Using spin-label W-band EPR to study membrane fluidity profiles in samples of small volume

Laxman Mainali, James S. Hyde, Witold K. Subczynski

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32 Scopus citations

Abstract

Conventional and saturation-recovery (SR) EPR at W-band (94 GHz) using phosphatidylcholine spin labels (labeled at the alkyl chain [n-PC] and headgroup [T-PC]) to obtain profiles of membrane fluidity has been demonstrated. Dimyristoylphosphatidylcholine (DMPC) membranes with and without 50 mol% cholesterol have been studied, and the results have been compared with similar studies at X-band (9.4 GHz) (L. Mainali, J.B. Feix, J.S. Hyde, W.K. Subczynski, J. Magn. Reson. 212 (2011) 418-425). Profiles of the spin-lattice relaxation rate (T1-1) obtained from SR EPR measurements for n-PCs and T-PC were used as a convenient quantitative measure of membrane fluidity. Additionally, spectral analysis using Freed's MOMD (microscopic-order macroscopic-disorder) model (E. Meirovitch, J.H. Freed J. Phys. Chem. 88 (1984) 4995-5004) provided rotational diffusion coefficients (R and R||) and order parameters (S0). Spectral analysis at X-band provided one rotational diffusion coefficient, R. T1-1, R, and R || profiles reflect local membrane dynamics of the lipid alkyl chain, while the order parameter shows only the amplitude of the wobbling motion of the lipid alkyl chain. Using these dynamic parameters, namely T1-1, R , and R||, one can discriminate the different effects of cholesterol at different depths, showing that cholesterol has a rigidifying effect on alkyl chains to the depth occupied by the rigid steroid ring structure and a fluidizing effect at deeper locations. The nondynamic parameter, S0, shows that cholesterol has an ordering effect on alkyl chains at all depths. Conventional and SR EPR measurements with T-PC indicate that cholesterol has a fluidizing effect on phospholipid headgroups. EPR at W-band provides more detailed information about the depth-dependent dynamic organization of the membrane compared with information obtained at X-band. EPR at W-band has the potential to be a powerful tool for studying membrane fluidity in samples of small volume, ∼30 nL, compared with a representative sample volume of ∼3 μL at X-band.

Original languageEnglish
Pages (from-to)35-44
Number of pages10
JournalJournal of Magnetic Resonance
Volume226
DOIs
StatePublished - Jan 2013
Externally publishedYes

Keywords

  • Cholesterol
  • Membrane fluidity
  • Saturation-recovery
  • Spin label
  • Spin-lattice relaxation rate
  • Spin-lattice relaxation time
  • W-band

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