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

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 be possible to perform EPR studies of membrane protein structure and dynamics in well-aligned bicelles at physiological temperatures and below.

A bifunctional spin label reports the structural topology of phospholamban in magnetically-aligned bicelles

“…With the addition of lanthanides (20% TmCl 3 or DyCl 3 , mol/mol long-chain lipid), we observed complete alignment of these bicelles at 298 K using 5-DOXYL-stearic acid as a reporter (Figure S2) [5]. Spectra of bicelles with the membrane normal aligned parallel ( n || B 0 ) and perpendicular ( n ⊥ B 0 ) to the field show substantial differences in outer splitting (Figure S2), clearly indicating alignment.…”

“…Spin-labeled PLB was reconstituted at 1400 long-chain lipids per PLB into 2/2/1 (mol/mol) DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine, 14:0 PC) / POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 16:0–18:1 PC) / DHexPC (1,2-dihexanoyl-sn-glycero-3-phosphocholine, 6:0 PC) bicelles containing 13% cholesterol (mol/mol long-chain phospholipid) and 20% TmCl 3 or DyCl 3 (mol/mol long-chain phospholipid) [5, 7, 32]. This lipid composition was shown previously to optimize bicelle orientation at X-band magnetic field strength and 298 K [5].…”

“…This lipid composition was shown previously to optimize bicelle orientation at X-band magnetic field strength and 298 K [5]. To prepare bicelles, lipid and cholesterol stocks in chloroform (Avanti Lipids) were combined with PLB, dried under N 2 gas, and stored overnight in a vacuum desiccator.…”

“…The field strength was then raised to 1.1 T and the temperature gradually increased to 298 K at a rate of 2 K per minute. Samples were then incubated for 20 min at 298 K and 1.1 T before the field strength was decreased for EPR measurements [5]. Spectra at 298 K were acquired using 1 G peak-to-peak amplitude modulation, 5 mW microwave power, and a 120 G sweep width, while spectra at 200 K were acquired using 1 G peak-to-peak amplitude modulation, 0.1 mW microwave power and a 120 G sweep width.…”

“…Similarly, membrane protein alignment in EPR can resolve the g -factor and hyperfine coupling anisotropies of nitroxides introduced via site-directed spin labeling, and optimization of long-chain lipid composition can produce excellent bicelle alignment in EPR, despite much lower magnetic field values than in NMR [5]. EPR offers a substantial advantage over NMR in sensitivity and can be applied to much larger proteins.…”