FIGURE 4.

Conformational heterogeneity in rWTX molecule associated with the cis-trans-isomerization of the Arg-32–Pro-33 peptide bond. A and B, two-dimensional ¹H,¹⁵N-HSQC spectra of 0.5 mm rWTX and rWTX(P33A) (pH 3.0, 40 °C). Signals of rWTX conformers with trans- and cis-configuration of Arg-32–Pro-33 peptide bond are marked by red and blue lettering, respectively. The signals of the cis form are also marked by asterisks. C, normalized difference of ¹H^N, ¹H^α, and ¹⁵N^H chemical shifts (√(Δδ¹H^N)² + (Δδ¹H^α)² + (Δδ¹⁵N^H/5)²) between “trans” and “cis” forms of rWTX. The data for proline residues and for Arg-37 and Tyr-38 (crosses) were not calculated. Signals of Arg-37 and Tyr-38 residues from the trans form of rWTX were not assigned. D, analysis of conformational heterogeneity in rWTX mutants. The fragments of one-dimensional ¹H NMR spectra containing H^Nϵ1 signals of the Trp-36 side chain are shown. The relative content (%) of the trans form is shown above each spectrum.