This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was attached to the pore-lining transmembrane domain (M2δ) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14° was calculated for the M2δ peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15 N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000 fold more sensitive than 15 N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 μg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique.

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This paper reports the EPR spectroscopic characterization of a recently developed magnetically oriented spin-labeled model membrane system. The oriented membrane system is composed of a mixture of a bilayer forming phospholipid 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 (Tm 3+ ) were added to align the bicelles such that the bilayer normal is collinear with the direction of the static magnetic field. Optimal bicelle alignment was obtained when the temperature was increased slowly (approximately 15 min) from 298 K (gel phase) to 318 K (L R phase) at 0.64 T. The nitroxide spin probe 3β-doxyl-5R-cholestane (cholestane) was used to demonstrate the effects of macroscopic bilayer alignment through the measurement of orientational dependent hyperfine 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 dependence of the loss of orientation upon cessation of the magnetic field has been characterized. Power saturation EPR experiments indicate that for the sample compositions described here, the lanthanide ions do not induce spectral line broadening of the cholestane EPR signal in DMPC-rich lipid bilayers. Recently, there has been a great deal of excitement over the use of magnetically oriented systems for both solution and solid-state NMR spectroscopy. This 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-oriented highly hydrated model membrane system whose preparation is amenable to the reconstitution of labile membrane components such as integral membrane proteins.

A series of copper complexes employing the neutral, bidentate, pyrazole‐based ligand diphenyldipyrazolylmethane was synthesized and fully characterized by various techniques, including elemental analysis, EPR, and structure elucidation by single‐crystal X‐ray diffraction. The structures of compounds 3−8 reveal variable coordination environments at the copper center, ranging from square pyramidal to tetrahedral, depending on the steric bulk at the pyrazole periphery and on the identity of the counterion. Cyclic voltammetry data on these compounds show a positively shifted reduction potential as compared to those observed employing the negatively charged pyrazolylborates or β‐diketiminates. These results indicate that these compounds may lead to (thiol)copper complexes that more accurately reproduce the reduction potentials observed during electron‐transfer processes in blue copper proteins. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

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The magnetic alignment behavior ofbicelles (magnetically alignable phospholipid bilayered membranes) as a function of the q ratio (1,2-dihexanoyl-sn-glycerol phosphatidylcholine/1,2-dimyristoyl-sn-glycerol phosphatidylcholine mole ratio) and temperature was studied by spin-labeled X-band electron paramagnetic resonance (EPR) spectroscopy and solid-state 2H and 31P NMR spectroscopy. Well-aligned bicelle samples were obtained at 45 degrees C for q ratios between 2.5 and 9.5 in both the EPR and NMR spectroscopic studies. The molecular order of the system, S(mol), increased as the q ratio increased and as the temperature decreased. For higher q ratios (> or = 5.5), bicelles maintained magnetic alignment when cooled below the main phase transition temperature (approximately 30 degrees C when in the presence of lanthanide cations), which is the first time, to our knowledge, that bicelles were magnetically aligned in the gel phase. For the 9.5 q ratio sample at 25 degrees C, S(mol) was calculated to be 0.83 (from 2H NMR spectra, utilizing the isotopic label perdeuterated 1,2-dimyristoyl-sn-glycerol phosphatidylcholine) and 0.911 (from EPR spectra utilizing the spin probe 3beta-doxyl-5alpha-cholestane). The molecular ordering of the high q ratio bicelles is comparable to literature values of S(mol) for both multilamellar vesicles and macroscopically aligned phospholipid bilayers on glass plates. The order parameter S(bicelle) revealed that the greatest degree of bicelle alignment was found at higher temperatures and larger q ratios (S(bicelle) = -0.92 for q ratio 8.5 at 50 degrees C).

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.