H-2 and C-13 nuclear magnetic resonance study of N-palmitoylgalactosylsphingosine (cerebroside)/cholesterol bilayers

C-13- and H-2-NMR experiments were used to examine the phase behavior rand dynamic structure of N-palmitoylgalactosylsphingosine palmitoylgalosylsphingosine (NPGS) (cerebroside) and cholesterol (CHOL) in binary mixtures. C-13 spectra of C-13=O- labeled and H-2 spectra of [7,7-H-2(2)] chain-labeled NPGS as well as 3 alpha-H-2(1) CHOL indicate that cerebroside and CHOL are immiscible in binary mixtures at temperatures less than 40 degrees C. In contrast, at 40 degrees C<T less than or equal to T-c (NPGS), up to 50 mol% CHOL can be incorporated into melted cerebroside bilayers, In addition, C-13 and H-2 spectra of melted NPGS/CHOL bilayers show a temperature and cholesterolaa concentration dependence, An analysis of spectra obtained from the melted C-13=O NPGS bilayer phase suggests that the planar NH-C=O group assumes an orientation tilted 40(d)egrees-55 degrees down from the bilayer interface. The similarity between the orientation of the amide group relative'co the bilayer interface in melted bilayers and in the crystal structure of cerebuoside suggests that the overall crystallographic conformation of cerebroside is preserved to a large degree in hydrated bilayers. Variation of temperature from 73 degrees C to 86 degrees C and CHOL concentration from O to 51 mol% results in small changes in this general orientation of the amide group, H-2 spectra of chain-labeled NPGS and labeled CHOL in NPGS/CHOL bilayer demonstrate that molecular exchange between the gel and liquid-gel (LG) phases is slow on ''the H-2 time scale, and this facilitates the simulation of the two component 2H spectra of [7,7-H-2(2)]NPGS/CHOL mixtures. Simulation parameters are used to quantitate the fractions of gel and LG cerebroside. The quadrupole splitting of [7,7-(2)H(2)INPGS/CHOL mixtures and 2H simulations allows the LG phase bilayer fraction to be characterized as an equimolar mixture ofcerebroside and CHOL.