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The reduction in EPR signal intensity of nitroxide spin-labels by ascorbic acid has been measured as a function of time to investigate the immersion depth of the spin-labeled M2δ AChR peptide incorporated into a bicelle system utilizing EPR spectroscopy. The corresponding decay curves of n-DSA (n=5, 7, 12, and 16) EPR signals have been used to (1) calibrate the depth of the bicelle membrane and (2) establish a calibration curve for measuring the depth of spin-labeled transmembrane peptides. The kinetic EPR data of CLS, n-DSA (n=5, 7, 12, and 16), and M2δ AChR peptide spin-labeled at Glu-1 and Ala-12 revealed excellent exponential and linear fits. For a model M2δ AChR peptide, the depth of immersion was calculated to be 5.8Å and 3Å for Glu-1, and 21.7Å and 19Å for Ala-12 in the gel-phase (298K) and L(α)-phases (318K), respectively. The immersion depth values are consistent with the pitch of an α-helix and the structural model of M2δ AChR incorporated into the bicelle system is in a good agreement with previous studies. Therefore, this EPR time-resolved kinetic technique provides a new reliable method to determine the immersion depth of membrane-bound peptides, as well as, explore the structural characteristics of the M2δ AChR peptide., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This paper reports the EPR spectroscopic characterization of a model membrane system that magnetically aligns with a variety of different lanthanide ions in the applied magnetic field (<1 T) of an X-band EPR spectrometer. The ability to align phospholipid bilayer systems is valuable because the anisotropic spectra provide a more detailed and complete description of the structural and motional properties of the membrane-associated spin label when compared to randomly dispersed EPR spectra. The nitroxide spin probe 3beta-doxyl-5alpha-cholestane (cholestane or CLS) was inserted into the bilayer discs to demonstrate the effects of macroscopic bilayer alignment through the measurement of orientational dependent hyperfine splittings. The effects of different lanthanide ions with varying degrees of magnetic susceptibility anisotropy and relaxation properties were examined. For X-band EPR studies, the minimal amounts of the Tm(3+), Yb(3+), and Dy(3+) lanthanide ions needed to align the phospholipid bilayers were determined. Power saturation EPR experiments indicate that for the sample compositions described here, the spin-lattice relaxation rate of the CLS spin label was increased by varying amounts in the presence of different lanthanide (Gd(3+), Dy(3+), Er(3+), Yb(3+), and Tm(3+)) ions, and in the presence of molecular oxygen. The addition of Gd(3+) caused a significant increase in the spin-lattice relaxation rate of CLS when compared to the other lanthanide ions tested.