Figure 5. Titration of CVN^mutDB with mannose sugars.

(A) Isothermal titration of CVN^mutDB with Man-3. The titration data for 25 automated injections are shown in the top panel and the total heat released as a function of the molar ratio of Man-3 versus CVN^mutDB is displayed in the bottom panel (black). For comparison, previous titration data obtained for CVN^mutDB with Man-9 are shown by a dotted line (Barrientos et al., 2006). (B) Isothermal titration of CVN^mutDB with Man-2. The titration data for 20 automated injections are shown in the top panel and the total heat released as a function of the molar ratio of Man-2 versus CVN^mutDB is displayed in the bottom panel. The continuous line represents the nonlinear least-squares best fit to the experimental data using a one site model. (C) NMR titration of CVN^mutDB with Man-2. The top right hand panel shows the ¹H-¹⁵N HSQC chemical shift titration curve (bound – free state) versus ligand/ protein molar ratio. The chemical shift difference is defined as: Δδ = [(Δδ _HN) ² + (Δδ_N × 0.17) ²]^1/2 The bottom panel shows the superposition of ¹H-¹⁵N HSQC spectra without and in the presence of 12 equivalents of Man-2 in black and magenta, respectively. Selected resonances that experience large shifts upon addition of the disaccharide are labeled with residue name and number in an expanded view in the two top left panels.