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Originally published as Biophys J. BioFAST on December 30, 2005.
doi:10.1529/biophysj.105.070441
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Biophysical Journal 90:2051-2061 (2006)
© 2006 The Biophysical Society

Characterization of the Liquid-Ordered State by Proton MAS NMR

Ivan V. Polozov and Klaus Gawrisch

Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892

Correspondence: Address reprint requests to Klaus Gawrisch, NIAAA, NIH, 5625 Fishers Ln., Rm. 3N-07, Bethesda, MD 20892-9410. Tel.: 301-594-3750; Fax: 301-594-0035; E-mail: gawrisch{at}helix.nih.gov.

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 (lo) are distinctly different from liquid-disordered (ld) and solid-ordered (so) membrane regions. At a MAS frequency of 10 kHz the methylene proton resonance of hydrocarbon chains in the ld phase has a linewidth of ~50 Hz. The corresponding linewidth is ~1 kHz for the lo phase and several kHz for the so phase. According to results of 1H NMR dipolar echo spectroscopy, the broadening of MAS resonances in the lo 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 lo. In spectra recorded as a function of temperature, the onset of lo domain (raft) formation is seen as a sudden onset of line broadening. Formation of small domains yielded homogenously broadened resonance lines, whereas large lo domains (diameter >0.3 µm) in an ld environment resulted in superposition of the narrow resonances of the ld phase and the much broader resonances of lo. 1H MAS NMR may be applied to detection of rafts in cell membranes.




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