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

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.

Conformational Analysis of TOAC‐Labelled Alamethicin F50/5 Analogues

“…-Alamethicin analogues, labelled at specific sequence position(s) by incorporation of a stable, rigidly linked, nitroxyl free radical (the TOAC residue) [3], are expected to contribute to a better understanding of numerous details related to the interaction of this long (19-mer) channel-forming peptaibol with model membranes [1] [2] by exploitation of EPR techniques [42]. In the analogues investigated in this work, TOAC replaced Aib residues in the natural sequence.…”

Solvent Dependence of the Rotational Diffusion of TOAC‐Spin‐Labeled Alamethicin

“…Proceeding inwards from the external aqueous phase, the polarity at the interfacial region is comparable to that of MeOH, whereas further in, the central membrane core has a polarity approaching that of CHCl 3 or toluene [15] [16]. Nonetheless, recent EPR experiments on TOAC-labeled alamethicins have shown that this peptaibiotic is present predominantly as a monomer in fluid phospholipid membranes, as in the alcohols studied here, even if the hydrophobic span of the lipid molecule does not match that of the peptide [6] [17]. The assembly of ion-conducting alamethicin channels is, therefore, a transient process facilitated by the rapid translational diffusion that characterizes lipid-membrane dynamics [18] [19].…”

“…Part). They are not consistent with isotropic rotation; in principle, they are compatible with y-axis rotation, but from the known orientation of the TOAC ring in these alamethicin analogues [6] [7], they are expected to correspond more closely to zaxis rotation. The mean values of the correlation time t R ?…”

Site-Directed Spin Labeling and Electron Paramagnetic Resonance (EPR) Spectroscopy: A Versatile Tool to Study Protein-Protein Interactions

“…Being the most popular one, the paramagnetic -amino acid TOAC (4-amino-1-oxyl-2,2,6,6,-tetramethyl-piperidine-4-carboxylic acid) (Rassat and Rey, 1967) is characterized by only one degree of freedom, the conformation of the six-membered ring (Fig. 2) The nitroxide is rigidly coupled to the peptide backbone, thereby providing the possibility to obtain direct information about the orientation of secondary structure elements, and has for example been used to study the secondary structure of small peptides in liquid solution (Anderson et al, 1999;Hanson et al, 1996;Marsh et al, 2007), and has also been successfully incorporated into the -melanocyte stimulating hormone without loss of function (Barbosa et al, 1999).…”