TOAC spin labels in the backbone of alamethicin: EPR studies in lipid membranes

¹ Max-Planck-Institut für biophysikalische Chemie
² University of Padova, 35131 Padova, Italy

^* To whom correspondence should be addressed. E-mail: dmarsh{at}gwdg.de.

Submitted on July 4, 2006
Revised on August 14, 2006
Accepted on 14 September 2006

	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-labelled in the peptide backbone have been synthesised by replacing residue 1, 8 or 16 with TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance (EPR) 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-labelled 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 EPR, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times less than 30 ns. Thus alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.

Key Words: 2,2,4,4-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC), EPR, alamethicin, isotropic hyperfine coupling, order parameter, spin label

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