhibited a distinct current noise behavior that forms a basis foranalyte discrimination. Finally, we observed unusually high as-sociation rate constants for the interaction of Pim kinases withtheir consensus substrate Pimtide (∼107 to 108 M–1·s–1), the re-sult of electrostatic enhancement, and propose a cellular rolefor this phenomenon.

substrate binding kinetics | single-molecule sensor |Coulombic interaction | phosphorylation

Stochastic sensing is a powerful single-molecule approach forthe detection of a wide range of analytes (1–3). Sensing is

achieved by the modulation of ionic current flowing under anapplied potential through an individual protein pore, such as theheptameric α-hemolysin (αHL) pore, reconstituted in an artifi-cial lipid bilayer. The versatility and specificity of stochasticsensing have been enhanced by introducing sensing elements(analyte binding sites) into the αHL pore by protein engineering,chemical modification, or the use of adapter molecules (1, 4, 5).Through such approaches, the stochastic detection of ions (1, 6),small molecules (1, 7, 8), reactive molecules (9, 10), and protein–ligand interactions has been achieved (1, 11–14). The stochasticdetection of proteins has also been achieved with functionalizedsolid-state nanopores (15). Polymer molecules passing througha protein pore can be detected by ionic current modulation(1, 16–19), which has provided a basis for polynucleotide se-quencing (20–25). Further, this approach can be applied to theanalysis of heterogeneous populations of nucleic acids for de-termination of purity, phosphorylation state, and chemical in-tegrity (26). The threading of polynucleotides has also been usedto study DNA–protein complexes (27). Finally, recent studies ofprotein translocation suggest that nanopore proteomics may bepossible (19, 28).Efforts have been made to apply stochastic sensing to protein

kinases (11, 14). Protein kinases comprise one of the largest genefamilies in eukaryotes, with 518 kinase domains identified in thehuman genome (29). As most cellular and physiological pro-cesses are mediated or modulated by protein kinases, aberrantkinase activity can have transformative effects, leading, for ex-ample, to cancers. Protein kinases are hence under intense in-

vestigation as therapeutic targets. The stochastic sensing ofprotein kinases might, in addition to allowing the study of thefundamental kinetics of substrate interactions or the detection ofkinases in different activation states, be extended to the screeningof inhibitors. The label-free, single-molecule nature of the tech-nique provides potential advantages over existing methods thatlargely rely on radiometric or fluorescence detection.Sensors for the catalytic subunit of cAMP-dependent protein

kinase (PKA) were previously engineered either by chemicallyattaching to the trans mouth of the αHL pore a peptide com-prising residues 5–24 of the heat-stable protein inhibitor of PKA(14) or by genetically fusing this sequence within a single transloop of the β-barrel domain (11). However, chemical modifica-tion requires time-consuming synthesis and does not achieve fullmodification (in the case cited: ∼70%). There again, fusion ofthe sensor element within the trans loop conformationally con-strains the peptide, which may alter binding kinetics in contrastwith a linear peptide (11).In the present work, we aimed to (i) design a stochastic sensor

for the Pim family of protein kinases (30) and (ii) investigatealternative engineering strategies that might circumvent the lim-itations of previous methods. The Pim kinase family comprisesthree serine/threonine protein kinases that are constitutively ac-tive monomers not requiring phosphorylation of the activationloop for activity. They lack regulatory domains and so are regu-lated at the transcriptional and translational level, for example bycytokine signaling through the Janus kinase/signal transducersand activators of transcription (JAK/STAT) pathway (31, 32).They have roles in survival signaling and cell proliferation, andtheir up-regulation can contribute to the progression of leukemias

Significance

The modulation of ionic current flowing through an individualpore can be used for the stochastic sensing of a wide variety ofanalytes. Here we have produced protein pores by insertingpeptide sequences that remain attached in an unconstrainedconformation after limited proteolysis. The engineered poreswere used for the label-free, single-molecule discrimination ofPim protein kinases. Further, we found unusually high, elec-trostatically enhanced, association rates for the Pim kinasesand a consensus substrate, which suggests an additional ele-ment of control between cell-signaling pathways.

Author contributions: L.H., S.C., and H.B. designed research; L.H. and S.C. performed research;L.T.A. and S.K. contributed new reagents/analytic tools; L.H. analyzed data; and L.H. and H.B.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1Present address: Department of Pharmacology, Alberta Diabetes Institute, University ofAlberta, Edmonton, AB, T6G 2E1, Canada.

2To whom correspondence should be addressed. E-mail: hagan.bayley@chem.ox.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1312739110/-/DCSupplemental.

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and tumors (33–37). Pim kinases possess an unusual ATP-bindingpocket, widened by an insertion in the hinge region and thepresence of a proline residue, which lacks one of two typicallyconserved residues that form backbone hydrogen bonds to theadenine ring of ATP (38–42). Their unusual ATP-binding sitesand roles in cancer have prompted the investigation of potentialPim-selective inhibitors for cancer therapy (30, 43).We first constructed a trans loop-constrained, genetically

encoded sensor for Pim kinases, analogous to that previouslymade for the detection of PKA (11). We chose the Pim kinaseconsensus substrate Pimtide (ARKRRRHPS*GPPTA) as thesensor element, which had been identified by a peptide libraryscreen and been shown to bind all three Pim kinases with highaffinity (38, 44). Because Pimtide is a substrate for Pim kinases ratherthan an inhibitory peptide, it also offers the prospect of extendingstochastic sensing to the measurement of catalytic activity.We then enhanced this approach by using site-specific, post-

translational cleavage of the loop to liberate one end of thesensor peptide. We also made an analogous sensor for PKA.These proteolytically cleaved sensors allowed us to study the ki-netics of kinase–substrate interactions independent of membranepotential. We also found differing noise characteristics for eachkinase when bound, which could form a basis for analyte dis-crimination. Further, we found that the association rates of thePim kinase family are electrostatically enhanced, and proposea possible role for this phenomenon in vivo. We hence demon-strated the utility of proteolytically cleaved αHL pores for thestudy of protein kinase substrate interactions.

ResultsInitial Pore Engineering and Optimization. We first sought to engi-neer a genetically encoded sensor for the Pim kinase family by usinga previously described strategy (11). In this approach, the Pimtidesensor element flanked by linker sequences of eight Ser/Gly resi-dues on either side is fused into the transmouth loop of one subunitof a heteroheptameric αHL pore (Fig. 1A). The heteromeric pore(αHL–PLM–D8)1(αHL–WT)6, [hereafter αHL–PLM, where PLM(Pimtide loop mutant) refers to the fused sensor element], wasproduced by in vitro transcription and translation (IVTT) and as-sembly on rabbit red blood cell membranes (Materials and Meth-ods). However, when αHL–PLMwas reconstituted into planar lipidbilayers, no alteration of its electrical behavior was observed uponthe addition of Pim-1 to the trans chamber; a conductance state thatreflected a single open pore continued to be observed at bothpositive and negative applied potentials (Fig. S1A).We hypothesized that the sensor loop may have been trapped

in a conformation that did not favor binding due to electrostaticinteractions between the highly basic Pimtide and the αHL transmouth charged residues Asp127, Asp129, and Lys131. We there-fore screened a number of charge neutralization mutants at thesepositions. One of these, D127N, when present in the heteromericpore (αHL–D127N–PLM–D8)1(αHL–D127N)6 (hereafter αHL–D127N–PLM), enabled a change in the electrical behavior of thepore upon the addition of Pim-1 to the trans chamber (Fig. 1 C andD). In the absence of Pim-1, and unlike αHL–PLM, αHL–D127N–PLM exhibited rapid transitions to a partially closed state of meanduration 0.41 ± 0.03 ms, with a mean interevent interval 7 ± 1 ms(both n= 3; Fig. S1B). The addition of Pim-1 to the trans chambercaused intermittent abolition of these closures, such that two dis-tinct mean interevent intervals were now apparent (Fig. 1D). Inthe presence of 91 nM Pim-1, the mean duration of the closureswas 0.46 ± 0.03 ms, and the mean interevent intervals were 4.7 ±0.2 and 100 ± 10 ms (all values n = 3; Fig. S1C).The two-state gating behavior exhibited by the pore at positive

potentials in the absence of Pim-1 is similar to that seen for otherpores modified with conformationally flexible attachments (11,45, 46), and we ascribe this behavior to transient blockade of thepore by the modified trans loop. When the kinase binds to the

loop, the motion is restrained, and the blockades are abolished.The addition of Pim-1 to the cis compartment did not producea change in pore behavior (Fig. 1E). Current–voltage relation-ships for the open states of (αHL–D127N)7 and αHL–D127N–

PLM show modest changes in rectification with respect to (αHL–WT)7, presumably because of alterations in charge distributionnear the trans mouth arising from the modifications (Fig. 1B).

Site-Specific Proteolytic Cleavage Yields Unconstrained Peptide SensorElements. We subsequently sought to produce pores bearing a sen-sor peptide attached by a single terminus, as these would betterresemble the synthetic, linear peptides often used in other methodsfor measuring kinase substrate interactions (38, 47, 48). This hadpreviously been achieved by forming an αHL pore with one subunitchemically coupled through a cysteine residue at the transmouth toa peptide sensor element by using an S-pyridyl-functionalized tetra(ethylene glycol) linker (14). We sought a similar outcome througha genetically encoded procedure, thus avoiding the need for pep-tide synthesis, functionalization, and subunit modification. Genet-ically encoded sensors would also be amenable to the constructionof combinatorial peptide libraries.

10 pA

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Fig. 1. Design and characterization of an engineered αHL pore for Pim ki-nase detection with a trans loop fusion sensor element. (A) Illustration of theαHL–D127N–PLM pore indicating the position of the Pimtide sensor element(blue) flanked by serine/glycine linkers (green) and genetically fused into thetrans loop of one subunit (orange) of αHL–D127N. (B) Current–voltage rela-tionships for αHL–WT (□) and αHL–D127N (○) homoheptamers and the αHL–D127N–PLM (△) heteroheptamer. Error bars represent SDs (n = 3). Repre-sentative current recordings and dwell-time histograms for the open currentlevel with an applied potential of +50 mV are shown for (C) the αHL–D127N–PLM pore only, (D) the αHL–D127N–PLM pore with 91 nM Pim-1 present in thetrans chamber, and (E) the αHL–D127N–PLMporewith 91 nM Pim-1 present inthe cis chamber. Histograms were fitted with one- or two-component prob-ability density functions. All measurements were performed in 15 mM Mops,pH 6.8, 300 mM KCl, 5 mM DTT, and the filter corner frequency was 2 kHz.

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We introduced into the αHL–D127N–PLM–D8 polypeptidea tobacco etch virus (TEV) protease cleavage site (ENLYFQG) atthe C-terminal end of the Pimtide sequence and preceding the Ser/Gly linker, so that posttranslational, site-specific proteolytic cleav-age would liberate one end of the sensor peptide (Fig. 2 A and B).The cleaved subunit would then be mixed with αHL–D127Nmonomer, oligomerized, and the desired heteroheptamer purifiedby SDS/PAGE (Fig. 2C, Fig. S2, and SI Materials and Methods).Heteroheptamers, (αHL–D127N–PLM–TEV–D8)1(αHL–

D127N)6 (hereafter αHL–D127N–PLM–TEV), were successfullyformed with both cleaved (“cut”) and uncleaved (“uncut”) sensorsubunits (Fig. 2C). Gel-purified, cut αHL–D127N–PLM–TEVwas denatured by heating at 95 °C for 20 min and analyzed bySDS/PAGE alongside nondenatured, cut αHL–D127N–PLM–

TEV (Fig. 2D). In the heated sample, two bands were seen ataround half the apparent molecular mass of the monomer band,consistent with the expected sizes of the cleavage products of the

sensor subunit, confirming that the cleaved sensor subunits hadbeen incorporated into the heteroheptamers.

Single-Channel Characterization of Pores. We then characterizedthe electrical properties of both cut and uncut αHL–D127N–

PLM–TEV. The open state current–voltage relationships wereindistinguishable and both exhibited altered rectification com-pared with αHL-D127N homoheptamer, which may be due to thepresence of additional charged residues near the trans mouth (Fig.2E). In the absence of Pim kinase, uncut αHL–D127N–PLM–TEVdisplayed large amplitude blockades at both +50 and –50 mV (Fig.2F). At +50 mV, the blockade duration histogram was best fit witha two-component probability density function, giving meanblockade durations of 0.49 ± 0.03 and 3.7 ± 0.9 ms (n = 3; Fig.S3A). The mean interevent interval was 32 ± 5 ms (n = 3; Fig.S3A). Analysis at –50 mV was complicated by a large number ofpoorly resolved events, so the mean durations may possess sub-

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Fig. 2. Design and characterization of a TEV protease-cleavable αHL trans loop fusion pore for Pim kinase detection. (A) Illustration of the protease-cleavedform of αHL–D127N–PLM–TEV indicating the positions of the Pimtide sensor element (blue), the serine/glycine linkers (green), and the TEV protease rec-ognition site (“TEV RS,” red). (B) Scheme for the TEV protease-cleavable trans loop fusion in αHL–D127N–PLM–TEV. (C) Preparative SDS/PAGE showinga reference homoheptameric αHL marker (“Ref” lane) and lanes containing mixtures of (αHL–D127N)x(αHL–D127N–PLM–TEV)y where the αHL–D127N–PLM–

TEV subunits were cleaved before assembly (cut) or uncleaved (uncut) by TEV protease. (D) Confirmation of the incorporation of the proteolytically cleavedαHL–D127N–PLM–TEV subunit into (αHL–D127N)6(αHL–D127N–PLM–TEV)1. Gel-purified (αHL–D127N)6(αHL–D127N–PLM–TEV)1 was electrophoresed with(+Δ) and without (–Δ) heating before loading. (E) Current–voltage relationships for the αHL–D127N (○) homoheptamer and the uncut (▽) and cut (◇)forms of the αHL–D127N–PLM–TEV heteroheptamer. Error bars represent SDs (n = 3). Representative current traces for the uncut (F) and cut (G) forms of theαHL–D127N–PLM–TEV pore at applied potentials of ±50 mV. All measurements were performed in 15 mM Mops, pH 6.8, 300 mM KCl, 5 mM DTT, and thefilter corner frequency was 2 kHz.

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stantial systematic error. The blockade duration histogram was bestfit with a two-component probability density function, giving meanblockade durations of 0.49 ± 0.09 and 3 ± 1 ms, and the meaninterevent interval was 100 ± 60 ms (all n = 3; Fig. S3A).Protease-cleaved αHL–D127N–PLM–TEV also exhibited block-

ades in the absence of Pim kinase at both positive and negativeapplied potentials, but with altered kinetics. At +50 mV, the meaninterevent interval was 50 ± 10 ms (n = 3; Fig. S3B). The blockadeduration histogram was best fit with a three-component probabilitydensity function, giving mean durations 0.22 ± 0.05, 2.2 ± 0.9, and18 ± 7 ms (all n = 3; Fig. S3B). At –50 mV, the mean intereventinterval was 1.4 ± 0.2 ms, and the mean blockade duration was 8 ± 2ms (both n = 6; Fig. S3B).We also produced a sensor similar to αHL–D127N–PLM–

TEV but with the TEV protease cleavage site placed at theN-terminal side of the Pimtide sequence. However, whenreconstituted into planar lipid bilayers, the protease-cleaved

pore gave a very noisy current signal without defined con-ductance states and so was not pursued further. From here on,the properties of the protease-cleaved form of αHL–D127N–

PLM–TEV are further discussed.

Single-Molecule Detection and Kinetic Analysis of Pim Kinase Binding.The addition of 91 nM Pim-1 to the trans chamber under anapplied potential of –50 mV altered the αHL–D127N–PLM–

TEV pore’s pattern of blockades (Fig. 3 A and B). Specifically,an additional population of longer events appeared in the du-ration histogram for the open current level (interevent intervals;Fig. 3B). In the presence of 91 nM Pim-1, the mean intereventinterval for the additional population was 100 ± 20 ms (n = 3;Fig. S4A). The mean blockade and interevent interval durationswere unchanged, within error, and were 8 ± 2 and 1.5 ± 0.2 ms,respectively (n = 3; Fig. S4A). We ascribe the long-durationintervals at the open current level to the binding of Pim-1 to the

k'+2 = [Kinase]

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Fig. 3. Stochastic detection of protein kinases using TEV protease-cleaved αHL trans loop fusion pores. Representative current traces of the αHL–D127N–PLM–

TEV (cut) pore under an applied potential of –50 mV before (A) and after (B) the addition of 91 nM Pim-1 to the trans chamber are shown together withrepresentative dwell-time histograms for the open-pore current level. (C) Kinetic model for the observed current signal from proteolytically cleaved sensors inthe presence of a protein kinase. The state B1 corresponds to the blocked current level of the pore, which is assumed to be due to obstruction of the conductivechannel by the peptide attached at the transmouth. State O1 corresponds to the open pore, where the pore is not bound to a kinase, and state O2 correspondsto the open pore, where kinase is bound to the pore through the attached peptide. Note: an additional blocked state B2 connected to O2 was included for Pim-3to account for an additional population of blockades that occur when Pim-3 is bound to the pore (Materials and Methods). Plots of the concentration de-pendence of the pseudo-first-order kinase association rate constants k′

þ2 and first-order kinase dissociation rate constants k–2 are shown for the interaction ofthe αHL–D127N–PLM–TEV (cut) pore with (D) Pim-1, (E) Pim-2, (F) Pim-3, and (G) for the interaction of the αHL–PKIP–TEV (cut) pore with cAMP-dependentprotein kinase (PKA). Error bars represent SDs (Pim kinases: n = 4; PKA: n = 3). Association rate constant plots were fitted to the equation y = mx, where thegradient,m, gave the final value for the biomolecular association rate constant, and dissociation rate constant plots were fitted to the equation y = c, where theintercept, c, gave the final value for the dissociation rate constant (Table 1). Measurements were performed in 15 mMMops, pH 6.8, 300 mM KCl for PKA and in15 mM Mops, pH 6.8, 300 mM KCl, 5 mM DTT for the Pim kinases. The applied potential was –50 mV, and the filter corner frequency was 2 kHz.

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sensor peptide, which prevents the peptide from blocking thepore. Pim-1 that had been denatured by heating did not producea change in pore behavior (Fig. S4B).We studied the kinetics of kinase association and dissociation

with a model in which it is assumed that the kinase binds onlywhen the peptide is not blocking the pore, i.e., from the openstate only (Fig. 3C). We measured the Pim-1 concentration de-pendence of the dissociation rate constant, k–2, and the observedpseudo-first-order association rate constant, k′þ2. k–2 was foundto be independent of Pim-1 concentration, consistent withunimolecular dissociation, and k′þ2 was found to be linearly de-pendent on Pim-1 concentration, consistent with bimolecularassociation of Pim-1. We also applied this analysis to measure-ments of the interactions of Pim-2 and Pim-3 with αHL–D127N–

PLM–TEV (Fig. 3 E and F and Fig. S5 A and B). Pim-3 requireda modified kinetic model due to additional resolved “noise”events in the kinase-bound state (below andMaterials and Methods).From these analyses, we determined the dissociation rate constants,k–2, and bimolecular association rate constants, k+2, and hencethe equilibrium dissociation constants, Kd = k–2/k+2, for all threePim kinase family members (Table 1).We applied the same kinetic analysis to the interaction of Pim-1

with our original loop–fusion sensor αHL–D127N–PLM and de-termined rate constants k+2 = (1.90 ± 0.09) × 108 M–1·s–1 and k–2 =6.8±0.6 s–1 and the equilibriumdissociation constantKd=36±4nM(all mean ± SE). Differences in rate constants were also observedwhen comparing previous chemically modified and loop-constrainedsensors for PKA (11, 14), but these differences and those in thepresent work are too small to be attributed with confidence toconformational constraints.

Stochastic Detection of cAMP-Dependent Protein Kinase. To test thegeneral applicability of the approach, we constructed an analo-gous sensor for the catalytic subunit of cAMP-dependent proteinkinase (PKA) by placing a TEV protease cleavage site afterresidues 5–24 of the heat-stable protein kinase inhibitor (PKIP5–24) (49) in the αHL–PKIP pore used previously for the stochasticdetection of PKA (11). We also introduced a lysine immediatelyafter PKIP5–24 to maintain the net neutral charge of the sensor

peptide (an additional negative charge is introduced by the TEVprotease cleavage site).The TEV–protease cleaved form of (αHL–PKIP–TEV)1(αHL–

WT)6 (hereafter αHL–PKIP–TEV) exhibited similar blockadebehavior at ±50 mV in the absence of PKA compared with thePimtide-based sensor in the absence of Pim kinase, but with al-tered kinetics. This reinforces the conclusion that the blockadesobserved in the absence of kinase arise from motion of the sensorpeptide element and not the vestigial Ser/Gly linker that remainson the adjacent strand after proteolytic cleavage (Fig. S5C). At+50 mV the duration histogram for the blocked current level wasbest fit by a two-component probability density function, givingmean blockade durations of 0.25± 0.07 and 2.8± 0.7ms; themeaninterevent interval was 120 ± 20 ms (all n = 3; Fig. S5C). At –50mV, the mean interevent interval was 4.5 ± 0.3 ms, and the meanblockade duration was 2.5 ± 0.4 ms (both n = 3; Fig. S5C).At –50 mV and in the presence of 128 nM PKA catalytic

subunit, the mean blockade duration was 2.6 ± 0.4 ms, the meaninterevent interval duration was 5.0 ± 0.2 ms, and the meanevent duration due to kinase binding was 140 ± 10 ms (all n = 3;Fig. S5C). The rate constants and equilibrium dissociation con-stant for the association of PKA with αHL–PKIP–TEV weredetermined as for the Pim kinases (Fig. 3G and Table 1).To investigate substrate selectivity between PKA and Pim-1,

we looked for signs of interaction between Pim-1 and αHL–PKIP–TEV and between PKA and αHL–D127N–PLM–TEV.For both αHL–PKIP–TEV in the presence of 182 nM Pim-1 andfor αHL–D127N–PLM–TEV in the presence of 128 nM PKA,binding was not observed (Fig. S6).

Voltage Independence of Kinase Binding. As the kinetics of asso-ciation and dissociation were measured under an applied trans-membrane potential, it was important to examine the voltagedependence of the rate constants and equilibrium constants. ForPim-1 interacting with αHL–D127N–PLM–TEV (Fig. S7 A–C),there was no statistically significant voltage dependence from –20to –80 mV for k+2, k–2, or Kd [t(26) = –0.55, P = 0.59; t(26) =–1.66, P = 0.11; t(26) = 0.18, P = 0.86; respectively, where thenull hypothesis was that the slope of the linear regression waszero]. This is an improvement over previous kinase stochastic

Table 1. Association and dissociation rate constants and equilibrium dissociation constants for several proteinkinase-peptide interactions

Source Pore Kinase k+2 (M–1·s–1) k–2 (s–1) Kd (μM)

This work αHL–D127N–PLM–TEV (cut) Pim-1 (1.34 ± 0.03) × 108 10.9 ± 0.6 0.081 ± 0.005Pim-2 (1.76 ± 0.08) × 107 30 ± 1 1.7 ± 0.1Pim-3 (5.3 ± 0.3) × 108 14.7 ± 0.5 0.028 ± 0.002

αHL–PKIP–TEV (cut) PKA (3.4 ± 0.1) × 106 7.1 ± 0.1 2.09 ± 0.07

Method Kinase k+2 k–2 Kd

From the literature ITC* Pim-1 — — 0.058ITC* Pim-2 — — 0.64ITC* Pim-3 — — 0.039Gel filtration† PKA — — 2.3SPR‡ PKA (+MgATP) 1.5 × 106 7.6 × 10−4 0.0005Stochastic sensing (14) PKA (2.5 ± 0.5) × 106 0.19 ± 0.05 0.08 ± 0.01Stochastic sensing (11) PKA (1.5 ± 0.1) × 106 0.2 ± 0.1 0.13 ± 0.01

Constants in this work were determined under an applied potential of –50 mV, in 15 mM Mops, pH 6.8, 300 mM KCl, and 5 mM DTT,except for PKA, where the buffer did not contain DTT. Values are mean ± SE, determined from curve fitting as shown in Fig. 3, wherethe fitted means were derived from n = 4 for the three Pim kinases, and n = 3 for PKA. αHL, α-hemolysin; ITC, isothermal titrationcalorimetry; PKIP, protein kinase inhibitor peptide (residues 5–24); PLM, Pimtide loop mutant; SPR, surface plasmon resonance; TEV,tobacco etch virus.*Pim kinase affinities for Pimtide peptide (30).†Interaction of full-length heat-stable protein kinase inhibitor (PKI) with PKA catalytic subunit (48).‡Interaction of GST–PKI fusion with PKA catalytic subunit in the presence of 1 mM ATP and 10 mM MgCl2 (47).

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sensors, which did exhibit voltage-dependent interactions (14).At higher negative potentials, we found that the αHL–D127N–

PLM–TEV pore closed with increasing frequency. The closureswere reversed if the polarity of the applied potential was alter-nated, but they prohibited experiments at potentials more neg-ative than –80 mV.

Discrimination of Pim Kinase Family Members. Stochastic sensingoffers the potential for analyte discrimination based on blockadeamplitude and/or the mean duration of binding. In the presentwork, binding was not associated with current blockade. We did,however, observe differences with respect to the noise in thekinase-bound conductance states; current traces correspondingto the kinase-bound state of the Pim kinases showed additionalcurrent noise compared with those for PKA-bound levels withαHL–PKIP–TEV, and the magnitude of the additional noisevaried between the three Pim kinases [Fig. 4 A and B; current–trace SDs: 1.78 pA (Pim-1), 2.07 pA (Pim-2), 2.45 pA (Pim-3),and 1.19 pA (PKA)]. In the case of Pim-3, the noise was suffi-ciently resolved that it was necessary to include an additionalstate in the kinetic model for the determination of rate constants(Materials and Methods). Exploiting these noise differencesenables an alternative means for analyte discrimination.

Association Rate of Pim-1 for Pimtide is Electrostatically Enhanced.The association rate constants determined for the Pim kinases aresignificantly higher than that determined for PKA, particularly in thecases of Pim-1 and Pim-3, which both have association rate constantsaround two orders of magnitude higher than PKA. Orientationrequirements usually limit protein–protein association rate constantsto 105–106 M–1·s–1 (50). However, it has been shown that rate con-stants as high as 109 M–1·s–1 can be achieved through long-rangeelectrostatic interactions between binding partners, which acceleratethe formation of a loosely associated “transient complex” precedingthe formation of the native complex (51). Such electrostatic en-hancement can be modulated by varying the ionic strength of thesolution. A significant decrease in an electrostatically enhanced as-sociation rate constant (k+2) occurs with increasing ionic strength,and a linear relationship is observed when log k+2 is plotted againstlog f p± , where f p± is the mean activity coefficient of the electrolytegiven by the extended Debye–Hückel equation (52):

log f p± ¼ −Ajz1z2j

ffiffi

Ip

1þ Baffiffi

Ip

log f p± is related to the electrostatic potential between the twobinding partners and log k+2 to the activation energy for associ-ation (52).We measured the association rate constant for Pim-1 with

αHL–D127N–PLM–TEV at ionic strengths of 150–1,000 mM.We observed a strong linear dependence for log f p± versus logk+2 (R2 = 0.97), which suggests that the association of Pim-1with its substrate is electrostatically enhanced (Fig. 5). Extrap-olation to infinite ionic strength allows a rough approximation ofthe basal association rate constant in the absence of electrostaticforces. The value for Pim-1, ∼8 × 105 M–1·s–1, is similar in mag-nitude to other basal association rate constants estimated forelectrostatically enhanced protein–protein interactions (51). Wecalculated electrostatic potential maps for Pim-1 and Pim-2 usingAdaptive Poisson-Boltzmann Solver (APBS) (53) and visualizedthem inVisualMolecular Dynamics (VMD) (54) (Fig. S9A andB).The maps clearly illustrate strong negative electrostatic potentialsacross the peptide-binding pockets that would form long-rangeelectrostatic interactions with the highly positively charged Pimtidesubstrate, consistent with the phenomenon described above. Incontrast, PKA does not have this feature (Fig. S9C).

DiscussionStochastic sensors for the catalytic subunit of cAMP-dependentprotein kinase (PKA) have previously been described (11, 14).These either relied upon chemical attachment of a peptidesensor element to αHL (14) or the fusion of a peptide sensorelement within the trans loop of αHL (11). Here we have de-scribed an engineering strategy that combines advantages fromeach approach while eliminating several weaknesses. We therebyextended the scope of stochastic sensing of protein kinases andused the approach to gain insight into the kinetics of substrateinteractions for the Pim kinase family.

Proteolytically Cleaved trans Loops in αHL as Stochastic Sensors. Bydesigning αHL trans loop sensor element fusions that can be site-specifically cleaved by TEV protease, we were able to producestochastic sensors for the Pim kinase family and for PKA that donot require chemical modification and which bear a sensor pep-tide attached through a peptide bond to a single terminus. Theprocedure is fast, comprising only a simple additional step withinthe existing method for producing heteromeric αHL pores. As thesensor element is genetically encoded, it should be possible toconstruct combinatorial sensor libraries by this approach. Poresbearing peptides attached to the trans mouth could also haveother applications. For example, peptides containing genetically

10 pA

100 ms

Pim-1 Pim-2

Pim-3 Pim-3 PKA

0 pA

0 pA

A

B

Fig. 4. Power spectra of the kinase-bound states for four different proteinkinases. (A) Power spectral densities of events corresponding to Pim-1, Pim-2,or Pim-3 bound to the αHL–D127N–PLM–TEV pore and the catalytic subunitof cAMP-dependent protein kinase (PKA) bound to the αHL–PKIP–TEV pore.For clarity, coincident spikes present in all four spectra at odd harmonics ofthe power-line frequency (50 Hz) have been removed by notch filtering. Theunfiltered spectra are shown in Fig. S8. (B) Representative traces showingindividual events that correspond to the kinase-bound states as describedabove, where the thick bars indicate the duration of binding. A downwarddeflection corresponds to a negative current. Measurements were per-formed in 15 mM Mops, pH 6.8, 300 mM KCl for PKA and with the additionof 5 mM DTT for the Pim kinases. The applied potential was –50 mV, and thefilter corner frequency was 2 kHz.

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encoded affinity tags could be used to immobilize pores on a solidsubstrate, or to attach them within a solid-state nanopore for theformation of hybrid nanopore devices (55).

Stochastic Detection of Protein Kinases. We determined the associ-ation and dissociation rate constants, and hence the equilibriumdissociation constants, for the interaction of all three Pim kinaseswith their consensus substrate peptide Pimtide by using the sensorαHL–D127N–PLM–TEV (cut) and for PKA with the inhibitorypeptide PKIP5–24 by using the sensor αHL–PKIP–TEV (cut)(Table 1). Our findings with Pim-1 show that measured con-stants were independent of transmembrane potential, allowingdirect comparison with values determined in bulk solution, incontrast with previous studies (14). Kinase binding occurs withouta reduction in pore current, unlike previously described sensors (11,14). This suggests that binding and dissociation occur sufficiently farfrom the pore and lipid bilayer to not be influenced by ionic flowthrough the pore or the transmembrane electric field.Equilibrium dissociation constants, but not rate constants, for

Pim kinases with Pimtide have previously been determined byisothermal titration calorimetry (ITC) (38). Values determinedhere correspond well with those from ITC (Table 1), givensomewhat different determination conditions (ITC: 50 mMHepes, pH 7.5, 150 mM NaCl, 1 mM DTT, at 10 °C; this work:15 mM Mops, pH 6.8, 300 mM KCl, 5 mM DTT, at 21 °C), andthe presence of the TEV recognition site adjacent to Pimtide inthe sensor peptide. Pim-3 bound strongest (this work: 28 nM;ITC: 39 nM), with Pim-1 binding slightly more weakly (this work:81 nM; ITC: 58 nM), and Pim-2 binding much more weakly (thiswork: 1.7 μM; ITC: 640 nM).For the PKA–PKIP5–24 interaction, the measured association

rate constant (3.4 × 106 M–1·s–1) is consistent with previous valuesfrom stochastic detection (2.5 × 106 M–1·s–1, 1.5 × 106 M–1·s–1)(11, 14) and a value determined by surface plasmon resonance(SPR) (1.5 × 106 M–1·s–1) for a GST–PKI fusion with a PKAcatalytic subunit (but in the presence of MgATP, and where PKIis the full-length heat-stable protein kinase inhibitor) (56).However, the dissociation rate constant differs from those pre-

viously determined by stochastic sensing (7.1 s–1 in the presentwork versus 0.19 and 0.2 s–1) (11, 14). The affinities determined inthe previous studies also differed substantially from that de-termined by analytical gel filtration (80 and 130 nM versus 2.3μM) (57), whereas the Kd value determined here (2.09 μM)corresponds well, suggesting that our determination of the dis-sociation rate constant is reliable. The origin of the discrepanciesin previous stochastic sensing studies is unclear.An intriguing property of the sensors is that the electrical

noise in the kinase-bound states depends on the identity of thekinase (Fig. 4). This phenomenon might be exploited as a meansto enhance analyte identification. Inspection of the 3D structuresof Pim-1, Pim-2, and PKA (Protein Data Bank ID codes 2BIL,2IWI, and 2CPK, respectively) suggests these differences may bedue to interactions between unstructured regions of the Pimkinase C termini and the pore, although it is debatable whetherdynamic behavior can be inferred from static structures. Pim-1and Pim-2 have C termini orientated on the side of the kinasesthat is expected to face the pore in the bound state, with the last7 and 24 residues, respectively, unresolved in the structures. Incontrast, PKA has a longer C-terminal tail that wraps around thesurface of the enzyme, contains the turn and hydrophobic motifscommon to AGC family kinases, and terminates on the oppositeside of the kinase, away from the pore and with no unresolvedterminal residues (58). Noise differences between Pim familymembers may arise from differences of length or charge distri-bution in their unstructured C termini.

Comparison with Other Methods. Stochastic detection has severaladvantages over traditional methods for determining rate andequilibrium constants. Our method does not use spectroscopicphenomena, is label-free, and may be more amenable to par-allelization/high throughput than stopped-flow spectroscopy.However, the engineering strategy may limit the scope of inter-actions studied, as one binding partner must be encoded withinthe αHL trans loop.SPR experiments must be performed carefully to avoid rebinding

and mass transport effects. Reliable determination of very highassociation rate constants (>106 M–1·s–1, depending on analyte size)and dissociation rate constants outside the range 10−5 to 1 s–1 isdifficult (59). In contrast, our method uses a single sensor moleculeunder steady-state conditions and so does not suffer these limi-tations. Notably, we were able to determine very rapid associationrate constants for Pim kinases (∼107–108 M–1·s–1).Although our method determines equilibrium constants in-

directly from the association and dissociation rate constants, thesimultaneous determination of rate and equilibrium constants isitself advantageous. If droplet interface bilayers are used, wherecompartment volumes are typically 200 nL or less (60), analyteconsumption in stochastic sensing can be very low (a few ng in200 nL droplets) compared with ITC (61). Our method mightalso be coupled with high-throughput single-channel recordingmethods (62–65). Finally, it is possible to estimate the entropicand enthalpic contributions to the change in free energy uponbinding by measuring the temperature dependence of the equi-librium dissociation constant according to the van’t Hoff equa-tion (66–68).

Electrostatic Enhancement of Pim-1–Pimtide Association. The mea-sured association rate constants for all three Pim kinases withPimtide were one to two orders of magnitude higher than that ofPKA for PKIP5–24 and were near the top of the range typicallymeasured for protein–protein interactions (51). Studies on othervery fast protein–protein interactions have concluded that thedominant factor that enhances association rates is the presenceof long-range electrostatic interactions between binding partners(51). We observed a strong decrease in the Pim-1–Pimtide as-sociation rate constant with increasing ionic strength, as seen for

Fig. 5. Association of Pim-1 with the αHL–PLM–TEV pore at different ionicstrengths. Log k+2 (where k+2 is the second-order association rate constant)was plotted against log fp± (where fp± is the mean activity coefficient, cal-culated from the extended Debye–Hückel equation). At the limit [KCl] → ∞,log fp± ¼ − 0:3423, and k+2 (basal) ∼8 × 105 M–1·s–1. Measurements wereperformed in 15 mM Mops, pH 6.8, with the ionic strength adjusted withKCl. The ionic strength contribution from the Mops was ∼4 mM. The appliedpotential was –50 mV, and the filter corner frequency was 2 kHz. Error barsrepresent SDs (n = 5). Dotted lines indicate the 95% prediction interval forthe linear regression.

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other protein–protein interactions with electrostatically enhancedassociation rates (52).Pimtide is a highly optimized Pim kinase substrate obtained

from a peptide library screen, whereas known natural substratesbind with lower affinity. Some do, however, possess high netcharge similar to Pimtide [e.g., p21tide: RKRRQTSMTD, PAP-1tide: KKRKHKASKSS (38)]. Compared with Pim-1, PKA doesnot possess the same extensive distribution of negative electro-static potential across its binding pocket (Fig. S9), and PKIP5–24has low net charge. Pim kinases may therefore be distinctlycapable of potentially rapid electrostatically enhanced substrateassociation rates.We propose that substrate association rates and their elec-

trostatic modulation may be important when considering sig-naling pathways at the systems level where several proteins orpathways compete. For instance, the transient expression of akinase with higher association rate for a particular shared sub-strate than another kinase could lead to the faster kinase out-competing the slower for that substrate, driving the slower kinasetoward a “second-preference” substrate, and hence effectinga shift between signaling pathways. However, very few kinasesubstrate-binding rate constants have so far been determined.Our approach could enable a detailed investigation of the con-tribution of electrostatic enhancement to the overall control ofsignaling through the study of binding kinetics for a range ofprotein kinases and natural substrates.In conclusion, we have developed a versatile, label-free method

for measuring the kinetics and affinities of kinase–substrateinteractions and have applied it to the interaction of Pim kinaseswith the consensus substrate Pimtide and the interaction of PKAwith the inhibitory peptide PKIP5–24. We thus identified elec-trostatic enhancement in Pim-1–Pimtide association, a phenome-non that may be important in the cellular context.We envisage several extensions of the method. First, it may be

possible to specifically screen for type II kinase inhibitors, astheir stabilization of the inactive “DFG-out” kinase conforma-tion (where DFG refers to a conserved motif in the activationloop) should interfere with substrate binding. Type I inhibitorswould be expected to occupy the ATP-binding pocket but notsignificantly interfere with substrate binding. Second, it should bepossible to study phosphorylation of the Pimtide sensor elementwhen MgATP is present. In addition, it would be desirable toestablish turnover of the substrate by the addition of a suitableprotein phosphatase. Finally, it should be possible to generatea pseudosubstrate sensor element by mutation of the Pimtidephosphoacceptor serine residue and hence study the interactionof Pim kinases with the sensor in the presence of nucleotides, butin the absence of phosphorylation. Previous measurements withPKA have shown a synergistic effect between MgATP and pep-tide binding (11, 14, 57), and it may be possible to measure themodulation of this effect by ATP-competitive (type I) inhibitors.

Materials and MethodsAll chemicals, reagents, and oligonucleotides were from Sigma Aldrich unlessotherwise indicated.

Pore Engineering. pT7–αHL was described previously (69). pT7–αHL–PLM–D8was prepared from pT7–αHL–RL4–D8 (11) by cassette mutagenesis. A DNAcassette encoding the Pimtide sequence, flanking glycine/serine linkers,and AgeI and StuI sites at the 5′ and 3′ ends, respectively, was prepared byassembly PCR using the following oligonucleotides: 5′-GCGCGCACCGGT-GATGATAG-3′, 5′-GCCGCTACTGCCGCTTCCGCTGCTATCATCACCGGTGCGC-3′, 5′-AGCGGCAGTAGCGGCGCACGTAAACGTCGTCGTCATCCGA-3′, 5′-GCTAC-CCGCGGTCGGTGGGCCGCTCGGATGACGACGACGT-3′, 5′-CCGACCGCGGGTA-GCTCCGGGAGCGGCTCTAGCAAAATTG-3′, and 5′-CCCGGGAGGCCTCCAATTT-TGCTAGAGCCGCTC-3′. The cassette was digested with AgeI and StuI (NewEngland Biolabs) and ligated into pT7–αHL–RL4–D8 that had also beendigested with AgeI and StuI.

pT7–αHL–D127N was generated from pT7–αHL by in vivo homologousrecombination (70, 71). Two PCR reactions were performed using pT7–αHL asthe template. For the first reaction, the template was linearized by NdeI(New England Biolabs) before PCR using the forward mutagenic primer: 5′-GTTACTGGTAATGATACAGGAAAAATTGGC-3′ and the reverse nonmutagenicprimer (SC47): 5′-CAGAAGTGGTCCTGCAACTTTAT-3′. The second PCR usedtemplate linearized by HindIII; the reverse mutagenic primer was: 5′-GCC-AATTTTTCCTGTATCATTACCAGTAAC-3′, and the forward nonmutagenic primer(SC46) was 5′-ATAAAGTTGCAGGACCACTTCTG-3′. PCR was performed withPhusion Flash HF Master Mix (New England Biolabs) using the cycling conditionsof themanufacturer. Equal volumes of each PCR (typically 5 μL) were thenmixedand transformed into chemically competent XL-10 Gold cells (Novagen), whichwere subsequently spread onto LB (Lysogeny Broth) carbenicillin plates and in-cubated at 37 °C overnight. Plasmid DNA was isolated from several coloniestaken from the plates. Successful constructs were confirmed by DNA sequencing.

pT7–αHL–D127N–PLM–D8 was generated from pT7–αHL–PLM–D8 by invivo homologous recombination. The forward mutagenic primer was 5′-GTCACCGGTAATGATAGCAGCGGAAG-3′, and the reverse mutagenic primerwas 5′-GCTTCCGCTGCTATCATTACCGGTGAC-3′. The nonmutagenic primerswere SC47 and SC46 as above.

pT7–αHL–D127N–PLM–TEV–D8was generated from pT7–αHL–D127N–PLM–D8by in vivo homologous recombination. The forward mutagenic primer was 5′-GAAAATCTGTATTTTCAAGGGGGTAGCTCCGGGAGCGGC-3′, and the reverse mu-tagenic primer was 5′-CCCTTGAAAATACAGATTTTCCGCGGTCGGTGGGCCGCT-3′.The nonmutagenic primers were SC47 and SC46 as above.

pT7–αHL–PKIP–TEV–D8was generated frompT7–αHL–PKIP–D8 (11) by in vivohomologous recombination. The forward mutagenic primer was 5′-AAAG-AAAATCTGTATTTTCAAGGGGGCAGCAGCGGCAGCGGC-3′, and the reversemutagenic primer was 5′-CCCTTGAAAATACAGATTTTCTTTATCATGAATCGC-ATTGCGACG-3′. The nonmutagenic primers were SC47 and SC46 as above.

Pore Synthesis. Engineered αHL pores were produced using the S30 T7 High-Yield Protein Expression System (Promega), a coupled IVTT system. A stan-dard reaction comprises: 500 ng plasmid DNA, 10 μL S30 Premix Plus (sup-plied with kit), 9 μL S30 extract (supplied with kit), 1 μL L-[35S]methionine(1,175 Ci·mmol–1, 10 mCi·mL–1, MP Biomedicals), and sufficient nuclease-freewater to achieve a total reaction volume of 25 μL. To suppress endogenousexpression, the S30 extract was pretreated with rifampicin (1 μg·mL–1 final)before addition to the IVTT reaction. Reaction mixtures were incubated at37 °C with shaking at 1,200 rpm in a Thermomixer Comfort (Eppendorf) for1 h. Mutant αHL homoheptamers were produced in 25-μL reactions supple-mented with 3 μL of rabbit erythrocyte membranes [∼1 mg (protein)·mL–1].After incubation, reactions were centrifuged for 10 min at 25,000 × g. Thesupernatant was removed and the pellet resuspended in 200 μL MBSA buffer(10 mMMops, pH 7.4, 150 mMNaCl, 1 mg·mL–1 BSA). The suspension was thencentrifuged and washed once more before being centrifuged a final time. Theresulting pellet was resuspended in 50 μL of 1X Laemmli sample buffer [62.5mM Tris·HCl, pH 6.8, 2.3% (wt/vol) SDS, 5% (vol/vol) β-mercaptoethanol, 10%(wt/vol) glycerol] and then electrophoresed in a 5% SDS/PAGE gel at 80 Vovernight (∼18–20 h).

To produce heteroheptamers, 100-μL-scale reactions were set up with thetwo different plasmids present in a 6:2 ratio, where the higher-concentra-tion plasmid is the αHL mutant that does not contain the sensing element,and the lower-concentration plasmid encodes αHL bearing the sensor ele-ment and a C-terminal D8 tag for the separation of oligomers of differentstoichiometry by gel mobility shift (11, 72). The procedure was then the sameas for homoheptamers.

To extract homo- and heteroheptameric pores after electrophoresis, thegel was dried on Whatman 3M filter paper under vacuum for 3–4 h at roomtemperature. The dried gel was autoradiographed using Kodak BioMax MRfilm. With the aid of an aligned autoradiograph, the desired bands wereexcised from the gel with a scalpel. Homoheptamers appear as a single band.Heteroheptamers appear as a ladder of bands due to the varying stoi-chiometries of incorporated D8-tagged sensor subunits, which producea downward gel shift. Each excised gel slice was rehydrated in 500 μL TEbuffer (10 mM Tris·HCl, pH 8.0, 1 mM EDTA) for 1 h at room temperature.The backing filter paper was then removed, and the rehydrated gel wasmacerated with a pestle. The resulting mixture was filtered through a 0.2-μm cellulose acetate filter (Rainin Microfilterfuge tube). The filtrate wasaliquoted in 10-μL portions and stored at –80 °C.

To produce TEV protease-cleaved heteroheptamer pores, two IVTT reac-tions were first prepared: a 75-μL reaction containing DNA encoding thenonsensor subunit and a 25-μL reaction containing DNA encoding the pro-tease-cleavable sensor subunit. After incubation, as described above, trans-lation was stopped in both reactions by the addition of chloramphenicol to

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a final concentration of 34 μg·mL–1. AcTEV protease (10 units, Invitrogen)was added to the sensor subunit reaction, which was then incubated at 4 °Cfor 4 h. The two tubes were then mixed, followed by the addition of rabbiterythrocyte membranes (5 μL, 1 mg·mL–1). The resulting mixture was in-cubated at room temperature for 1 h before recovery of the membranes bycentrifugation for 10 min at 25,000 × g. The membrane pellet was thenwashed twice with MBSA before resuspension in Laemmli sample buffer andSDS/PAGE purification of the assembled αHL pores as described above.

Expression and Purification of Kinases. The catalytic subunit of murine cAMP-dependent protein kinase (α isoform) was purchased from New EnglandBiolabs and was supplied at a concentration of 0.13 mg·mL–1.

Pim kinases were expressed and purified from Escherichia coli by affinity andsize-exclusion chromatography (SI Materials and Methods). Pim kinase concen-trations were obtained from their absorbances at 280 nm as determined witha NanoDrop 1000 (Thermo Scientific), using the following molecular weights(Mr) and extinction coefficients («): Pim-1 Mr = 35,546 Da, « = 48,930 M–1·cm–1;Pim-2 Mr = 34,277 Da, « = 47,440 M–1·cm–1; Pim-3 Mr = 36,830 Da, « =47,440 M–1·cm–1. All measurements were made in quadruplicate.

Single-Channel Recording. Single-channel recording was performed by usingthe method ofMontal andMueller (73). A 25-μm-thick Teflon film containingan aperture 60 μm in diameter separated two Delrin chambers. Before bi-layer formation, the aperture was pretreated with 1 μL of 1% (vol/vol)hexadecane in pentane on each side. Each chamber was then filled with1 mL of buffer solution, which comprised 15 mM Mops, pH 6.8, 300 mMKCl, and 5 mM DTT, except where PKA was used, in which case DTT wasomitted. Mops-based buffers were titrated to the desired pH with 1 M KOH;1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in pentane (5 μL, 10mg·mL–1) was then dropped onto the buffer surface of each chamber. After5 min, lipid bilayers were folded across the Teflon aperture by the repeatedraising and lowering of the buffer level above and below the aperture ineach chamber by pipetting. Measurements were made at 21 ± 1 °C. Electricalcurrents were measured with two Ag/AgCl electrodes, each contained withinsalt bridges formed of 3 M KCl in 3% (wt/vol) low-melt agarose. The elec-trodes were connected to the headstage of a patch-clamp amplifier (Axo-patch 200B, Molecular Devices) operating in voltage-clamp mode. The cischamber was defined as the chamber connected to the grounded electrodeand to which αHL protein was added. The trans chamber was connected tothe working electrode. Current signals were filtered with a low-pass Besselfilter (80 dB/decade) with a corner frequency of 2 kHz. Signals were digitizedby using a Digidata 1320A digitizer (Molecular Devices), connected to acomputer running the pCLAMP 9.2 software suite (Molecular Devices).Current signals were sampled at a frequency of 10 kHz. To obtain the in-sertion of a single engineered αHL pore, 0.1–0.5 μL of protein (typically ∼1ng·μL–1) was added to the cis chamber. A potential of +150 mV was thenapplied with stirring of the cis chamber until pore insertion was observed asa step increase in current.

Single-Channel Current Analysis. Single-channel data were analyzed with QuB2.0 software (www.qub.buffalo.edu). Current traces were idealized by usingthe segmental k-means algorithm (74) of QuB, according to the kineticmodels described (Fig. 3C). Dwell time analysis and rate constant estimationwere performed by using the maximum interval likelihood algorithm of QuB(75), with retrospective application of a dead time of 300 μs for approximatecorrection of missed events due to low-pass filtering. In a few cases, wheresignificant numbers of events were near the limit of time resolution, ashorter dead time of 200 μs was used to minimize errors due to missedevents and improve histogram fitting. Additionally, a higher dead time of 1ms was used for voltage dependence data obtained at –20 and –30 mV tominimize the effect of short, false events caused by difficulties in idealizingtraces where the open pore current is very small.

Kinetic analysis of the interaction of Pim-3 with αHL–D127N–PLM–TEVrequired a modification of the model shown in Fig. 3C, because the noise(seen for all Pim kinase-bound states) was sufficiently resolved in this case toform transitions to an additional blocked level. Hence, we added a blockedstate, B2, which was connected to the kinase-bound open state O2. Theoverall kinetic model was of the form: B1 ⇔ O1 ⇔ O2 ⇔ B2, where states otherthan B2 are the same as in Fig. 3C.

Duration histograms were made from logarithmically binned durationswith square-root y ordinates (76). OriginPro 8.5.1 (OriginLab) was used forthe plotting, fitting, and presentation of data. Fitting was performedwithout error weighting. Numerical data are reported as mean ± SD, unlessotherwise indicated. All statistical tests were performed with an alpha valueof 0.05. Significance testing (Student’s t test) for the voltage dependence ofkinase binding was performed on constituent independent data pointsrather than the means that are shown in Fig. S7 A–C. t test values arereported with degrees of freedom in parentheses.

For spectral analysis, several hundred events corresponding to the kinase-bound state were manually extracted from a single recording by usingClampfit 10.3 (Molecular Devices). The extracted events were concatenatedto form a single continuous trace. The baseline was then adjusted by sub-tracting the mean current value of the concatenated trace. Power spectrawere calculated in Clampfit using segment lengths of 4,096 samples (spectralresolution 2.44 Hz), to which were applied Hamming window functions andwhich were averaged together with 50% window overlap. The resultingpower spectral densities for all four protein kinases were then plottedin OriginPro.

Ionic-Strength Dependence of Pim-1 Association. Experiments were performedin 15 mMMops, pH 6.8, with the ionic strength adjusted with KCl at 20 ± 1 °C.The ionic strength contribution from the Mops was ∼4 mM. The Pim-1 con-centration used in all cases was 182 nM. log fp± was calculated according tothe extended Debye–Hückel equation (77, 78):

log f p± ¼ −

Ajz1z2jffiffi

Ip

1þ Baffiffi

Ip

where I is the ionic strength of the solution, z1 and z2 are the charges of theanion and cation, A and B are constants dependent on the temperature anddielectric constant of the solution (78), and a is the ion size parameter.Values of 0.5046 dm3/2·mol–1/2 and 3.276 × 108 dm1/2·mol–1/2 were used for Aand B, respectively (79). A value of 4.5 Å was used for a (note: the units ofa are dm in the above equation). Schreiber et al. (52) used a value of 5.6 Åfor a, which is somewhat larger than literature values for the hydrateddiameters of Na+ and Cl–, but which gave the best fit to their data. To beconsistent, we calculated our value of a for KCl by scaling the NaCl value bya factor of 0.8 to account for the smaller hydrated diameter of K+ vs. Na+

(80). A justification for the use of the extended Debye–Hückel equation atthe high electrolyte concentrations used here is given in Schreiber et al. (52)and should hold for KCl as well as for NaCl.

Visualization of Electrostatic Potentials. Electrostatic potentials were calcu-lated with APBS (53) and visualized in VMD (54) (SI Materials and Methods).

ACKNOWLEDGMENTS. This work was supported by grants from the NationalInstitutes of Health and Oxford Nanopore Technologies. L.H. was supportedin part by a Biotechnology and Biological Sciences Research Council doctoraltraining grant. S.K. and L.T.A. are supported by the Structural GenomicsConsortium, a registered charity (1097737) that receives funds from AbbVie,Boehringer Ingelheim, the Canada Foundation for Innovation, the CanadianInstitutes for Health Research, Genome Canada, GlaxoSmithKline, Janssen, LillyCanada, the Novartis Research Foundation, the Ontario Ministry of EconomicDevelopment and Innovation, Pfizer, Takeda, and the Wellcome Trust.

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