Fig. 2.

The direct analysis of Aspergillus GMT interaction with GDP-Man, GDP and GMP using STD NMR spectroscopy. ¹H (a) and competition STD NMR spectra of Aspergillus Golgi-enriched fractions complexed with GDP-Man (b) followed by the addition of equimolar amounts of GMP (c) and GDP (d). Some STD signals were found to increase due to overlapping chemical shifts (e.g. the H1 ribose signal at 5.65 ppm), however the H8 guanine signal of the three ligands does not have the same chemical shift and therefore could be used to monitor the interaction of the GMT with GDP-Man, GDP and GMP. The H8 GuaGDP-Man signal (b) is reduced following the addition of GMP and GDP (c and d, respectively) with a corresponding appearance of H8 guanine signals associated with GMP and GDP. Specific mannose signals were reduced by ∼ 50% following the addition of equimolar GMP (c), and the signals after addition of GDP (d) showed a further reduction of ∼ 50% compared to (c).