Biochemistry Article Figure 4. Solution structure of kurtoxin. (A) Stereopairs of backbone heavy atoms (N, Cα, and C) for the 20 converged structures of kurtoxin. These are the results of the best-fit superposition of the backbone heavy atoms of the molecule. N and C indicate N- and C-terminal positions, respectively. (B) Ribbon structure of kurtoxin. Schematic diagram of kurtoxin illustrating the location of the β-strands (cyan), α-helix (red and yellow), and disulfide bonds (numbered ball and stick). Long loop I and long loop II correspond to residues Tyr5−Asn20 and Pro50−Ala63, respectively. (C) Tubular representation of kurtoxin illustrating its motional properties. The diameter of the tube is proportional to the atomic rmsds of the backbone atoms: red for residues whose backbone rmsds are >1.0, cyan for those with β-strands, and yellow for those with an α-helix. (D) Surface profile of kurtoxin. The surface hydrophobic patches and charged residues are indicated. The molecular surface of kurtoxin is shown in color according to the electrostatic potential: red for negatively charged amino acids, blue for positively charged amino acids, and white for uncharged or hydrophobic amino acids. The left and right figures are rotated 180° with respect to one another about a vertical axis. These figures were generated using MOLMOL. was also refolded in a redox solution containing 30% CH3CN,59 suggesting the nativelike conformation of kurtoxin is stabilized in an aqueous solution containing CH3CN. A small number of additional minor peaks were observed in NMR spectra, including 1H−15N HSQC spectra in the presence and absence of CD3CN (Figure 3). The intensity ratio of the minor peaks is <5−10% compared to that of the major peaks according to relevant NMR signals. Unfortunately, we failed to assign these minor peaks because the signals were very weak and it was hard to sequentially connect these minor peaks. In a recent study, two stable conformations of BmκαTx11, a kurtoxin-homologous scorpion α-toxin, were identified by using NMR spectroscopy.90 We assume that the minor peaks of kurtoxin also come from a possible minor conformation similar to BmκαTx11. NMR Spectroscopy. Sequence-specific assignments for the backbone atoms of kurtoxin were obtained from an analysis of heteronuclear 3D NMR spectra [HNCACB, CBCA(CO)- NNH, HNCA, and HN(CO)CA] recorded in 25% CD3CN at pH 4.0 and 288 and 278 K using a uniformly 15N- and 13C- labeled protein. All backbone Cα atoms were assigned except those of Cys12, Arg14, Ile15, Asp25, Leu26, Ala32, and Trp40. The backbone and side chain protons were obtained from 2D NMR spectra (DQF-COSY, TOCSY, and NOESY) and 3D 15N NOESY-HSQC and 3D 15N TOCSY-HSQC spectra recorded in 25% CD3CN at pH 4.0 and 288 K. The assignments of all backbone and side chain protons were complete except for those of the Hζ atoms of Lys13 and Lys21, Hδ2 of Asn19 and Asn20, and Hδ and Hε of Tyr22. For all Pro residues (Pro6 and Pro50), strong sequential dα,δ and no dα,α were observed in the NOESY spectra, indicating the toxin’s proline residues are all in the trans configuration. Identification of Secondary Structure Elements. As summarized in Figure S2 of the Supporting Information, the pattern of observed NOEs and chemical shift index (CSI) values for Hα was ultimately interpreted in terms of the secondary structure of the molecule. The weak 3JHNα coupling constants, strong dNN NOE peaks and dαN(i,i+3) and dαβ(i,i+3) NOE correlations, and the CSI value of −1 all indicate an α- helical conformation for residues Lys21−Leu30. The extent of the β-strands and their relative orientations within the β-sheet structure were determined using standard criteria: large 3JHNα coupling constants (Ile2, Asp3, Try36, Cys37, Tyr45, Cys46, and Gln47), strong sequential dαN, interstrand NH−NH and NH− C α H connectivities, and slowly exchanging amide protons (Ile2, Gly4, Gly35, Tyr36, Cys44, Tyr45, Cys46, Gln47, and Leu49). 1865 dx.doi.org/10.1021/bi201633j | Biochemistry 2012, 51, 1862−1873