Supporting Information
Tilley et al. 10.1073/pnas.1406876111
SI Materials and Methods
Peptide Synthesis and Folding. All mutants were made in a back-
ground where the methionine at position 35 of guangxitoxin-1E
was replaced by its isostere norleucine to avoid any complications
of methionine oxidation. Norleucine35 variants are used in all
experiments, and referred to as GxTX. Linear peptides were
synthesized on an AAPTEC Apex 396 peptide synthesizer using
an Fmoc (N-(9-fluorenyl)methoxycarbonyl) methodology. Peptides were assembled stepwise on 0.1–0.3 mmol resin (Fmoc-ProNovaSyn TGT, Novabiochem) in N-methyl-2-pyrrolidone, 0.4 M
Fmoc amino acids, 0.44 M N-hydroxybenzotriazole, and 10%
(vol/vol) N,N′-diisopropylcarbodiimide. The side chain protecting groups for amino acids were triphenylmethyl or acetamidomethyl (Acm) for cysteine and asparagine, tert-butyloxycarbonyl
for tryptophan and lysine, and tert-butyl for serine. Removal
of Fmoc groups with 20% 4-methylpiperidine in dimethylformamide (DMF) preceded 2-h coupling steps. Resin was washed
five times with DMF after coupling and Fmoc removal. Linear
peptides were cleaved and deprotected with trifluoracetic
acid (TFA): triisopropylsilane:1,2-ethanedithiol:thioanisole:H2O
(85:2.5:2.5:5:5 by volume) for 2–4 h at room temperature, with
removal of deprotecting groups monitored by MALDI-TOF
mass spectrometry. Cleaved peptide was separated from resin by
filtration. Peptide was precipitated with cold diethyl ether; pellet
was washed once with ether and dried under a stream of N2.
Peptide pellet was dissolved in water with (by volume) 50%
acetonitrile (ACN) or 50% acetic acid (AcOH), injected onto
a preparatory C18 column (Vydac 218TP101522), and eluted
with an increasing concentration of ACN with 0.1% TFA. Recovered peptides were lyophilized, dissolved in 50% (vol/vol)
ACN, diluted to 50 μM, and folded by air oxidation in 1 M
guanidinium HCl, 0.1 M ammonium acetate, 2.5 mM GSH, 0.25
mM GSSG, 1% ACN, pH 8 with ammonia. Oxidation was
monitored by mass spectrometry (Applied Biosystems SCIEX
TF4800 MALDI TOF-TOF). Upon completion (3 d), the visible
aggregates that had formed in solution were removed by filtration, and 0.1% TFA added. This solution was pumped onto
a C18 column, eluted as above, and lyophilized.
His6-GxTX Expression. Oligonucleotides encoding the 36 amino
acid-GxTX sequence were ligated into a pET-30a(+) plasmid
between the N-terminal hexahistidine tag and an enterokinase
cleavage site at the C terminus of Venus yellow fluorescent
protein. The His6-GxTX-Venus fusion protein was expressed in
Escherichia coli strain BL21 (DE3). Cells were grown using 2×
YT culture medium in a shaker incubator at 37 °C until an optical density of 0.5 was reached, and 1 mM isopropyl-1-thio-β-Dgalactopyranoside was added to induce expression. After 4 h,
cells were pelleted, resuspended in 20 mL of PBS, and then lysed
by heating to 80 °C for 7 min. The lysate was cooled on ice, and
1 mM PMSF, 1 mM MgCl2, and 0.1 mg DNaseI were added.
After 20 min, cells were centrifuged at 25,000 × g to remove
precipitate, and the supernatant concentrated by spin dialysis
(Amicon Ultra 30-kDa molecular weight cutoff). The fusion
protein was purified using nickel affinity FPLC, eluting with an
imidazole gradient. Imidazole was removed by spin dialysis, and
the GxTX domain, as part of the fusion protein, was refolded by
air oxidation as above. Refolded His6-GxTX was cleaved from
Venus using recombinant enterokinase (1 U/50 μg substrate, Novagen), then subsequently purified by size exclusion FPLC to obtain
pure His6-GxTx (Sequence: MHHHHHHSTS EGECGGFWWK
Tilley et al. www.pnas.org/cgi/content/short/1406876111
CGSGKPACCP KYVCSPKWGL CNFPAPDLGT DDDDK,
m/z = 6098.8).
Toxin Conjugation. Acm deprotection was achieved with TFA +
1% anisole for a final concentration of 10 mg/mL (2.5 mM)
GxTX. Silver acetate was added to 30 mg/mL (0.18 M), protected from light, mixed via slow rotation for 2 h, and monitored
by MALDI-TOF. Peptides were pelleted with diethyl ether as
above. The remaining silver was removed by dissolving the pellet
in 50% (vol/vol) AcOH, adding 1 volume of 2 M guanidinium
HCl, incubating for 10 min in room temperature, and pelleted by
centrifugation. The resultant GxTX Cys13 peptide was purified
by HPLC as above. GxTX Cys13 was labeled with a maleimido–
fluorophore, either tetramethylrhodamine maleimide (Life Technologies T6027) or DyLight 550 Maleimide (Thermo 62290), to
yield GxTX-TMR or GxTX-dy550, respectively. GxTX Cys13
lyophilisate was brought to 300 μM in 50% (vol/vol) ACN + 1
mM Na2EDTA. One part 200 mM Tris, 20 mM Na2EDTA (pH
6.8 with HCl) was mixed with this solution. A 2.5-mM solution of
the maleimido–fluorophore in DMSO was added to 20% (vol/vol)
final DMSO concentration. The reaction was agitated in a polypropylene tube for 2 h at 20 °C. The conjugate was purified by
HPLC, as above with a C18 column (Thermo 72105–254630).
For confocal imaging experiments lyophilisate was resuspended
in 50% (vol/vol) ACN and aliquots stored at −80 °C. GxTX-dy550
lyophilisate appeared sparingly soluble in 50% ACN and prone to
aggregation. To enhance solubility and prevent aggregation in
subsequent electrophysiology experiments it was resuspended in
1 M arginine HCl, 50 mM glutamic acid, pH 5 with NaOH, and
aliquots were stored at −80 °C.
GxTX Pra13 was conjugated to methoxy-PEG-Azide with an
average mass of 5 kDa (Creative PEGWorks PLS-2024) using
copper(I)-catalyzed azide-alkyne cycloaddition in the presence of
the catalytic ligand BTTAA (bis[(tertbutyltriazoyl)methyl]-[(2carboxymethyltriazoyl)methyl]-amine) (1). Reagents were added
sequentially to a polypropylene tube: 5 μL 1 M sodium phosphate buffer, pH 7; 24 μL 2.5 mM CuSO4; 15 mM BTTAA; then
15 μL 1 mM methoxy-PEG-azide; 15 μL DMSO; 15 μL 1.5 mM
GxTX Pra13; 10 μL 150 mM sodium ascorbate. The reaction
mixture was briefly vortexed after each addition. The reaction
was shielded from light, mixed with 1,000 rpm shaking at 25 °C
for 4 h before quench with 50 μL 10 mM EDTA and HPLC
purification as described above.
Purity of conjugates was further confirmed by Tris/Tricine SDS/
PAGE (Fig. S1D). Five hundred ng of each peptide or 0.7 μl
polypeptide standard (Bio-Rad 161–0326) was diluted in tricine
sample buffer (Bio-Rad 161–0739) with 2% (vol/vol) 2-mercaptoethanol and denatured at 95 °C for 5 min. Samples were loaded
into a 10–20% (wt/vol) polyacrylamide gel (Bio-Rad 456–3116)
with 100 mM Tris, 100 mM Tricine, 0.1% SDS, pH 8.3 running
buffer. Gels were run at 30 V until dye entered gel (∼10 min),
then 100 V until dye reached the bottom of the gel (∼45 min).
Gels were fixed in 10% acetic acid, 40% (vol/vol) ethanol for 60
min, washed twice for 5 min in water, and stained overnight in
a colloidal Coomassie stain (2) composed of 0.12% Coomassie
G-250, 10% (wt/vol) ammonium sulfate, 17% (wt/vol) o-phosphoric acid, and 20% (vol/vol) methanol. Gels were destained in
water at 4 °C, and imaged digitally (Bio-Rad 170–8270).
Synthesis of the azide beads was initiated by swelling amine
functionalized (0.26 mmol/g) dendrimetic resin beads (Rapppolymere S30902) with DMF for 12 h in a polypropylene tube.
Azido-PEG4-NHS (Conju-Probe), 3 equiv, and N,N-diisopropyl1 of 7
treated polystyrene dishes (BioLite, Thermo) 37 °C in a 5% CO2
atmosphere in Ham’s F-12 media (Corning MT-10–080-CV)
containing 10% FBS (GemCell 100–500), and 1% penicillin–
streptomycin solution (Life Technologies 15140–122). Transfections were achieved with Lipofectamine LTX (Life Technologies 15338–100) following manufacturer’s instruction, with 2 μL
Lipofectamine, 1 μg DNA per ml Ham’s F-12 media; media was
replaced 4–6 h after transfection, and cells used for experiments
2 d later. A CHO-K1 cell line expressing rat Kv2.1 (3) was cultured with 1 μg/mL blasticidin, 25 μg/mL zeocin to retain transfected vectors. Before experiments, 1 μg/mL tetracycline was
added to media to induce a desirable amount of channel expression: 1–2 h for most electrophysiology, or overnight for imaging. The BFP cell line was generated from the blue fluorescent
protein variant EBFP2 with a nuclear localization sequence (4),
kind gift of Michael W. Davidson, Florida State University,
Tallahassee, FL. BFP positive cells were subcloned following
selection with 1 mg/mL G418. The cell line was expanded and
maintained with 100 μg/mL G418, 10 μg/mL blasticidin, and 250
ug/mL zeocin. The GFP-Kv2.1 expression vector (5) was the kind
gift of James Trimmer, University of California, Davis. Rat
hippocampal neurons were prepared as described (6).
and digitized at 100 kHz. All recordings were made after addition
of 0.1% BSA. Toxins were added by flushing 100 μL through
a low-volume recording chamber (Warner R-24N). For dissociation rates, the chamber was under constant perfusion of the external solution at 2 mL/min and 200 μL of toxin was perfused
through the recording chamber while holding at −100 mV. Toxin
binding rate and affinity with a −100 mV holding potential was
measured from the change in current level at the end of 100-ms
steps to 0 mV. These test pulses were repeated every 2 s. Toxin
binding rate and affinity with a 0-mV holding potential was
measured by continuous recording at 0 mV after inactivation had
reached steady state. P/N subtraction was not used with these
protocols. Cells were induced longer to have more channels for
these experiments. It is not known whether toxin interacts differently with activated vs. inactivated states. Toxin dissociation
with a −100 mV holding potential was measured from the change
in current level at the end of 100 ms steps to 0 mV. These test
pulses were repeated every 10 s. To measure the effect of 0-mV
stimulus, 5-s pulses to 0 mV were given every 10 s, with the
−100-mV interval sufficient to recover from inactivation.
For experiments with Pra13 GxTX variants, whole-cell patchclamp experiments were performed with a QPatch-16 automated
electrophysiology platform (Sophion Biosciences) using disposable 16-channel planar patch chip plates (QPlates; patch hole
diameter ∼1 μm, resistance 2.00 ± 0.02 MΩ). Intracellular solution was same as for manual patch clamp, Qpatch external
solution contained (in mM): 10 Hepes, 3.5 KCl, 155 NaCl,
1 MgCl2, 1.5 CaCl2, adjusted to pH 7.4 with NaOH. Cells were
grown to 60–80% confluency in 175-cm2 flasks and harvested by
incubating in 2 mL detachin (Genlantis T100100) for 10–15 min
at 37 °C. Five mL of resuspension solution (in mM: 10 Hepes,
2 KCl, 150 NaCl, 1.5 CaCl2, 1 MgCl2, 10 glucose, pH 7.4 with
NaOH), was added, then cells were pelleted at 200 × g for 5 min,
and suspended in 1 mL of resuspension solution. The resuspended cells were then added to the centrifuge of the Qpatch
and pelleted at 200 × g for 5 min before commencing automated
electrophysiology protocols. Cell positioning and sealing parameters were set as follows: positioning pressure −100 mbar,
resistance increase for success 750%, minimum seal resistance
0.05 GΩ, holding potential −90 mV, holding pressure −20 mbar.
Access was obtained with the following sequence: suction pulses
in 25-mbar increments from −250 mbar to −475 mbar; suction
ramp to −450 mbar; 10 × 10 ms −40- mV voltage zaps. Vehicle
for toxin application included 0.1% BSA and 5 μM tetrodotoxin.
Electrophysiological current traces shown were digitally smoothed
with a 1-kHz Gaussian filter for presentation.
Electrophysiology. Whole-cell voltage-clamp recordings were used
to measure currents from Kv2.1 channels. Cells were harvested by
scraping in divalent-free PBS with 1 mM EDTA, pelleted at 1,000 g
for 2 min, resuspended in CHO-SFMII media (Life Technologies
12052–114) supplemented with 25 mM Hepes (pH 7.3), and rotated in a polypropylene tube at room temperature until use.
Aliquots of cell suspension were added to a recording chamber
and rinsed with external solution 5 or more minutes before recording. The external (bath) solution contained (in mM): 50
Hepes, 20 KOH, 155 NaCl, 2 CaCl2, 2 MgCl2, 0.1 Mg-EDTA,
adjusted to pH 7.3 with HCl. The internal (pipet) solution contained (in mM): 50 KF, 70 KCl, 35 KOH, 5 EGTA, 50 Hepes,
adjusted to pH 7.3 with HCl. A calculated liquid junction potential
of 6.6 mV was corrected. Pipette tip resistances with these solutions were less than 3 MΩ. Recordings were at room temperature (22–24 °C). Voltage clamp was achieved with an EPC-10
amplifier run by Patchmaster software (HEKA). Holding potential was −100 mV. Series resistance compensation was used when
needed to constrain voltage error to less than 10 mV. Unless
otherwise indicated, capacitance and Ohmic leak were subtracted
using a P/5 protocol. Recordings were low-pass filtered at 10 kHz
Live Cell Imaging. Cells were plated in chambered coverglass (Nunc
155409) and imaged in 5 mM K+ ringer (in mM: 5 KCl, 135 NaCl,
2 CaCl2, 2 MgCl2, 0.1 MgEDTA, 50 Hepes, 20 NaOH, pH 7.3 with
HCl) with 0.1% BSA. Cells were incubated in 100 nM GxTX-dy550
or 120 nM GxTX-TMR. Confocal images were obtained using an
inverted Zeiss LSM510 system with a 1.4 N.A. 63× apochromat oil
immersion objective. The GFP-Kv2.1 was stimulated using the 488nm line from an argon laser with an HFT 488/543-nm main dichroic
beam splitter and BP 505–530-nm emission filter. For GxTX-550 and
TMR, a 543-nm helium–neon laser was used with the 488/543-nm
dichroic and an LP 56-nm emission filter. For time-lapse imaging,
unless otherwise noted, a 530-nm LED light source (Zeiss Colibri)
with 520/28-nm excitation filter, 538-nm dichroic, and 550-nm longpass emission filter set were used. Images were collected with an
EMCCD camera (Photometrics QuantEM 512SC) camera using
a Zeiss 40× 0.95 N.A. apochromat air objective, run by the Micromanager software suite 1.4.14 (7). To increase K+ concentration,
a solution with K+ replacing Na+ was added manually. For patchclamp fluorometry, the pipet solution contained (in mM): 155 Nmethyl-D-glucamine, 50 HF, 5 EGTA, 50 Hepes, adjusted to pH 7.3
with HCl; a tube lens magnification of 2.5× was used and the camera
ethylamine (3 equiv), dissolved in DMF, were added to the suspended resin beads. The tubes were placed on a rotator for 8 h
until negative Kaiser tests confirmed complete coupling. The obtained azide-functionalized beads were washed with dichloromethane, methanol, and DMF, respectively, three times each, then
dried and stored at 4 °C. For conjugation, beads were swollen for
1 h in DMF, rinsed three times each with 1:1 DMF:water, water,
then 0.5 M sodium phosphate, pH 7. GxTX Pra13 was conjugated
to beads by copper-mediated azido-alkyne condensation. Azidefunctionalized beads were reacted with alkyne-functionalized
molecules. To 2-mg beads in 122 μl 0.5 M sodium phosphate,
pH 7, in a 1.5-mL polypropylene tube, reagents were added and
briefly vortexed after each addition: 20 μl of 200 μM GxTX Pra13;
8 μl 2.5 mM CuSO4, 15 mM BTTAA; 30 μl DMSO; 10 μL 150
mM sodium ascorbate. For fluorophore labeled beads, GxTX solution was substituted with water, and DMSO included 10 mM alkyne-PEG3-5(6)-carboxytetramethylrhodamine (Click Chemistry
Tools TA108). After 2 h of slow rotation protected from light,
the reaction was quenched with 1 mM EDTA. Beads were rinsed
with water, 50% (vol/vol) DMF, then DMF, slowly rotated for
5 min, rinsed with 50% (vol/vol) DMF, three times with water,
and stored in 70% (vol/vol) ethanol at 4 °C until use.
Cell Culture. CHO-K1 cells were maintained in tissue-culture
Tilley et al. www.pnas.org/cgi/content/short/1406876111
2 of 7
exposure, light source, and patch-clamp recordings were synchronized using the electrophysiology software; in Fig. 4 D and F, an LDC apochromat 63×/1.15 water immersion objective and images were
collected with an EMCCD camera (QImaging Rolera Thunder)
camera, run by ZEN 2012 (Zeiss). Fig. 4 D–G and Movie S1 were
obtained using a pipet solution that contained (in mM): 5 KOH, 30
CsOH, 70 CsCl, 50 NaF, 50 Hepes, 5 EGTA, adjusted to pH 7.3
with HCl. In Fig. 4G, action potential trains were approximated by
epochs stimulating voltage steps from a holding potential of −80
mV to +40 mV at a frequency of 100 Hz for 50 s.
Bead Assays. GxTX-bead conjugates were rinsed three times with
water, then added to suspended CHO cells. To assess bead binding
to cells, CHO-K1 cells or the CHO-K1 cell line expressing Kv2.1
were grown to ∼80% confluency on 14-cm–round tissue culture
dishes. Cells were incubated with 1 μg/mL tetracycline for 1 d
before experiments to express Kv2.1. Cells were harvested as described and each dish resuspended in 2 mL media in a 2-mL
polypropylene tube. GxTX-coated and rhodamine control beads
were added to cell suspensions, mixed by inverting the tube, and
allowed to settle and make adhesive contacts. Tubes were incubated at room temperature and inverted every 15 min to allow
new bead–cell contacts to form. After 1 h, aliquots of beads were
removed for visual inspection. Incubations were continued up to
3 h, until a majority of GxTX coated beads were bound to Kv2.1
cells. At this point, bead-coated cells were split into different saline solutions, control (in mM: 5 KCl, 155 NaCl, 2 CaCl2, 2 MgCl2,
0.1 MgEDTA, 50 Hepes, 20 NaOH, pH 7.3 with HCl + 0.1%
BSA) or high [K+] (in mM: 160 KCl, 2 CaCl2, 2 MgCl2, 0.1
MgEDTA, 50 Hepes, 20 KOH, pH 7.3 with HCl + 0.1% BSA),
and placed on a tube rotator. Following at least 20-min rotation,
beads were plated into a multiwell plate for quantitation. Beads
were scored as having cells bound if four or more cells could be
seen adhering to beads. Experimenter was blinded to identity of
cell type and saline solution when assessing cell binding. Fluorescence overlays were obtained with a 550/25ex, 605/70em filter
set (Zeiss 43HE).
Data Analysis. Electrophysiology analysis and graphing were per-
formed with IgorPro software (Wavemetrics), which performs
nonlinear least-squares fits using a Levenberg–Marquardt algorithm. Unless indicated otherwise, means are geometric; error bars
indicate SEs; SDs are reported in text from individual fits; statistical comparisons are with a two-tailed Mann–Whitney two-sample
rank-sum test. Conductance values were determined from current
level at the end of 100-ms voltage steps to the indicated values
normalized by the Nernst potential for K+. Conductance data were
fit using the fourth power of a Boltzmann distribution function:
GK = A
1
1 + e½−ðV − Vsubunit ÞzF=RT
4
;
[S1]
where GK is Kv2.1 conductance, A is maximum amplitude, Vsubunit
is the activation midpoint of each of four subunits, z is elementary
charge, F the Faraday constant, R the ideal gas constant, and T
absolute temperature. The midpoint (Vmid) was the value of V
when A reaches half-maximum.
The Kd of GxTX was determined by
0
IK
IKmax
B
=B
@1 −
14
1
1 + Kd=½GxTX
C
C ;
A
[S2]
where IK/IK max is the fraction of current remaining after application of GxTX at the end of a 100-ms voltage step to 0 mV, Kd
Tilley et al. www.pnas.org/cgi/content/short/1406876111
is the dissociation constant for GxTX, and [GxTX] the concentration of GxTX applied.
Toxin association (kon) and dissociation (koff) rates were determined by
8
"
>
>
<
IK
1
1
= 1−
+
IKmax >
1
+
k
1
+
k
off k ½GxTX
off k ½GxTX
>
:
on
on
∞
0
!
−
9
=
4
#>
>
1
e−ðkon ½GxTX∞ + koff Þt
;
>
1 + koff k ½GxTX
>
;
on
∞
[S3]
where IK/IK max is fraction of current remaining after application
of GxTX with respect to time, Kd is the dissociation constant for
GxTX, [GxTX]0 is the initial concentration of GxTX, and
[GxTX]∞ is the steady-state concentration of GxTX. For dissociation, kon[GxTX] = 0, and koff values were adjusted for the
fraction of time channels were stepped to 0 mV. For association,
koff was constrained to the mean value measured in dissociation
experiments.
Fluorescent images were analyzed using ImageJ 1.47 software
(8). The colocalization Pearson’s coefficient was calculated using
the JACoP plugin (9). Epifluorescent regions of interest (ROIs)
were selected manually. Background was defined as the mean
fluorescence intensity of a region lacking cells. Reported fluorescence intensity and changes (ΔF) were calculated from
ROI mean fluorescence intensity minus background. Time dependence of fluorescence intensity was fit with a single exponential decay:
ΔF
= Fo + Ae−koff t ;
F
[S4]
where F corresponds to the maximum fluorescence intensity.
Fo is the remaining fluorescent signal after attenuation from
voltage activity. The variables A, t, and koff correspond to
the amplitude, time, and rate constant, respectively. When
indicated, fluorescence intensity was fit with a double-exponential decay:
ΔF
= Fo + A − Fo fe−kfast t + 1 − f e−kslow t ;
F
[S5]
where f corresponds to the ratio of the dominant amplitude over
the sum of the two components (f = Afast/[Afast +Aslow]). Interestingly, at 0 mV, fluorescence decay data over longer intervals
revealed a second, slow component that was fit well with a double-exponential decay (Eq. S5; dashed blue line in Fig. 4B). The
fast component k0 mV fast = 0.20 ± 0.10 s−1 of these doubleexponential fits was similar to the monoexponential (Eq. S4)
fit to the first 20 s of decay, k0 mV = 0.20 ± 0.11 s−1, n = 5 cells.
A minor kinetic component was slower, k0 mV slow = 0.021 ±
0.003, n = 5 and had an amplitude consistently 26 ± 4% of
k0 mV fast. The rate of k0 mV slow was also significantly faster than
k−100 mV (P = 0.008 two-tailed Mann–Whitney two-sample ranksum test), indicating that this fluorescence component is also
voltage sensitive. The presence of a slow fluorescence component was not predicted by electrophysiology. Its physical origins
are unknown, and could potentially result from complexities in
GxTX binding to cells or self-quenching of multiple VST–fluorophores bound to a single channel. To reduce the number of fit
parameters, we used rates from monoexponential fits (Eq. S4) to
3 of 7
the initial fluorescence change to report activation of ion channels with this VST probe.
The voltage dependence of kΔF was fit with the following
Boltzmann distribution:
1
;
[S6]
kΔF = kΔFmin + A
1 + e½−ðV −V1=2 ÞzF=RT
where kΔF is the rate of change of fluorescence, kΔF min the minimum value of the function, V1/2 the midpoint of the function, and
the maximum value of the function kΔF max = kΔF min + A.
Structural Modeling of the Kv2.1 VSD and GxTX. Homology modeling
of the voltage-sensing domain of rat Kv2.1 channel was performed
using the Rosetta-Membrane method (10–13). The Kv2.1 sequence was aligned with the Kv1.2–Kv2.1 chimera channel se1. Besanceney-Webler C, et al. (2011) Increasing the efficacy of bioorthogonal click reactions
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quence [Protein Data Bank (PDB) ID code 2R9R] using the
ClustalX software (14); 10,000 models of the voltage-sensing
domain were generated from the homology template and the
lowest-scoring model was chosen as the best model. The GxTX
NMR structure (PDB ID code 2WH9) was minimized and
docked to the lowest-scoring Kv2.1 VSD model. The GxTX
centroid was placed within 20 Å of the centroid of the Kv2.1
residues I273, F274, L275, T276, E277, and S278 (15). Translational and rotational perturbations were induced by Monte
Carlo methods and side-chain conformations allowed to relax to
energetic minima; 10,000 docking complexes were generated
using Rosetta Dock with an implicit membrane and the lowest
ΔΔG score model was chosen as the best (16, 17). All structural
modeling images were rendered with the UCSF Chimera software package (18).
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4 of 7
Fig. S1. Chemoselective side chains retain tarantula toxins bioactivity and permit ligation. (A) Conductance–voltage relations from GxTX variant with Cys(Acm)
insertions at indicated position. Data points are from single cells normalized to maximal amplitude of Eq. S1 fit to vehicle control data from same cell.
Lines are fits of Eq. S1. Acm1: Vmid = 76 ± 3 mV, z = 0.8 ± 0.2e+; Acm13: Vmid = 66 ± 2 mV, z = 1.4 ± 0.3e+; Acm15: Vmid = 84.2 ± 0.3 mV, z = 0.95 ± 0.02e+;
Acm17: Vmid = 84 ± 3 mV, z = 0.7 ± 0.2e+; Acm21: Vmid = 77.5 ± 0.5 mV, z = 1.24 ± 0.04e+; 300 nM Acm27: Vmid = 6 ± 3 mV, z = 1.7 ± 0.2e+; 1 uM Acm27: Vmid =
55 ± 3 mV, z = 1.4 ± 0.2e+; 1 uM Acm32: Vmid = 8 ± 2 mV, z = 1.0 ± 0.1e+; 3.8 μM His6: Vmid = 31 ± 3 mV, z = 0.7 ± 0.1e+. Dotted lines are fits to vehicle and 1 μM
GxTX from Fig. 1B. (B) Reaction scheme of azide–PEG conjugation to a propargylglycine residue to form GxTX–PEG conjugate. (C) Reaction scheme of Cys(Acm)
deprotection and subsequent tetramethylrhodamine maleimide conjugation to form GxTX conjugate. Proprietary structure of Dylight-550 maleimide could
not be shown. (D) Coomassie stained SDS/PAGE gel indicates formation of expected conjugates and lack of contaminants. PEG mobility is less than predicted by
molecular weight.
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Fig. S2. Dylight550 modestly alters GxTX binding to Kv2.1. (A) Normalized dose–response profile of GxTX-dy550 (red circles), −100-mV holding potential. Red
line is fit of Eq. S2; where Kd = 30.0 ± 3.9 nM. Black line is fit of Eq. S2 to GxTX dose–response from Fig. 3B. (B) Summary of association rates at −100 mV for GxTX
and GxTX-dy550. kon = 1.84 × 105 ± 3.03 × 104 M-1·s−1. (C) Summary of dissociation rates at −100 mV for GxTX and GxTX-dy550. koff = 6.53 × 10−3 ± 8.33 × 10−4 s−1.
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Movie S1. Complete frame set of fluorescence images used in Fig. 4 D and E. CHO-K1 cells expressing Kv2.1 channels bathed in 10 nM GxTX-dy550. Cell on
right is clamped at voltages indicated.
Movie S1
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