Measuring the Magnitude of Internal Motion in a Complex Hexasaccharide

For the development of a scheme for quantitative experimental estimation of internal motion in the complex human milk hexasaccharide lacto-N-di-fuco hexose I (LNDFH I), we measured a large number of experimental residual dipolar couplings in liquid crystal orienting media. We present a total of 40 C-13-H-1 and H-1-H-1 dipolar coupling values, each representing distinct directions of internuclear vectors. The NMR data were interpreted with established methods for analysis of rigid subdomains of the oligosaccharide as well as a novel method in which dipolar couplings were calculated over an ensemble of conformers from a solvent Molecular Dynamics trajectory using multiple linear regression analysis. The Lewis(b) epitope region of LNDFH I assumed a single unique conformation with internal motion described by fluctuations of 5-10 degrees in glycosidic dihedral angles consistent with previous studies. Greater flexibility was observed for the remaining GlcNAc1 -> 3-beta-D-Gal and beta-D-Gal1 -> 4Glc linkages, with the former glycosidic linkage existing in a conformational exchange among three states. The results were also supported by similar results of calculations carried out with conformers obtained from a simple Monte Carlo simulation without explicit solvent. (C) 2010 Wiley Periodicals, Inc. Biopolymers 95: 39-50, 2011.