Figure 1. Determination of longitudinal backbone amide ¹⁵N paramagnetic relaxation enhancements in Cys-EDTA-Cu²⁺ GB1 mutants by solid-state NMR spectroscopy.

a, Pulse scheme used to determine residue-specific longitudinal ¹⁵N rate constants, R₁, from a series of 2D ¹⁵N-¹³CO correlation spectra recorded with different values of the relaxation delay, τ_relax. See Supplementary Fig. S1 for the complete pulse scheme description. b, Ribbon diagram of GB1 (PDB entry 2GI9) with the locations of the non-native Cys-EDTA-Cu²⁺/Zn²⁺ tags in point mutants investigated in this study (residues 8, 19, 28, 42, 46 or 53) indicated by blue spheres. c, Small regions of 2D ¹⁵N-¹³CO spectra recorded for ¹³C,¹⁵N-labeled 8EDTA-Zn²⁺ (green contours) and 8EDTA-Cu²⁺ (magenta contours) mutants with longitudinal ¹⁵N relaxation delays of 100 μs and 4 s, as indicated in the bottom left corner of each spectrum. The resonance assignments have been established previously,³⁵ and cross-peaks are labeled according to the amide ¹⁵N frequency of the residue involved. d, Representative site-resolved ¹⁵N longitudinal relaxation trajectories for residues T51 and T53 in 8EDTA-Cu²⁺ and Zn²⁺. Experimental trajectories for 8EDTA-Zn²⁺ (green circles) and 8EDTA-Cu²⁺ (magenta circles) correspond to integrated cross-peak intensities in a series of 2D NCO spectra recorded as a function of the relaxation delay. Simulated best-fit trajectories to decaying single exponentials used to extract the longitudinal ¹⁵N relaxation rate constants are shown as solid lines of the corresponding color. The ¹⁵N PREs are calculated by taking the difference between the ¹⁵N R₁ values obtained for the EDTA-Cu²⁺ and EDTA-Zn²⁺ proteins. e, Same as panel c for 28EDTA-Zn²⁺ (blue contours) and 28EDTA-Cu²⁺ (red contours). f, Same as panel d for residues Q32 and A48 in 28EDTA-Zn²⁺ (blue circles/lines) and 28EDTA-Cu²⁺ (red circles/lines).