Characterization of the Liquid-Ordered State by Proton MAS NMR

Abstract

We investigated if magic angle spinning (MAS) 1H NMR can be used as a tool for detection of liquid-ordered domains (rafts) in membranes. In experiments with the lipids SOPC, DOPC, DPPC, and cholesterol we demonstrated that 1H MAS NMR spectra of liquid-ordered domains (l o) are distinctly different from liquid-disordered (l d) and solid-ordered (s o) membrane regions. At a MAS frequency of 10 kHz the methylene proton resonance of hydrocarbon chains in the l d phase has a linewidth of ∼50 Hz. The corresponding linewidth is ∼1 kHz for the l o phase and several kHz for the s o phase. According to results of 1H NMR dipolar echo spectroscopy, the broadening of MAS resonances in the l o phase results from an increase in effective strength of intramolecular proton dipolar interactions between adjacent methylene groups, most likely because of a lower probability of gauche/ trans isomerization in l o. In spectra recorded as a function of temperature, the onset of l o domain (raft) formation is seen as a sudden onset of line broadening. Formation of small domains yielded homogenously broadened resonance lines, whereas large l o domains (diameter >0.3 μm) in an l d environment resulted in superposition of the narrow resonances of the l d phase and the much broader resonances of l o. 1H MAS NMR may be applied to detection of rafts in cell membranes.