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. 2015 Jan:250:71-75.
doi: 10.1016/j.jmr.2014.09.026. Epub 2014 Oct 13.

Optimization of bicelle lipid composition and temperature for EPR spectroscopy of aligned membranes

Jesse E McCaffrey  1 , Zachary M James  1 , David D Thomas  2
Affiliations

Optimization of bicelle lipid composition and temperature for EPR spectroscopy of aligned membranes

Jesse E McCaffrey et al. J Magn Reson. 2015 Jan.

Abstract

We have optimized the magnetic alignment of phospholipid bilayered micelles (bicelles) for EPR spectroscopy, by varying lipid composition and temperature. Bicelles have been extensively used in NMR spectroscopy for several decades, in order to obtain aligned samples in a near-native membrane environment and take advantage of the intrinsic sensitivity of magnetic resonance to molecular orientation. Recently, bicelles have also seen increasing use in EPR, which offers superior sensitivity and orientational resolution. However, the low magnetic field strength (less than 1 T) of most conventional EPR spectrometers results in homogeneously oriented bicelles only at a temperature well above physiological. To optimize bicelle composition for magnetic alignment at reduced temperature, we prepared bicelles containing varying ratios of saturated (DMPC) and unsaturated (POPC) phospholipids, using EPR spectra of a spin-labeled fatty acid to assess alignment as a function of lipid composition and temperature. Spectral analysis showed that bicelles containing an equimolar mixture of DMPC and POPC homogeneously align at 298 K, 20 K lower than conventional DMPC-only bicelles. It is now possible to perform EPR studies of membrane protein structure and dynamics in well-aligned bicelles at physiological temperatures and below.

Keywords: Bicelle; EPR; Lipid composition; Spectral simulation; Temperature.

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Figures

Figure 1
Figure 1
EPR spectra of 5-DSA in conventional DMPC vesicles (top) and bicelles with membrane normal oriented parallel to the instrument magnetic field (bottom) aligned at 318 K (left) and 298 K (right). All samples contain 13% cholesterol (mol cholesterol / mol long-chain lipid). Structural formula for 5-DSA (bottom) indicating that probe principal magnetic axis (zP) is parallel with probe principal diffusion axis (zD).
Figure 2
Figure 2
EPR spectra of 5-DSA in POPC/DMPC vesicles (left) and bicelles (right) as a function of POPC:DMPC molar ratio (left text). All samples were aligned at 298 K, and all spectra were acquired at 298 K. All samples contain 13% cholesterol (mol cholesterol / mol long-chain lipid). The relative amount of 5-DSA in uniformly oriented bicelles is indicated as fraction aligned (right text).
Figure 3
Figure 3
EPR spectra for 5-DSA in DMPC-only vesicles (left) and bicelles (right) as a function of alignment temperature (left text). All samples contain 13% cholesterol (mol cholesterol / mol long-chain lipid). The relative amount of 5-DSA in uniformly oriented bicelles is indicated as fraction aligned (right text).
Figure 4
Figure 4
EPR spectra for 5-DSA in 1:1 POPC:DMPC vesicles (left) and bicelles (right) as a function of alignment temperature (left text). All samples contain 13% cholesterol (mol cholesterol / mol long-chain lipid). The relative amount of 5-DSA in uniformly oriented bicelles is indicated as fraction aligned (right text).
Figure 5
Figure 5
EPR spectra of 5-DSA in 1:1 POPC/DMPC bicelles with 13% cholesterol (mol cholesterol / mol long-chain lipid) as a function of time (20 min intervals between adjacent spectra) after alignment and reduction of magnetic field strength from 11000 G to 3356 G for spectral acquisition. All spectra were acquired at 298 K.
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