Application of the out-of-phase absorption mode to separating overlapping EPR signals with different T1 values

Abstract

The use of 90 degrees-out-of-phase first-harmonic absorption (V1'-) EPR to resolve the spectra from nitroxide spin labels with differing T1-relaxation times is described. Non-linear V1'-EPR spectra recorded under moderate saturation have sharper lines compared with the in-phase V1-EPR spectra, and amplitudes that preferentially enhance components with longer T1-relaxation. Discrimination between V1'-spectral components can be increased further by means of selective paramagnetic relaxation enhancement agents. Examples are given of biophysical applications to double labelling in single-component membranes and phase separation in two-component membranes, to lipid-peptide complexes, and to binding of spin-labelled reagents. It is concluded that optimal resolution in V1'-EPR spectroscopy is obtained at relatively low Zeeman modulation frequencies (20-30 kHz) and moderate saturation (H1 approximately 0.2-0.3 G).

Spin-label CW microwave power saturation and rapid passage with triangular non-adiabatic rapid sweep (NARS) and adiabatic rapid passage (ARP) EPR spectroscopy

“…In another experiment, NARS spectra were collected at a moderately slow sweep rate while slowly increasing the microwave power to investigate saturation behavior across the entire spectrum, and various displays were created by the method of differential saturation [20]. Figure 5A shows raw NARS spectra collected under non-saturating conditions (-32 dB, ~P 1/2 *0.05), moderately saturating conditions (-20 dB, ~P 1/2 ), and saturating conditions (-8 dB, ~P 1/2 *20).…”

Phase Diagram of Ternary Cholesterol/Palmitoylsphingomyelin/Palmitoyloleoyl-Phosphatidylcholine Mixtures: Spin-Label EPR Study of Lipid-Raft Formation

“…Coexisting phases are expected to be characterized by populations of spin-labeled lipids with different intrinsic spin-lattice relaxation times (T 1,e ) (35), or with different relaxation enhancements induced by paramagnetic relaxants such as oxygen (36) or Ni perchlorate (37,30). Therefore, in addition to normal linear V 1 -EPR spectra, nonlinear out-of-phase V 0 1 -EPR spectra (29) were also studied in the putative coexistence regions.…”

“…Therefore, in addition to normal linear V 1 -EPR spectra, nonlinear out-of-phase V 0 1 -EPR spectra (29) were also studied in the putative coexistence regions. Because the V 0 1 -EPR intensities of both spectral components depend directly on T 1,e (29), in regions of phase coexistence, one expects a change in overall V 0 1 -EPR lineshape, relative to that of the conventional in-phase low-power V 1 -EPR spectrum (30).…”

“…Out-of-phase V 0 1 -EPR spectra were recorded under conditions of microwave power saturation at hH 2 1 i 1=2 ¼ 18 mT, in phasequadrature with the 100-or 30-kHz field modulation. Procedures for recording the nonlinear V 0 1 -EPR signals are described in references (29,30). The static magnetic field was measured with a Bruker ER 035M NMR magnetometer and the microwave frequency with a digital counter incorporated in the Bruker ER 041 XK-D bridge.…”

“…In the present work, spin-label EPR techniques are used to study the phase behavior and dynamics of the PSM/POPC/chol system. Conventional EPR is augmented by nonlinear EPR because the out-of-phase, first-harmonic, absorption spectra are able to separate overlapping EPR signals with differing spinlattice relaxation times (29,30). Particular attention is paid to detecting possible L a ÀL o phase coexistence, especially at low PSM content where the published phase diagrams differ.…”