The Secondary Structure of a Membrane-Modifying Peptide in a Supramolecular Assembly Studied by PELDOR and CW-ESR Spectroscopies

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Abstract
The new technique of pulsed electron−electron double resonance in electron spin−echo (PELDOR) in combination with the CW-ESR method has been used to investigate the secondary structure of a double spin-labeled peptide (the [TOAC-1,8]-analogue of the peptaibol antibiotic trichogin GA IV) that is hidden into a tetrameric supramolecular assembly of unlabeled peptide molecules. The magnetic dipole−dipole relaxation of spin labels has been experimentally studied in glassy solutions of the double-labeled peptide frozen to 77 K in a mixture of chloroform−toluene with an excess of unlabeled peptide. The PELDOR signal oscillations have been observed at high degrees of dilution with unlabeled peptide. The intramolecular distance between the spin labels of the peptide molecule in the aggregate has been determined from the oscillation frequency to be 15.7 Å which is close to the value of ≅14 Å calculated for a 310-helical structure. Estimation of the fraction of this ordered secondary structure shows that about 19% of the peptide molecules in aggregates are folded in the 310-helical conformation. The present experimental results are consistent with our molecular model presented in J. Am. Chem. Soc.2000, 122, 3843−3848, wherein four amphiphilic 310-helical peptide molecules form a vesicular system with the polar amino acid side chains pointing to the interior, and the apolar side chains, to the exterior of the cluster. The experimental data were compared with the results obtained with other techniques.