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. 2017 May 3;139(17):6210-6216.
doi: 10.1021/jacs.7b01929. Epub 2017 Apr 21.

Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy

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Exploiting Uniformly 13C-Labeled Carbohydrates for Probing Carbohydrate-Protein Interactions by NMR Spectroscopy

Gustav Nestor et al. J Am Chem Soc. .

Abstract

NMR of a uniformly 13C-labeled carbohydrate was used to elucidate the atomic details of a sugar-protein complex. The structure of the 13C-labeled Manα(1-2)Manα(1-2)ManαOMe trisaccharide ligand, when bound to cyanovirin-N (CV-N), was characterized and revealed that in the complex the glycosidic linkage torsion angles between the two reducing-end mannoses are different from the free trisaccharide. Distances within the carbohydrate were employed for conformational analysis, and NOE-based distance mapping between sugar and protein revealed that Manα(1-2)Manα(1-2)ManαOMe is bound more intimately with its two reducing-end mannoses into the domain A binding site of CV-N than with the nonreducing end unit. Taking advantage of the 13C spectral dispersion of 13C-labeled carbohydrates in isotope-filtered experiments is a versatile means for a simultaneous mapping of the binding interactions on both, the carbohydrate and the protein.

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Figures

Figure 1
Figure 1
(a) Structural formula of Man3 (Manα(1–2)Manα(1–2)ManαOMe). (b) Anomeric region and (c) ring proton region of the 1H,13C-CT-HSQC spectrum of free Man3 (red/magenta) superimposed on the 1H,13C-CT-HSQC spectrum of CV-N-bound Man3 (blue/cyan). An unidentified cross-peak is marked with an asterisk (*).
Figure 2
Figure 2
(a) 1H NMR spectrum of Man3 in D2O buffer. (b) Selected region of the 1H,13C-CT-HSQC spectrum of Man3 bound to CV-N. (c) 1D 1H,1H-DPFGSE-T-ROESY spectrum of Man3 with selective excitation on H1″ and a mixing time of 300 ms. (d) Selected region of a 1H,13C-HSQC-NOESY spectrum of CV-N-bound Man3 with a mixing time of 60 ms. (e-f) PANIC plots of Ij/Ii versus τmix, where Ij is the intensity of the NOE peak, -Ii is the intensity of the target peak (selective experiments) and Ii is the average intensity of the diagonal peaks (2D experiments). Plots are shown for H1″-H2 (red squares), H1′-H2″ (blue circles), and H1-H1″ (green diamonds) of (e) free and (f) bound Man3. Cross-relaxation rates were obtained from the slopes of the lines.
Figure 3
Figure 3
(a) Superposition of the 2D version of the 13C-filtered NOESY-HSQC (red, 80 ms mixing time) and the 1H,13C-CT-HSQC (grey) spectra of the CV-N/Man3 complex. (b) Side-chain region of the 1H,13C-HSQC-NOESY spectrum of the CV-N/Man3 complex (60 ms mixing time). Intermolecular NOEs between sugar ring protons (correlated through 13C in f1) and protein side-chain protons are labeled.
Figure 4
Figure 4
(a) Amide proton region of the 1H,13C-HSQC-NOESY spectrum (60 ms mixing time) of the CV-N/Man3 complex. Intermolecular NOEs between sugar ring protons and protein amide protons are labeled. (b) 1H,15N 2D version of the CNH-NOESY experiment (red, 20 ms mixing time) superimposed onto a 1H,15N-HSQC spectrum (grey). Amide proton resonances exhibiting NOEs to 13C-attached sugar ring protons are connected by dashed lines.

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