Biochemistry torsion angle was constrained in the ranges of −60 ± 30°, 60 ± 30°, and 180 ± 30°, respectively.75 Quantitative determination of the cross-peak intensities was based on the counting levels. Observed NOE data were classified into four distance ranges (1.8−2.7, 1.8−3.5, 1.8−5.0, and 1.8−6.0 Å) that corresponded to strong, medium, weak, and very weak NOE values, respectively. Pseudoatoms were used for the methyl protons or the nonstereospecifically assigned methylene protons.76 Correcting factors for the use of pseudoatoms were added to the distance constraints, and 0.5 Å was added to the distance constraints involving methyl protons.77 For each disulfide bond, we used three distance constraints, S(i)−S(j), S(i)−Cβ(j), and S(j)−Cβ(i), whose target values were set to 2.02 ± 0.02, 2.99 ± 0.5, and 2.99 ± 0.5 Å, respectively.78 The hydrogen bond acceptors for the slowly exchanged amide protons were identified by analyzing the preliminarily calculated structures.79,80 The distance restraints on the hydrogen bonds were added as target values of 1.8−2.3 Å for NH(i)−O(j) and 2.8−3.3 Å for N(i)−O(j) bonds. All calculations were conducted on an SGI Octane2 workstation using the X-PLOR version 3.851.81 The three- dimensional structures were calculated on the basis of the experimentally derived distance and torsion angle constraints using a dynamically simulated annealing protocol starting from a template structure with randomized backbone ϕ and ψ torsion angles. The final 20 structures with the lowest energy and smallest Lennard-Jones van der Waals energy were chosen. The convergence of the calculated structures was evaluated in terms of the structural parameters. There were root-mean- square deviations (rmsds) from the experimental distances and dihedral constraints, from the energetic statistics (FNOE, Ftor, Frepel, and EL‑J), and from the idealized geometry. The structures were analyzed using the PROCHECK_NMR,82 PROMOTIF,83 MOLMOL,84 and MolProbity.85,86 The dis- tributions of the backbone dihedral angles in the final converged structure were evaluated by representation of the Ramachandran dihedral pattern, which indicated the deviations from the allowed ϕ and ψ angle limits. The degrees of angular variation among the converged structures were further assessed using an angular order parameter.87 The solvent-accessible surface areas for the side chains of the amino acid residues were calculated with a solvent radius of 1.4 Å. Structural figures were generated using MOLMOL and INSIGHT II 2000 (Accelrys Inc.). ■ RESULTS AND DISCUSSION NMR Sample Preparation. Kurtoxin was insoluble in aqueous solution, even at a concentration of <1 mM. We therefore tested whether kurtoxin could be solubilized using 0− 30% acetonitrile (CH3CN) without disrupting the protein’s intrinsic structure. Figure 2 shows that the CD spectra for kurtoxin recorded in the absence or presence of 10, 15, 20, 25, and 30% CH3CN are nearly identical in the far-UV region (200−250 nm), indicating that the backbone structure of kurtoxin is affected little by the addition of CH3CN. Moreover, we obtained high-quality NMR spectra with 1 mM kurtoxin in the presence of 25% deuterated acetonitrile (CD3CN), strongly suggesting that the addition of CD3CN effectively prevented the aggregation of kurtoxin (Figure 3). In the 1H−15N HSQC spectra recorded in pure water, several hydrophobic peaks, including Tyr17, Tyr18, Trp38, Gly39, Trp40, and Leu42, could not be detected, most likely because of the line broadening in the absence of CD3CN (Figure 3). This appeared to be due to the Article Figure 2. CD spectra of 0.05 mM kurtoxin in H2O containing 0, 10, 15, 20, 25, or 30% CH3CN [0.01 M sodium phosphate (pH 7.0)] at 20 °C. Figure 3. 1H−15N HSQC spectra of kurtoxin in the presence (black) and absence (green) of 25% CD3CN at 288 K. The resonance assignments are indicated by the one-letter amino acid codes and residue numbers. The dotted circles indicate additional and unassigned peaks. self-aggregation of kurtoxin molecules. On the other hand, in the 1H−15N HSQC spectra recorded in the presence of 25% CD3CN, all of the backbone 1H−15N cross peaks were identifiable, and the intensities of nearly all the peaks were increased without large chemical shift variations (Figure S1 of the Supporting Information). Addition of CH3CN can weaken the hydrophobic interactions between surface hydrophobic regions without altering the three-dimensional structure of the proteins.88 Nonetheless, CH3CN can influence chemical shift variations by changing the polarity of the accessible atom environments; of those, the chemical shifts located in the flexible N- and C- terminal regions (Ala63 of kurtoxin in Figure S1 of the Supporting Information) and loops are most sensitive.89 Comparison of the CD and 1H−15N HSQC spectra recorded in the presence and absence of 25% CD3CN indicates that CD3CN prevented the hydrophobic-related aggregation of kurtoxin without disrupting its overall topology. Linear kurtoxin 1864 dx.doi.org/10.1021/bi201633j | Biochemistry 2012, 51, 1862−1873