NMR analysis of apoNCS in various concentrations of GdnHCl. (A) 1H-15N HSQC spectra of apoNCS obtained at 25°C in 0, 1.2, and 4 M GdnHCl. Residues which show maximum perturbation in the intermediate state in 1.2 M GdnHCl are boxed. (B) The weighted average (of 15N and 1H) chemical shift perturbation of residues in 1.2 M GdnHCl. ApoNCS samples (720 μM) were prepared in 10 mM phosphate buffer (pH 3) in 90% H2O and 10% D2O. The β-strands in the protein are shown in roman numerals (see Fig. 1).