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. 2018 Nov 23;29(1):45–58. doi: 10.1093/glycob/cwy100

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© The Author(s) 2018. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fig. 6. — Analysis of GtfC_100-23 and GtfC₅₃₆₀₈ ligand specificity. (A–F) Differential scanning fluorimetry (DSF) analysis. (A) Melt curve of GtfC₅₃₆₀₈ in the presence of increasing concentrations of UDP-GlcNAc. (B) Tm of GtfC₅₃₆₀₈ in the presence of increasing concentrations of UDP, UDP-Gal, UDP-Glc and UDP-GlcNAc. Error bars represent the standard error of the mean of four technical replicates. (C) Melt curve of GtfC₅₃₆₀₈ in the presence of 4 mM UDP-GlcNAc, UDP-Glc, UDP-Gal and UDP. (D) Melt curve of GtfC_100-23C in the presence of 4 mM UDP-GlcNAc, UDP-Glc, UDP-Gal and UDP. (E) Melt curves of GtfC₅₃₆₀₈ in the presence of 5 mM Mn²⁺ (left), or 5 mM Mn²⁺ and 4 mM UDP-GlcNAc. (F) Melt curves of GtfC_100-23C in the presence of 5 mM Mn²⁺ (left), or 5 mM Mn²⁺ and 4 mM UDP-Glc. Since no significant difference was observed between the different divalent ions, only Mn²⁺ is shown. (G–L) Saturation Transfer Difference (STD) NMR analysis. (G), (H), (I) Binding epitope maps for the complexes of GtfC_100-23 with UDP-GlcNAc, UDP-Glc and UDP-Gal, respectively. Bottom row, (J), (K), (L) binding epitope maps for the complexes of GtfC₅₃₆₀₈ with UDP-GlcNAc, UDP-Glc and UDP-Gal, respectively. See also Table I and Figure S2 for the competition assays of the sugar nucleotides against GtfC_100-23 and GtfC₅₃₆₀₈.