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Supplemented with 25 Mm Hepes (Ph 7.3), and Ro- Or 120 Nm Gxtx-TMR

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Supplemented with 25 Mm Hepes (Ph 7.3), and Ro- Or 120 Nm Gxtx-TMR Supporting Information Tilley et al. 10.1073/pnas.1406876111 SI Materials and Methods CGSGKPACCP KYVCSPKWGL CNFPAPDLGT DDDDK, Peptide Synthesis and Folding. All mutants were made in a back- m/z = 6098.8). ground where the methionine at position 35 of guangxitoxin-1E + was replaced by its isostere norleucine to avoid any complications Toxin Conjugation. Acm deprotection was achieved with TFA of methionine oxidation. Norleucine35 variants are used in all 1% anisole for a final concentration of 10 mg/mL (2.5 mM) experiments, and referred to as GxTX. Linear peptides were GxTX. Silver acetate was added to 30 mg/mL (0.18 M), pro- synthesized on an AAPTEC Apex 396 peptide synthesizer using tected from light, mixed via slow rotation for 2 h, and monitored an Fmoc (N-(9-fluorenyl)methoxycarbonyl) methodology. Pep- by MALDI-TOF. Peptides were pelleted with diethyl ether as tides were assembled stepwise on 0.1–0.3 mmol resin (Fmoc-Pro- above. The remaining silver was removed by dissolving the pellet NovaSyn TGT, Novabiochem) in N-methyl-2-pyrrolidone, 0.4 M in 50% (vol/vol) AcOH, adding 1 volume of 2 M guanidinium Fmoc amino acids, 0.44 M N-hydroxybenzotriazole, and 10% HCl, incubating for 10 min in room temperature, and pelleted by (vol/vol) N,N′-diisopropylcarbodiimide. The side chain protect- centrifugation. The resultant GxTX Cys13 peptide was purified by HPLC as above. GxTX Cys13 was labeled with a maleimido– ing groups for amino acids were triphenylmethyl or acetamido- fluorophore, either tetramethylrhodamine maleimide (Life Tech- methyl (Acm) for cysteine and asparagine, tert-butyloxycarbonyl nologies T6027) or DyLight 550 Maleimide (Thermo 62290), to for tryptophan and lysine, and tert-butyl for serine. Removal yield GxTX-TMR or GxTX-dy550, respectively. GxTX Cys13 of Fmoc groups with 20% 4-methylpiperidine in dimethylform- lyophilisate was brought to 300 μM in 50% (vol/vol) ACN + 1 amide (DMF) preceded 2-h coupling steps. Resin was washed mM Na EDTA. One part 200 mM Tris, 20 mM Na EDTA (pH five times with DMF after coupling and Fmoc removal. Linear 2 2 6.8 with HCl) was mixed with this solution. A 2.5-mM solution of peptides were cleaved and deprotected with trifluoracetic the maleimido–fluorophore in DMSO was added to 20% (vol/vol) acid (TFA): triisopropylsilane:1,2-ethanedithiol:thioanisole:H2O – final DMSO concentration. The reaction was agitated in a poly- (85:2.5:2.5:5:5 by volume) for 2 4 h at room temperature, with propylene tube for 2 h at 20 °C. The conjugate was purified by removal of deprotecting groups monitored by MALDI-TOF HPLC, as above with a C18 column (Thermo 72105–254630). mass spectrometry. Cleaved peptide was separated from resin by For confocal imaging experiments lyophilisate was resuspended filtration. Peptide was precipitated with cold diethyl ether; pellet in 50% (vol/vol) ACN and aliquots stored at −80 °C. GxTX-dy550 was washed once with ether and dried under a stream of N2. lyophilisate appeared sparingly soluble in 50% ACN and prone to Peptide pellet was dissolved in water with (by volume) 50% aggregation. To enhance solubility and prevent aggregation in acetonitrile (ACN) or 50% acetic acid (AcOH), injected onto subsequent electrophysiology experiments it was resuspended in a preparatory C18 column (Vydac 218TP101522), and eluted 1 M arginine HCl, 50 mM glutamic acid, pH 5 with NaOH, and with an increasing concentration of ACN with 0.1% TFA. Re- aliquots were stored at −80 °C. covered peptides were lyophilized, dissolved in 50% (vol/vol) GxTX Pra13 was conjugated to methoxy-PEG-Azide with an μ ACN, diluted to 50 M, and folded by air oxidation in 1 M average mass of 5 kDa (Creative PEGWorks PLS-2024) using guanidinium HCl, 0.1 M ammonium acetate, 2.5 mM GSH, 0.25 copper(I)-catalyzed azide-alkyne cycloaddition in the presence of mM GSSG, 1% ACN, pH 8 with ammonia. Oxidation was the catalytic ligand BTTAA (bis[(tertbutyltriazoyl)methyl]-[(2- monitored by mass spectrometry (Applied Biosystems SCIEX carboxymethyltriazoyl)methyl]-amine) (1). Reagents were added TF4800 MALDI TOF-TOF). Upon completion (3 d), the visible sequentially to a polypropylene tube: 5 μL 1 M sodium phos- aggregates that had formed in solution were removed by filtra- phate buffer, pH 7; 24 μL 2.5 mM CuSO4; 15 mM BTTAA; then tion, and 0.1% TFA added. This solution was pumped onto 15 μL 1 mM methoxy-PEG-azide; 15 μL DMSO; 15 μL 1.5 mM a C18 column, eluted as above, and lyophilized. GxTX Pra13; 10 μL 150 mM sodium ascorbate. The reaction mixture was briefly vortexed after each addition. The reaction His6-GxTX Expression. Oligonucleotides encoding the 36 amino was shielded from light, mixed with 1,000 rpm shaking at 25 °C + acid-GxTX sequence were ligated into a pET-30a( ) plasmid for 4 h before quench with 50 μL 10 mM EDTA and HPLC between the N-terminal hexahistidine tag and an enterokinase purification as described above. cleavage site at the C terminus of Venus yellow fluorescent Purity of conjugates was further confirmed by Tris/Tricine SDS/ protein. The His6-GxTX-Venus fusion protein was expressed in PAGE (Fig. S1D). Five hundred ng of each peptide or 0.7 μl × Escherichia coli strain BL21 (DE3). Cells were grown using 2 polypeptide standard (Bio-Rad 161–0326) was diluted in tricine YT culture medium in a shaker incubator at 37 °C until an op- sample buffer (Bio-Rad 161–0739) with 2% (vol/vol) 2-mercap- tical density of 0.5 was reached, and 1 mM isopropyl-1-thio-β-D- toethanol and denatured at 95 °C for 5 min. Samples were loaded galactopyranoside was added to induce expression. After 4 h, into a 10–20% (wt/vol) polyacrylamide gel (Bio-Rad 456–3116) cells were pelleted, resuspended in 20 mL of PBS, and then lysed with 100 mM Tris, 100 mM Tricine, 0.1% SDS, pH 8.3 running by heating to 80 °C for 7 min. The lysate was cooled on ice, and buffer. Gels were run at 30 V until dye entered gel (∼10 min), 1 mM PMSF, 1 mM MgCl2, and 0.1 mg DNaseI were added. then 100 V until dye reached the bottom of the gel (∼45 min). After 20 min, cells were centrifuged at 25,000 × g to remove Gels were fixed in 10% acetic acid, 40% (vol/vol) ethanol for 60 precipitate, and the supernatant concentrated by spin dialysis min, washed twice for 5 min in water, and stained overnight in (Amicon Ultra 30-kDa molecular weight cutoff). The fusion a colloidal Coomassie stain (2) composed of 0.12% Coomassie protein was purified using nickel affinity FPLC, eluting with an G-250, 10% (wt/vol) ammonium sulfate, 17% (wt/vol) o-phos- imidazole gradient. Imidazole was removed by spin dialysis, and phoric acid, and 20% (vol/vol) methanol. Gels were destained in the GxTX domain, as part of the fusion protein, was refolded by water at 4 °C, and imaged digitally (Bio-Rad 170–8270). air oxidation as above. Refolded His6-GxTX was cleaved from Synthesis of the azide beads was initiated by swelling amine Venus using recombinant enterokinase (1 U/50 μg substrate, No- functionalized (0.26 mmol/g) dendrimetic resin beads (Rapp- vagen), then subsequently purified by size exclusion FPLC to obtain polymere S30902) with DMF for 12 h in a polypropylene tube. pure His6-GxTx (Sequence: MHHHHHHSTS EGECGGFWWK Azido-PEG4-NHS (Conju-Probe), 3 equiv, and N,N-diisopropyl- Tilley et al. www.pnas.org/cgi/content/short/1406876111 1of7 ethylamine (3 equiv), dissolved in DMF, were added to the sus- and digitized at 100 kHz. All recordings were made after addition pended resin beads. The tubes were placed on a rotator for 8 h of 0.1% BSA. Toxins were added by flushing 100 μL through until negative Kaiser tests confirmed complete coupling. The ob- a low-volume recording chamber (Warner R-24N). For dissocia- tained azide-functionalized beads were washed with dichloro- tion rates, the chamber was under constant perfusion of the ex- methane, methanol, and DMF, respectively, three times each, then ternal solution at 2 mL/min and 200 μL of toxin was perfused dried and stored at 4 °C. For conjugation, beads were swollen for through the recording chamber while holding at −100 mV. Toxin 1 h in DMF, rinsed three times each with 1:1 DMF:water, water, binding rate and affinity with a −100 mV holding potential was then 0.5 M sodium phosphate, pH 7. GxTX Pra13 was conjugated measured from the change in current level at the end of 100-ms to beads by copper-mediated azido-alkyne condensation. Azide- steps to 0 mV. These test pulses were repeated every 2 s. Toxin functionalized beads were reacted with alkyne-functionalized binding rate and affinity with a 0-mV holding potential was molecules. To 2-mg beads in 122 μl 0.5 M sodium phosphate, measured by continuous recording at 0 mV after inactivation had pH 7, in a 1.5-mL polypropylene tube, reagents were added and reached steady state. P/N subtraction was not used with these briefly vortexed after each addition: 20 μl of 200 μMGxTXPra13; protocols. Cells were induced longer to have more channels for 8 μl2.5mMCuSO4, 15 mM BTTAA; 30 μlDMSO;10μL 150 these experiments. It is not known whether toxin interacts dif- mM sodium ascorbate. For fluorophore labeled beads, GxTX so- ferently with activated vs. inactivated states. Toxin dissociation lution was substituted with water, and DMSO included 10 mM al- with a −100 mV holding potential was measured from the change kyne-PEG3-5(6)-carboxytetramethylrhodamine (Click Chemistry in current level at the end of 100 ms steps to 0 mV.
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