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TOAC Spin Labels in the Backbone of Alamethicin: EPR Studies in Lipid Membranes

Derek Marsh ^*, Micha Jost , Cristina Peggion  and Claudio Toniolo 

^* Max-Planck-Institut für biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Germany; and Department of Chemistry, University of Padova, Padova, Italy

Correspondence: Address reprint requests to Dr. Derek Marsh, Tel.: 49-551-201-1285; E-mail: dmarsh{at}gwdg.de.

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 TOACⁿ alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic ¹⁴N-hyperfine couplings indicate that TOAC⁸ and TOAC¹⁶ are situated in the hydrophobic core of the membrane, whereas the TOAC¹ 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.

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