Quantitative conformational analysis of the core region of N-glycans using residual dipolar couplings, aqueous molecular dynamics, and steric alignment

A method is described for quantitatively investigating the dynamic conformation of small oligosaccharides containing an alpha(1 --> 6) linkage. It was applied to the oligosaccharide Man-alpha(1 --> 3) [Man-alpha(1 --> 6)] Man-alpha-O-Me, which is a core region frequently observed in N-linked glycans. The approach tests an aqueous molecular dynamics simulation, capable of predicting microscopic dynamics, against experimental residual dipolar couplings, by assuming that alignment is caused purely by steric hindrance. The experimental constraints were heteronuclear and homonuclear residual dipolar couplings, and in particular those within the alpha(1 --> 6) linkage itself. Powerful spin-state-selective pulse sequences and editing schemes were used to obtain the most relevant couplings for testing the model. Molecular dynamics simulations in water over a period of 50 ns were not able to predict the correct rotamer population at the alpha(1 --> 6) linkage to agree with the experimental data. However, this sampling problem could be corrected using a simple maximum likelihood optimisation, indicating that the simulation was modelling local dynamics correctly. The maximum likelihood prediction of the residual dipolar couplings was found to be an almost equal population of the gg and gt rotamer conformations at the alpha(1--> 6) linkage, and the tg conformation was predicted to be unstable and unpopulated in aqueous solution. In this case all twelve measured residual dipolar couplings could be satisfied. This conformer population could also be used to make predictions of scalar couplings with the use of a previously derived empirical equation, and is qualitatively in agreement with previous predictions based on NMR, X-ray crystallography and optical data.