Figure 4.

NMR titration of [CVN^ΔA]_ssm with ¹⁹F-Man2 recorded at 298 K and ¹⁹F-Man3 recorded at 280 K, respectively. A) Perturbed amide resonances in the [CVN^ΔA]_ssm spectrum clearly reside in domain B and are labeled by amino acid name and number. The left panel display a superposition of ¹H-¹⁵N HSQC NMR spectra of [CVN^ΔA]_ssm in the absence (black) and presence of 25 molar equivalents of ¹⁹F-Man2 (green). The right panel displays the titration curve (chemical shift difference versus ligand/protein molar ratio), with the chemical shift difference defined as: Δδ = [(Δδ_H)² + (Δδ_N × 0.17)²]^1/2, where Δδ_H and Δδ_N are the observed chemical shift changes for ¹H and ¹⁵N, respectively. The system is in fast exchange, as evidenced by a single signal corresponding to glycan free and glycan bound [CVN^ΔA]_ssm. B) Superposition of ¹H-¹⁵N HSQC spectra of 50 μM [CVN^ΔA]_ssm in the absence (black) and presence (green) of 3 molar equivalents of ¹⁹F-Man3 (left panel). The right panel displays the titration curve at glycan/protein molar ratios: 0:1, 0.5:1, 1:1, 1.5:1, 2:1, and 3:1. The system is in slow exchange, as evidenced by separate signals for glycan free and glycan bound [CVN^ΔA]_ssm. The binding isotherm is derived from the relative signal intensities (1-I_free/I₀) (right panel).