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.
Bibliographical noteFunding Information:
This work was supported in part by NIH Grants GM27906 and AR057220 to DDT. JEM was supported by NIH Training Grant AR007612 , and ZMJ was supported by NIH Training Grants AR007612 and GM008700 . EPR experiments were performed at the Biophysical Spectroscopy Center, and computational resources were provided by the Minnesota Supercomputing Institute. We thank Edmund Howard and Yuri Nesmelov for EPR simulation assistance, Kaustubh Mote for helpful bicelle preparation discussions, and Octavian Cornea for assistance in preparing the manuscript.
© 2014 Elsevier Inc. All rights reserved.
- Lipid composition
- Spectral simulation