Preferred conformations of the saccharide-ceramide linkage of glucosylceramides with different ceramide structures (normal and hydroxy fatty acids) were investigated by molecular mechanics (MM3) calculations and compared with conformational features obtained for glucosylglycerolipids (diacyl and dialkyl analogues). Relaxed energy map calculations with MM3 were performed for the three bonds (C1'-O1-C1-C2, torsion angles phi, psi, and theta 1) of the glucose-ceramide/diglyceride linkage at different values of the dielectric constant. For the phi torsion of the glycosidic C1'-O1 bond the calculations show a strict preference for the +sc range whereas the psi/theta 1 energy surface is dependent on the structure of the lipid moiety as well as on the dielectric constant (epsilon). Calculations performed on glucosylceramide with normal and hydroxy fatty acids at epsilon = 4 (bilayer subsurface conditions) show three dominating conformers (psi/theta 1 = ap/-sc, -sc/ap, and ap/ap). The ap/-sc conformer, which represents the global energy minimum, is stabilized by polar interactions involving the amide group. The +sc rotamer of theta 1 is unfavored in sphingolipids due to a Hassel-Ottar effect involving the sphingosine O3 and O1 oxygen atoms. Comparative calculations on glycosylglycerolipid analogues (ester and ether derivatives) show a distinct preference for the ap rotamer of theta 1. An evaluation of the steric hindrance imposed by the surrounding membrane surface shows that in a bilayer arrangement the range of possible conformations for the saccharide-lipid linkage is considerably reduced. The significance of preferred conformations of the saccharide-ceramide linkage for the presentation and recognition of the saccharide chains of glycosphingolipids at the membrane surface is discussed.
Conformations of two types of bovine brain cerebroside containing normal and alpha-hydroxy-fatty acids (NFA-CER and HFA-CER, respectively) in solution and in bilayers were investigated using 1H and 13C NMR in solution and in the solid state. The analysis of vicinal 1H-1H coupling constants and NOE measurements in solution indicated that in both cerebrosides the predominant conformation about the O1-C1, C1-C2, and C2-C3 bonds is ap/-sc/ap, respectively. The remarkable similarity in the 13C NMR chemical shifts in solution and in hydrated liquid-crystalline bilayers indicated that both cerebrosides in bilayers assume conformation essentially identical to those in solution. The obtained 13C NMR spectra in solution were used as a reference for comparison with the variable-temperature 13C CP-MAS NMR spectra in the metastable and stable gel phases. The lack of chemical shift changes of polar carbon atoms upon cooling the HFA-CER bilayers below the Tm strongly suggests that the liquid-crystalline-metastable gel transition is not associated with a conformational change of the head group. The observed line broadening can be interpreted in terms of the hydrocarbon chain crystallization and slow dynamics of the head group in the metastable phase. On the other hand, the relaxation of the metastable gel phase of HFA-CER caused profound changes in the 13C spectra, primarily of the signals of the galactose C1, the ceramide C2, C4, and C5, and the carbonyl group. These changes are interpreted using the known dependence of the chemical shifts of anomeric carbon on the conformation about the O1-C1 bond to suggest that the gel phase relaxation involves a significant reorientation of the galactose moiety caused by a change in the rotation of the O1-C1 bond from the ap to -sc conformer. Similar changes of chemical shifts were observed in the case of NFA-CER during the transition from the liquid-crystalline phase to the stable gel phase.
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