FIGURE 4.

Mapping Zn²⁺ binding to TTR by TROSY-HSQC measurements at pH 7.5. The chemical-shift perturbations (CSP) in the NMR spectrum of fully ¹⁵N-²H labeled WT-TTR (100 μm) were measured at different concentrations of ZnCl₂. CSP derived from differences between a reference spectrum of WT-TTR in the absence of Zn²⁺ and the spectra in the presence of Zn²⁺ were obtained for: (A) 100 μm ZnCl₂; (B) 200 μm ZnCl₂; (C) 300 μm ZnCl₂; (D) 400 μm ZnCl₂; (E) 900 μm ZnCl₂, and (F) 900 μm ZnCl₂ + 1 mm EDTA. Blue bars are residues whose signal vanished during the experiment; red bars are non-assigned residues; gray bars are prolines. Images at the right show where CSP occurred in WT-TTR at each Zn²⁺ concentration, marked in red in a ribbon representation of Zn²⁺:TTR pH 7.5 (PDB ID 3GRG). For clarity, only one of the four monomers is marked. Zn²⁺ atoms are represented as gray spheres. CSP was calculated for each assigned main-chain amide using Equation 1. Dotted lines show the threshold for considering a CSP significant (0.025 ppm).