Self-aggregation of spin-labeled alamethicin in ePC vesicles studied by pulsed electron-electron double resonance

The pulsed electron-electron double resonance technique was used to study the dipole-dipole interactions between 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin labels located at position 16 of an alamethicin analogue in ePC vesicles that were frozen to 77 K. We show that under these conditions the alamethicin molecules tend to form aggregates over the range of peptide concentrations 3.5 x 10(-3) to 1 x 10(-2) M. The number of molecules in the aggregate is found to be 4.2 +/- 0.2. A spin-label distance distribution function is also obtained with a maximum at a distance of 2.3 nm and a half-height width of 1.3 nm. We envisage that these data will permit us to generate a molecular model of cellular ion channel

Solvent effect on the distance distribution between spin labels in aggregated spin labeled trichogin GA IV dimer peptides as studied by pulsed electron-electron double resonance

Pulsed electron-electron double resonance (PELDOR) was used to study aggregate formation in frozen glassy solutions of mono- and double-spin labeled trichogin GA IV dimers in a toluene-methanol mixture. The modified method proposed and used for the distance distribution function calculation from the PELDOR data. Distance distribution functions between spin labels in the peptide molecules and their aggregates in solution were determined as a function of solvent composition. Double-labeled peptide molecules in aggregates in solutions with low methanol content display two types of structures, i.e. the alpha-helix with a 2.8 nm distance between labels and the 3(10)-helix with a 3.2 nm distance between labels. As the methanol content of the solvent increases, a part of conformations at 3.2 nm changes. An increase of the methanol content leads to disruption of the aggregates and a change to the peptide conformation as well. In pure methanol peptides fail to form aggregates and a wide distribution of distances between labels centered at 3 nm were observed

PELDOR Conformational Analysis of bis-Labeled Alamethicin Aggregated in Phospholipid Vesicles

Alamethicin (Alm) is a linear peptide antibiotic of great interest for its capability to form self-assembled ion channels in lipid membranes. Here, the pulsed electron electron double resonance technique was used to obtain unique conformational information on the aggregated peptide in the lipid membrane-bound state. Since a specific helical conformation implies a given length to the peptide molecule, a distance r was measured at the nanometer scale via the electron dipole-dipole interaction between two 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin labels synthetically incorporated at positions 1 and 16 of this 19-mer peptide. Two data sets were collected (at 77 K): (i) from aggregates of Alm in hydrated egg-yolk phosphocholine (ePC) vesicles (at peptide-to-lipid ratios of 1:200 and 1:75) and (ii) from nonaggregated Alm in pure (nonhydrated) ePC and in solvents of different polarity. The intramolecular distance between the two labels obtained in this manner is in excellent agreement with that calculated on the basis of an almost fully developed a-helical conformation for this peptide and is found to be independent of the molecular aggregated state and the environment polarity as well

Aggregation of spin-labeled alamethicin in low-polarity solutions as studied by PELDOR spectroscopy

Alamethicin is a 20-residue antibiotic peptide. Interest in studying alamethicin stems from its ability to form conducting channels in biological membranes, thus changing their permeability. Although such channels in artificial and biological membranes have been well documented (see, e.g., [1, 2] and references therein), available reliable data are still insufficient to elucidate the mechanism of action of alamethicin on the properties of membranes. Therefore, it is of interest to study the self-aggregation of alamethicin molecules in media mimicking the membrane surface and interfacial layer, for example, in polar and nonpolar media. One approach to studying peptide self-aggregation in solutions is the spin label method in combination with CW ESR and pulsed ESR electron\u2013electron double resonance (PELDOR). In this work, using these methods, we obtained the first reliable structural data on alamethicin aggregates in nonpolar media

Structure and spatial distribution of the spin-labelled lipopeptide trichogin GA IV in a phospholipid membrane studied by pulsed electron-electron double resonance (PELDOR)

The method of pulsed electron - electron double resonance (PELDOR) is exploited to study intra- and intermolecular dipole - dipole interactions between the spin labels of trichogin GA IV analogues. This lipopeptaibol antibiotic was studied in multilamellar membranes of dipalmitoylphosphatidylcholine frozen to 77 K. For mono-labelled trichogin analogues, the molecules are shown not to form aggregates in the lipid membranes studied. For the double-labelled trichogin analogues, a function of the distance distribution between the spin labels has been obtained. We determined that the distribution function has two main maxima located at distances of 1.25 nm and 1.75 nm. The value of 1.25 nm is close to the distance between labels of a alpha-helical structure. On the other hand, a distance of 1.75 nm corresponds to a mixed 3D-structure in which a 3(10)-helix is combined with a more elongated conformation

Supramolecular structure of self-assembling alamethicin analog studied by ESR and PELDOR

Three analogs of alamethicin F50/5, labelled with the TOAC (=‘2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid’) spin label at positions 1 (Alm1), 8 (Alm8), and 16 (Alm16), resp., were studied by Electron-Spin-Resonance (ESR) and Pulsed Electron–Electron Double-Resonance (PELDOR) techniques in solvents of different polarity to investigate the self-assembly of amphipathic helical peptides in membrane-mimicking environments. In polar solvents, alamethicin forms homogeneous solutions. In the weakly polar chloroform/toluene 1 : 1 mixture, however, this peptide forms aggregates that are detectable at 293 K by ESR in liquid solution, as well as by PELDOR in frozen, glassy solution at 77 K. In liquid solution, free alamethicin molecules and their aggregates show rotational-mobility correlation times τr of 0.87 and 5.9 ns, resp. Based on these values and analysis of dipole–dipole interactions of the TOAC labels in the aggregates, as determined by PELDOR, the average number N of alamethicin molecules in the aggregates is estimated to be less than nine. A distance-distribution function between spin labels in the supramolecular aggregate was obtained. This function exhibits two maxima: a broad one at a distance of 3.0 nm, and a wide one at a distance of ca. 7 nm. A molecular-dynamics (MD)-based model of the aggregate, consisting of two parallel tetramers, each composed of four molecules arranged in a ‘head-to-tail’ fashion, is proposed, accounting for the observed distances and their distribution

Conformational properties, membrane interaction, and antibacterial activity of the peptaibiotic chalciporin A: Multitechnique spectroscopic and biophysical investigations on the natural compound and labeled analogs

In this work, an extensive set of spectroscopic and biophysical techniques (including FT-IR absorption, CD, 2D-NMR, fluorescence, and CW/PELDOR EPR) was used to study the conformational preferences, membrane interaction, and bioactivity properties of the naturally occurring synthetic 14-mer peptaibiotic chalciporin A, characterized by a relatively low (approximate to 20%), uncommon proportion of the strongly helicogenic Aib residue. In addition to the unlabeled peptide, we gained in-depth information from the study of two labeled analogs, characterized by one or two residues of the helicogenic, nitroxyl radical-containing TOAC. All three compounds were prepared using the SPPS methodology, which was carefully modified in the course of the syntheses of TOAC-labeled analogs in view of the poorly reactive a-amino function of this very bulky residue and the specific requirements of its free-radical side chain. Despite its potentially high flexibility, our results point to a predominant, partly amphiphilic, a-helical conformation for this peptaibiotic. Therefore, not surprisingly, we found an effective membrane affinity and a remarkable penetration propensity. However, chalciporin A exhibits a selectivity in its antibacterial activity not in agreement with that typical of the other members of this peptide class