A comparative study of the effect of cholesterol on bicelle model membranes using X-band and Q-band EPR spectroscopy

Abstract

X-band and Q-band electron paramagnetic resonance (EPR) spectroscopic techniques were used to investigate the structure and dynamics of cholesterol containing phospholipid bicelles based upon molecular order parameters (S(mol)), orientational dependent hyperfine splittings and line shape analysis of the corresponding EPR spectra. The nitroxide spin-label 3-beta-doxyl-5-alpha-cholestane (cholestane) was incorporated into DMPC/DHPC bicelles to report the alignment of bicelles in the static magnetic field. The influence of cholesterol on aligned phospholipid bicelles in terms of ordering, the ease of alignment, phase transition temperature have been studied comparatively at X-band and Q-band. At a magnetic field of 1.25 T (Q-band), bicelles with 20 mol% cholesterol aligned at a much lower temperature (313 K), when compared to 318 K at a 0.35 T field strength for X-band, showed better hyperfine splitting values (18.29 G at X-band vs. 18.55 G at Q-band for perpendicular alignment and 8.25 G at X-band vs. 7.83 G at Q-band for the parallel alignment at 318 K) and have greater molecular order parameters (0.76 at X-band vs. 0.86 at Q-band at 318 K). Increasing cholesterol content increased the bicelle ordering, the bicelle-alignment temperature and the gel to liquid crystalline phase transition temperature. We observed that Q-band is more effective than X-band for studying aligned bicelles, because it yielded a higher ordered bicelle system for EPR spectroscopic studies.

Figure 1

A comparison between X-band and Q-band EPR spectra of cholestane incorporated into magnetically aligned bicelles at 318 K as a function of cholesterol mol% (0,5,10,15,20) with respect to DMPC. For the superimposed EPR spectra, the dashed line represents the randomly dispersed spectra (without lanthanides), solid line represents the Dy³⁺-doped perpendicular-aligned spectra and the dotted line shows the Tm³⁺-doped parallel-aligned spectra with respect to the static magnetic field.

Figure 2

Diagram showing the dependence of hyperfine splitting values on the concentration of cholesterol in the DMPC/DHPC phospholipids bilayers at 318 K. The filled circles and squares represent the hyperfine splitting values of Dy³⁺-doped perpendicular aligned bicelles spectra and open circles and squares represent the hyperfine splitting values of Tm³⁺-doped parallel aligned bicelles spectra. The solid lines and broken lines represent the hyperfine splitting values measured at Q-band and X-band, respectively.

Figure 3

Molecular order parameter (S_mol) values of X-band and Q-band EPR spectra at 318 K as a function of cholesterol concentration in bicelles.

Figure 4

Figure 4A: A comparison of Tm³⁺-doped bicelle EPR spectra at X-band and Q-band without cholesterol. The bicelle sample temperature was ramped from 298 K to 318 K for 16 minutes time in the static magnetic field (0.64 T for X-band and 1.25 T for Q-band) and allowed to equilibrate for 10 minutes at 318 K. The bicelle spectra were first recorded at 318 K. Then the temperature was lowered to 313, 308, 303 and 298 K, and the spectra were taken after a 5 minutes equilibration time in each step. Figure 4B: X-band and Q-band EPR spectra of Tm³⁺-doped bicelles containing 15 mol% cholesterol as a function of temperature

Figure 4

Figure 4A: A comparison of Tm³⁺-doped bicelle EPR spectra at X-band and Q-band without cholesterol. The bicelle sample temperature was ramped from 298 K to 318 K for 16 minutes time in the static magnetic field (0.64 T for X-band and 1.25 T for Q-band) and allowed to equilibrate for 10 minutes at 318 K. The bicelle spectra were first recorded at 318 K. Then the temperature was lowered to 313, 308, 303 and 298 K, and the spectra were taken after a 5 minutes equilibration time in each step. Figure 4B: X-band and Q-band EPR spectra of Tm³⁺-doped bicelles containing 15 mol% cholesterol as a function of temperature