Structure of Self-Aggregated Alamethicin in ePC Membranes Detected by Pulsed Electron-Electron Double Resonance and Electron Spin Echo Envelope Modulation Spectroscopies

• Published in: Biophysical journal 2009-04, Vol.96 (8), p.3197-3209
• Main Authors: Milov, Alexander D., Samoilova, Rimma I., Tsvetkov, Yuri D., De Zotti, Marta, Formaggio, Fernando, Toniolo, Claudio, Handgraaf, Jan-Willem, Raap, Jan
• Format: Article
• Language: English
• Subjects: Alamethicin - chemistry; Algorithms; Amino Acid Sequence; Channels, Receptors, and Electrical Signaling; Membranes, Artificial; Models, Molecular; Molecular Sequence Data; Phosphatidylcholines - chemistry; Protein Conformation; Protein Multimerization; Spectrum Analysis
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2009.01.026

PELDOR spectroscopy was exploited to study the self-assembled super-structure of the [Glu(OMe)7,18,19]alamethicin molecules in vesicular membranes at peptide to lipid molar ratios in the range of 1:70–1:200. The peptide molecules were site-specifically labeled with TOAC electron spins. From the magnetic dipole-dipole interaction between the nitroxides of the monolabeled constituents and the PELDOR decay patterns measured at 77 K, intermolecular-distance distribution functions were obtained and the number of aggregated molecules (n ≈ 4) was estimated. The distance distribution functions exhibit a similar maximum at 2.3 nm. In contrast to Alm16, for Alm1 and Alm8 additional maxima were recorded at 3.2 and ∼5.2 nm. From ESEEM experiments and based on the membrane polarity profiles, the penetration depths of the different spin-labeled positions into the membrane were qualitatively estimated. It was found that the water accessibility of the spin-labels follows the order TOAC-1 > TOAC-8 ≈ TOAC-16. The geometric data obtained are discussed in terms of a penknife molecular model. At least two peptide chains are aligned parallel and eight ester groups of the polar Glu(OMe)18,19 residues are suggested to stabilize the self-aggregate superstructure.