The interaction of GMCBM21 with carbohydrates was tested by NMR titration experiments using α-CD (Thermo Fisher Scientific) and β-CD (Acros Organics). To this end, 2D [1H,15N] HSQC spectra at 298 K were recorded at 500 MHz 1H Larmor frequency. Experiments were performed in 20 mM phosphate (pH 6.5), 50 mM NaCl, 10 mM DTT, and 90% H2O/10% D2O, with α-CD and β-CD titrated into 15N-labeled GMCBM21 at 1:1, 1:2, 1:5, 1:10, 1:20, and 1:40 (protein:sugar) molar ratios. 15N-GMCBM21 was used at concentrations of 300 μM for the 1:0.5, 1:1, and 1:2 titrations, 190 μM for the 1:20 titration, 100 μM for the 1:40 titration, and 40 μM for the 1:5 titration. In the 1:20 and 1:40 titration points, the concentration of GMCBM21 was limited by the maximum solubility of β-CD in this buffer (less than 10 mM). Saturation of sugar binding by GMCBM21 was achieved at a ratio of 1:20 for both α-CD and β-CD. Chemical shift differences (Δδ) between free GMCBM21 (no α-CD or β-CD) and sugar-bound GMCBM21 (1:20 molar ratio) spectra were calculated usingEmbedded Image

To test the interaction of carbohydrates with GM64–237:PP1α7–330, a 2D [1H,15N] TROSY spectrum was recorded with β-CD (1:20 molar ratio; for GMCBM21 alone, saturation was observed at this ratio). β-CD was chosen because of its stronger binding affinity to GMCBM21 when compared to α-CD. Chemical shift differences between GM64–237:PP1α7–330 and GM64–237:PP1α7–330:β-CD were calculated as described above. The data were recorded on a Bruker Avance NEO 800MHz 1H Larmor frequency equipped with a cryoprobe at 298 K.

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