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TOAC spin labels in the backbone of alamethicin: EPR studies in lipid membranes
Authors:Marsh Derek  Jost Micha  Peggion Cristina  Toniolo Claudio
Institution:Max-Planck-Institut für Biophysikalische Chemie, Abteilung Spektroskopie, G?ttingen, Germany. dmarsh@gwdg.de
Abstract:Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.
Keywords:Ac  acetyl  Aib  α-aminoisobutyric acid  DMPC  1  2-dimyristoyl-sn-glycero-3-phosphocholine  EDTA  N  N  N′  N′-ethylenediaminetetraacetic acid  EPR  electron paramagnetic resonance  Hepes  N-(2-hydroxyethyl)piperazine-N′-2-ethanesulphonic acid  NHtBu  tert-butylamino  OMe  methoxy  Phol  phenylalaninol  ST-EPR  saturation transfer EPR  TOAC  2  2  4  4-tetramethylpiperidine-1-oxy-4-amino-4-carboxylic acid  V1  first-harmonic absorption EPR spectrum detected in phase with respect to the static magnetic field modulation  V2  second-harmonic absorption EPR spectrum detected 90° out-of-phase with respect to the static magnetic field modulation  Z  benzyloxycarbonyl
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