HIGH-RESOLUTION NMR AND MOLECULAR MODELING STUDIES ON COMPLEX CARBOHYDRATES - CHARACTERIZATION OF O-BETA-D-GAL-(1-]3)-O-BETA-D-GAL-(1-]4)-O-BETA-D-XYL-(1-]0)-L-SER, A CARBOHYDRATE PROTEIN LINKAGE REGION FRAGMENT FROM CONNECTIVE-TISSUE PROTEOGLYCANS

High-resolution NMR spectroscopy and molecular mechanics calculations were employed to characterize the solution conformation of O-beta-D-Gal-(1 --> 3)-O-beta-D-Gal-(1 --> 4)-O-beta-D-Xyl-(1 --> 0)-L-Ser (G'GXS), a fragment from the carbohydrate-protein linkage region of serine-linked connective tissue proteoglycans. The H-1 and C-13 resonances were assigned by 2D NMR spectroscopy. The conformations of the individual sugars were determined from an analysis of vicinal proton coupling constants. Interresidue spatial constraints between the sugars and between xylose and serine were determined by 2D NOESY. A general molecular modeling methodology suitable for complex carbohydrates was described. It was used to generate families of conformations compatible with the NMR data. This methodology employs a set of starting conformations which are refined by molecular dynamics and energy minimization procedures together with NOESY derived distance constraints introduced in the form of a pseudoenergy function into the calculations. In the final stage, the structures are energy-minimized without NOE constraints to relieve any steric conflicts and bond length and bond angle distortions. On the basis of these calculations we have identified a single family of conformations for G'GXS that are compatible with the solution-phase NMR data.