Figure 3.
Figure 3. α-Scorpion toxins interact with the isolated rNav1.2a VSD IV paddle motif. (A) Shown are the CD spectra of the rNav1.2a VSD II and IV paddle peptides used in this paper. Analysis of both spectra revealed the presence of ∼75% structured (α-helix/β-sheet) and ∼25% unstructured peptide. Inset shows the crystal structure of the NavAb voltage sensor (3RVY) (Payandeh et al., 2011) in which the paddle motif is indicated in green; wheat shows S1 and S2 helices, and white indicates the portion of S3 and S4 helices outside the paddle. (B) Representative association and dissociation kinetic curves obtained using SPR after the application of varying concentrations of AaHII (15–2,000 nM) over a sensor chip to which 50 fmol of the rNav1.2a VSD IV paddle peptide was linked. Toxin was applied after obtaining a steady baseline. Colored traces represent toxin binding obtained after subtraction of the signal from the control flow cell. Green dotted lines depict a fit of the data to a heterogeneous surface ligand model, a typical SPR analysis method (Schuck and Zhao, 2010), which yielded a high affinity KD1 of 479 ± 241 nM (RUmax1 = 135) and a lower affinity KD2 of 747 ± 203 nM (RUmax2 = 23; n = 3; all results presented as mean ± SD). The respective contributions of RUmax1 (∼85%) and RUmax2 (∼15%) to the overall RUmax (100%) are reminiscent of the percent structured (∼75%) versus unstructured (∼25%) paddle peptide as observed in the CD spectrum. Toxin concentrations are indicated on the right in a shade of gray corresponding to the sensorgram. (C) Representative SPR traces after the application of 100 nM KTX, AaHI, AaHII, LqqV, BomIV, TsVII, and CssIV over a sensor chip to which the rNav1.2a VSD II (left) or IV (right) paddle peptide (500 fmol) was linked. Toxin was applied after obtaining a steady baseline. (D) Binding capacities of 100 nM α-scorpion toxins AaHI, AaHII, LqqV, the α-like scorpion toxin BomIV, and the β-scorpion toxins TsVII and CssIV to the VSD II and IV paddle motifs using SPR (error bars represent ± SEM). Y-axis represents the maximum RUs obtained after toxin application. Note that 500 fmol paddle peptide was used in C and D as opposed to 50 fmol in B. As a result, RUs differ by a factor of ∼10.

α-Scorpion toxins interact with the isolated rNav1.2a VSD IV paddle motif. (A) Shown are the CD spectra of the rNav1.2a VSD II and IV paddle peptides used in this paper. Analysis of both spectra revealed the presence of ∼75% structured (α-helix/β-sheet) and ∼25% unstructured peptide. Inset shows the crystal structure of the NavAb voltage sensor (3RVY) (Payandeh et al., 2011) in which the paddle motif is indicated in green; wheat shows S1 and S2 helices, and white indicates the portion of S3 and S4 helices outside the paddle. (B) Representative association and dissociation kinetic curves obtained using SPR after the application of varying concentrations of AaHII (15–2,000 nM) over a sensor chip to which 50 fmol of the rNav1.2a VSD IV paddle peptide was linked. Toxin was applied after obtaining a steady baseline. Colored traces represent toxin binding obtained after subtraction of the signal from the control flow cell. Green dotted lines depict a fit of the data to a heterogeneous surface ligand model, a typical SPR analysis method (Schuck and Zhao, 2010), which yielded a high affinity KD1 of 479 ± 241 nM (RUmax1 = 135) and a lower affinity KD2 of 747 ± 203 nM (RUmax2 = 23; n = 3; all results presented as mean ± SD). The respective contributions of RUmax1 (∼85%) and RUmax2 (∼15%) to the overall RUmax (100%) are reminiscent of the percent structured (∼75%) versus unstructured (∼25%) paddle peptide as observed in the CD spectrum. Toxin concentrations are indicated on the right in a shade of gray corresponding to the sensorgram. (C) Representative SPR traces after the application of 100 nM KTX, AaHI, AaHII, LqqV, BomIV, TsVII, and CssIV over a sensor chip to which the rNav1.2a VSD II (left) or IV (right) paddle peptide (500 fmol) was linked. Toxin was applied after obtaining a steady baseline. (D) Binding capacities of 100 nM α-scorpion toxins AaHI, AaHII, LqqV, the α-like scorpion toxin BomIV, and the β-scorpion toxins TsVII and CssIV to the VSD II and IV paddle motifs using SPR (error bars represent ± SEM). Y-axis represents the maximum RUs obtained after toxin application. Note that 500 fmol paddle peptide was used in C and D as opposed to 50 fmol in B. As a result, RUs differ by a factor of ∼10.

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