Spectroscopic characterization of spin-labeled magnetically oriented phospholipid bilayers by EPR spectroscopy

This paper reports the EPR spectroscopic characterization of a recently dev eloped magnetically oriented spin-labeled model membrane system. The orient ed membrane system is composed of a mixture of a bilayer forming phospholip id and a short chain phospholipid that breaks up the extended bilayers into bilayered micelles or bicelles that are highly hydrated (approximately 75% aqueous). Paramagnetic lanthanide ions (Tm3+) were added to align the bice lles such that the bilayer normal is collinear with the direction of the st atic magnetic field. Optimal bicelle alignment was obtained when the temper ature was increased slowly (approximately 15 mini from 298 K (gel phase) to 318 K (L-alpha phase) at 0.64 T. The nitroxide spin probe 3 beta-doxyl-5 a lpha-cholestane (cholestane) was used to demonstrate the effects of macrosc opic bilayer alignment through the measurement of orientational dependent h yperfine splittings that were close to A(yy). The EPR signals of cholestane inserted into oriented and randomly dispersed DMPC-rich bilayers have been investigated over the temperature range 298-348 K. Also, the time dependen ce of the loss of orientation upon cessation of the magnetic field has been characterized. Power saturation EPR experiments indicate that for the samp le compositions described here, the lanthanide ions do not induce spectral line broadening of the cholestane EPR signal in DMPC-rich Lipid bilayers. R ecently, there has been a great deal of excitement over the use of magnetic ally oriented systems for both solution and solid-state NMR spectroscopy. T his study demonstrates the feasibility of conducting bicelle experiments in the relatively low magnetic field of a conventional EPR spectrometer. The system offers the opportunity to carry out EPR studies using a well-oriente d highly hydrated model membrane system whose preparation is amenable to th e reconstitution of labile membrane components such as integral membrane pr oteins.