Table 1.
Oxidative folding yield of DkTx under different conditionsa.
Table 2.
Folding properties of DkTx and its analogues.
Figure 1.
(A) Design of a synthetic DkTx gene. (B) Sequence of the toxins used in the experiments. Note that DkTx has an N-terminal Gly residue because of the hydroxyl amine cleavage of Asn-Gly sequence added to the N-terminus.
Figure 2.
Expression of DkTx in E. coli.
SDS-PAGE gels (15%) were stained with Coomassie Blue. Lane M, molecular weight markers (kDa); lane T, total cell lysate; lane P, pellet fraction; lane S, soluble fraction.
Figure 3.
Purification and activity of the DkTx folding product.
(A) Purification of linear DkTx. Cleavage of the fusion protein and reduction of the disulfide bond were accomplished using hydroxylamine and DTT, respectively, as described in methods. (B) Linear DkTx was folded in 1 M NH4OAc (pH 8.0) buffer containing 1 M GdnHCl, 1 mM EDTA, 2.5 mM GSH and 0.25 mM GSSG for 5 days at 4°C. The toxins were purified using a linear gradient of 29–44% solvent B for 15 min at a flow rate of 14 ml/min, where solvent A was water containing 0.1% TFA and solvent B was acetonitrile containing 0.1% TFA. (C) Fraction 5 activated TRPV1 expressed in oocytes. Holding voltage was −60 mV.
Figure 4.
HPLC profiles of DkTx during the folding reaction.
The peptides were separated using a linear gradient of 5–65% solvent B for 30 min at a flow rate of 1 ml/min, where solvent A was water containing 0.1% TFA and solvent B was acetonitrile containing 0.1% TFA. (A) Oxidative folding of DkTx in redox buffer was monitored by HPLC. Asterisks indicate correctly folded DkTx. (B) HPLC chromatogram of purified DkTx. Dashed and solid arrows indicate the minor and major forms of DkTx, respectively. (C) The minor form was collected and re-injected after 75 min. (D) The major form was collected and re-injected after 175 min. (E) Co-injection of native and synthetic DkTx.
Figure 5.
Folding kinetics of K1, K2 and DkTx.
(A) HPLC chromatograms showing the folding of DkTx, K1 and K2. The left panel depicts the linear toxins. Arrows indicate correctly folded forms of the toxins. The toxins were eluted with a linear gradient of 5–65% solvent B for 30 min at a flow rate of 1 ml/min, where solvent A was water containing 0.1% TFA and solvent B was acetonitrile containing 0.1% TFA. (B) Comparison of folding kinetics of DkTx, K1 and K2. At each time point, aliquots were withdrawn and acidified by adding acetic acid. Quantification of the folding specifies was accomplished using HPLC. The correctly folded form (%) was determined based on the areas of the HPLC peaks. The curves represent fits of the data to a one-phase association.
Figure 6.
Activity of K1, K2 and DkTx on TRPV1 channels.
(A) Synthetic K1, K2 and DkTx activate TRPV1 channels. Oocytes were held at −60 mV and toxins were added to the recording chamber. (B) Concentration-dependence for activation of TRPV1 by the toxins.
Figure 7.
CD spectra of K1, K2 and DkTx.
The CD spectra were recorded in 0.01 M sodium phosphate (pH 7.0) at 20°C. K1+K2 depicts the added values of the CD spectra from K1 and K2.