Figure 5.

NOE cross-peaks between the C-peptide and the OB-domain. The spectra were recorded of a 0.37 mM solution of ¹⁵N/¹³C-labeled SSB at pH 3.4 and 25°C at a ¹H NMR frequency of 800 MHz. (A) Selected spectral regions of a 3D NOESY-¹³C-HSQC spectrum taken at δ₂(¹³C) = 38.37 (D173C^β) and 19.88 ppm (V29C^γ1). The NOE cross-peaks between D173H^β and V29H^γ are at the intersection of the dashed lines drawn at δ_1,3(¹H) = 2.84 (D173H^β1), 2.67 (D173H^β2) and 0.56 ppm (V29H^γ1). (B) Spectral region of the 3D NOESY-¹³C-HSQC spectrum taken at δ₂(¹³C) = 130.50 ppm (F171C^δ). The NOE cross-peaks between F171H^δ and V58H^γ are at the intersection of the dashed lines drawn at δ₂(¹H) = 7.11 (F171H^δ) and δ₁(¹H) = 0.76 (V58H^γ1) and 0.52 ppm (V58H^γ2). The cross-peak between F171H^δ and V58H^γ2 appears to be slightly shifted in the δ₁-dimension because of an overlapping NOE cross-peak between F171H^δ and V29H^γ or V101H^γ. (C) A 2D (H)CB(CGCC-TOCSY)H^ar spectrum correlating the ¹³C^β with the aromatic ¹H chemical shifts of phenylalanine residues (28). The dashed lines are drawn at δ₁(¹³C) = 38.93 (F171C^β) and δ₂(¹H) = 7.11 ppm (F171H^δ). This spectrum proves that the resonances marked by the dashed line belong to Phe171 rather than to Phe60, which also produces NOE cross-peaks to Val58. Only one of the aromatic signals of Phe171 is well resolved, whereas the others overlap with the stronger peaks from Phe147 and Phe177.