Figure 1.
Alignment of the primary structures of agitoxin-2 (AgTx2) and margatoxin (MgTx).
Conserved residues are highlighted with a grey background and cysteine residues are shown in red. The lines above the sequences indicate disulfide bonds. The secondary structure of AgTx2 (based on PDB file 1AGT) is shown above the sequences.
Figure 2.
Western blot using anti-His6 primary antibody showing expression and purification of recombinant (A) AgTx2 and (B) MgTx produced in P. pastoris.
The mass (in kDa) of the prestained molecular weight markers (“Stds”) is indicated. Lane 1, culture supernatant after methanol induction of toxin expression; Lane 2, recombinant peptide after purification using nickel affinity chromatography; Lane 3 recombinant peptide after RP-HPLC purification.
Figure 3.
Chromatograms showing purification of (A) rAgTx2 and (B) rMgTx using HPLC.
RP-HPLC was performed on a Vydac C18 column using a flow rate of 1 ml/min and a gradient of 20–30% acetonitrile over 40 min. The inset in each chromatogram is a MALDI-TOF spectrum showing the average mass of purified recombinant toxin.
Figure 4.
Inhibition of mouse KV channels and human T lymphocyte proliferation by rAgTx2, rMgTx, and rMgTX-K28A.
Inhibition of mKV1.3 currents in stably transfected L929 cells by (A) 200 nM rAgTX2, (B) 1 nM rMgTX, and (C) 10 nM MgTX-K28A. Dose-dependent inhibition of mKV1.3 (▪) and mKV1.1 (○) currents by (D) rAgTx2, (E) rMgTX, and (F) rMgTX-K28A (each data point is the mean of 3–5 determinations). Dose-dependent inhibition of [3H] thymidine incorporation by human T lymphocytes by (G) rAgTx2, (H) rMgTx, and (I) rMgTx-K28A. Error bars represent the SEM from three independent experiments with cells from different donors. Asterisks indicate statistically significant differences (***, p≤0.001; **, p<0.01).
Figure 5.
1H-15N HSQC spectra of recombinant (A) AgTx2 and (B) MgTx produced in P. pastoris.
Correlation peaks are labelled according to residue type and sequence number. The peaks connected by horizontal lines correspond to the side-chain NH2 groups of Gln and Asn residues. Peaks marked with a red asterisk correspond to residues from the N-terminal His6 tag.
Figure 6.
Amide-proton strips from 3D 15N-edited NOESY spectra of (A) rAgTx2 (G1–K38) and (B) rMgTx (T1–H39).
Sequential αN, βN, and NN connectivities that facilitated sequence-specific resonance assignments are indicated by red, green, and orange lines, respectively. Medium- and long-range dipolar connectivities that facilitated assignment of secondary structure are highlighted and labelled in blue.
Figure 7.
Secondary structure of recombinant (A) AgTx2 and (B) MgTx determined from NMR data.
Dipolar connectivities observed in 2D NOESY spectra are shown as double-sided red arrows. Cross-strand hydrogen bonds inferred from 1H-2H exchange rates are depicted as dashed orange lines. Green lines indicate disulfide-bonds inferred from NOE connectivities. The shaded arrows indicate the direction (N→C) of the three β strands.