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| | Diffusion Ordered Spectroscopy (DOSY) |
| Description: | Solutions of TS, TSY342H, and TSD247A in 20 mM Tris-HCl were exchanged with deuterated PBS using four cycles of concentration with Amicon Ultra (50,000 nominal molecular weight limit; Millipore) and diluted in deuterated PBS. One hundred microliters of 0.1 mM protein solution (TcTSs or BSA) was titrated with the sialoside (0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.6, and 2.0 mM) or lacto-N-neotetraose (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.9mM) as a negative control. The solution containing 0.1 mM protein plus sialoside was further titrated with Me-Lac (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, and 0.9 mM) in Shigemi tubes. NMR spectra were obtained at a probe temperature of 293 K on a Bruker DMX 600-MHz spectrometer equipped with a 5-mm self-shielded gradient triple resonance probe. All DOSY experiments were performed with a stimulated echo using bipolar gradient pulses for diffusion, one spoil gradient, and the 3-9-19 pulse sequence with gradients for water suppression according to the Bruker pulse program stebpgp1s19. The gradient duration was set to δ = 10 ms (protein-only experiments) and 20ms (protein⋅carbohydrate complexes experiments), and the diffusion time was set to Δ = 40 ms. Gradient strength was linearly varied in 16 steps from 0.6 to 6.7 G/cm. For each of the 16 gradient amplitudes, eight transients of 192 complex data points were acquired to a 10.0-ppm spectral width. The pulse repetition delay between each scan was 3 s. |
| Affinity data for this assay | |
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