Figure 4.

Metal binding studied by 2D-NMR. (A) The ¹H-¹⁵N HSQC spectra of EEDDQ at different Ca²⁺ concentrations. The Ca²⁺-induced chemical shift perturbations are indicated by the arrows. Several assigned resonances are labeled. (B) Determination of the Ca²⁺-binding affinity of EEDDQ by following the chemical shift perturbations as a function of Ca²⁺ concentration. D62 (solid circle); downfield resonance of Q64 sidechain amide (solid square); upfield resonance of Q64 sidechain amide (solid triangle); K66; ×, Q22 (inverted solid triangle). The solid lines are generated by assuming a 1:1 Ca²⁺ binding to EEDDQ. The results from different resonances are similar. (C) Overlay of the HSQC spectra of NENDN in the presence of 0.05 mM EGTA (red) or 13.1 mM Ca²⁺ (green).