Figure 2.

1D-¹⁹F NMR spectra of ¹⁹F-Man2/Man3 in the presence of [CVN^ΔA]_ssm, a variant that contains a single glycan binding site on domain B, CVN^mDB, a variant that contains a single glycan binding site on domain A, and CV-N^P51G that is wild-type-like with both glycan binding sites present, at 280 K. CV-N domains are represented by two elongated crescents, representing the two binding sites. Site 1 on domain B is colored pink and site 2 on domain A in green. A) ¹⁹F-1D NMR spectra of 200 μM ¹⁹F-Man2 in the presence of [CVN^ΔA]_ssm, CVN^mDB, and CV-N^P51G. B) ¹⁹F-1D NMR spectra of 50 μM ¹⁹F-Man3 in the presence of [CVN^ΔA]_ssm, CVN^mDB and CV-N^P51G. Bound-state and free-state signals are in slow exchange for [CVN^ΔA]_ssm in C) and CVN^mDB in D. In the titration curves (bottom panels) the bound fraction is derived from the signal intensity (1-I_free/I₀) during the titration, with I_free the intensity of the free ligand signal at each point in the titration, and I₀ the intensity of the free ligand signal at the beginning of the titration. In C) [CVN^ΔA]_ssm was titrated into 50 μM ¹⁹F-Man3 (top panel), with molar ratios of protein/glycan: 0:1, 0.5:1, 1:1, 1.5:1. In D) CVN^mDB was titrated into 50 μM ¹⁹F-Man3 (top panel), with protein/glycan molar ratios: 0:1, 0.5:1, 1:1, 2:1, and 3:1.