Yuri D. Tsvetkov

Pulsed electron double resonance (PELDOR) and its applications in free-radicals research

The papers related to the theoretical background and experimental investigations by pulsed electron double resonance (PELDOR) are reviewed. The main aim of this pulsed ESR application is to study the dipole-dipole spin interaction. In PELDOR the ESR spectrum is excited by two ESE pulses at frequencyωa and additional pumping pulse atωb. Decay functionV(T) of the ESE signal, when the time intervalT between the first ESE pulse and pumping pulse is varied, contains the information on dipole-dipole couplings in the spin system. The kinetics ofV(T) decay strongly depends upon distance, mutual orientation inside interacting spin pairs and space distribution of radicals throughout the sample. The distances between spins which were measured or estimated using PELDOR in the papers reviewed are in the range of 15 ÷ 130 Å. This pulsed ESR technique turns now to be a powerful supplement to conventional ESE in studying the free radicals space distribution..

PULSED ELDOR IN SPIN-LABELED POLYPEPTIDES

Abstract The pulsed electron–electron double-resonance (PELDOR) technique was applied to obtain information about the structure of the synthetic polypeptide–biradical in a frozen glassy solution. From the concentration dependence of the PELDOR signal, the effects of intermolecular and intramolecular interactions were separated. It was found that the intramolecular dipole–dipole interactions in the biradical peptide led to the modulation effects in the PELDOR signal decay. This may be attributed to the existence of a conformational population having a distance between the two unpaired electrons of ∼20 A with a distribution of (∼2 A). Its fraction is estimated as about 25%.

Weakly coupled radical pairs in solids: ELDOR in ESE structure studies

Possibilities of the structure determination of radical pairs having fixed geometry with the help of ELDOR in ESE technique are considered. It is demonstrated that one can obtain information on relative orientation of paramagnetic centers in weakly coupled pairs in addition to the energy parameters of the spin Hamiltonian. Appropriate requirements for such experiments are formulated.

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

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 approximately 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 approximately 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 approximately 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.

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 alpha-helical conformation for this peptide and is found to be independent of the molecular aggregated state and the environment polarity as well.

Molecular dynamics and spatial distribution of TOAC spin-labelled peptaibols studied in glassy liquid by echo-detected EPR spectroscopy

Abstract2,2,6,6-tetramethylpiperidine-l-oxyl-4-carboxylic acid (TOAC) spin-labelled analogues of the Aib-rich peptide (peptaibol) Trichogin GA IV are investigated in a glassy methanol/glycerol (70/30 v/v%) system, using conventional CW EPR and electron spin echo spectroscopy. Echo-detected (ED) EPR spectra indicate that the labels undergo restricted orientational motion (libration). Comparison with the small molecular spin probe Tempone shows that the dynamics of peptide molecules is determined by their structure and not by the surrounding medium. At the terminal positions (position 1 and 8) TOAC residues were found to be more flexible than at the central 4th position. Instantaneous diffusion mechanism in electron spin echo, which also contributes to the ED EPR spectra, was employed for deriving the local concentration of peptaibols. This approach may serve as a tool for investigation of peptide aggregation. In the studied model glassy solution the peptaibols were found to be randomly distributed.

Review conformation, self-aggregation, and membrane interaction of peptaibols as studied by pulsed electron double resonance spectroscopy

Pulsed EPR methods, in particular pulsed electron double resonance (PELDOR) [or double electron–electron resonance (DEER)], are very sensitive to the dipole ··· dipole interaction between electron spins in a pair of free radicals. Using PELDOR, the conformations of a number of double radical‐containing biomolecules have been determined. In this review article, we focused our attention on the application of this spectroscopy to nitroxide‐labeled peptaibols. This is an emerging class of naturally occurring, relatively short, linear, helical peptide molecules endowed with hydrophobic character, capability to interact with and to alter the structure of membranes, and antibiotic activity. We extracted detailed information on the secondary structures of specifically site‐directed, double nitroxide‐labeled peptaibols under a variety of experimental conditions, including biologically relevant environments. Moreover, we examined in‐depth peptaibol clustering, related to the marked propensity of these molecules to undergo self‐association in model and whole‐cell membrane systems, using mainly mono‐nitroxide‐containing synthetic analogs. Finally, based on the PELDOR data accumulated, we proposed models of supramolecular (quaternary) structures of peptaibols and their binding modes to membranes.

Dipole-dipole interactions of high-spin paramagnetic centers in disordered systems

Dipole-dipole interactions between distant paramagnetic centers (PCs) where at least one PC has spinS>1/2 are examined. The results provide a basis for the application of pulsed electron-electron double resonance method to the measurement of distances between PCs involving high-spin species. A projection operator technique based on spectral decomposition of the secular Hamiltonian is used to calculate electron paramagnetic resonance (EPR) line splitting caused by the dipole coupling. This allows calculation of operators projecting an arbitrary wave function onto high-spin PC eigenstates when the eigenvectors of the Hamiltonian are not known. The effective spin vectors — that is, the expectation values for vector spin operators in the PC eigenstates — are calculated. The dependence of these effective spin vectors on the external magnetic field is calculated. There is a qualitative difference between pairs having at least one integer spin (non-Kramers PC) and pairs of two half-integer (Kramers PC) spins. With the help of these effective spin vectors, the dipolar line shape of EPR lines is calculated. Analytical relations are obtained for PCs with spinS=1/2 and 1. The dependence of Pake patterns on variations of zero-field splitting, Zeeman energy, temperature and dipolar coupling are illustrated.

Synthesis and conformational properties of a TOAC doubly spin-labeled analog of the medium-length, membrane active peptaibiotic ampullosporin a as revealed by cd, fluorescence, and EPR spectroscopies

We describe the challenging solid‐phase synthesis of the medium‐length (14 amino‐acid residues) peptaibiotic ampullosporin A, originally extracted from the fungus Sepedonium ampullosporum, and an analog doubly spin labeled (at positions 3 and 13) with the stable nitroxyl free‐radical 4‐amino‐1‐oxyl‐2,2,6,6‐tetramethylpiperidine‐4‐carboxylic acid (TOAC). The results of a circular dichrosim investigation in methanol strongly support the view that both peptides are essentially right‐handed helical, in particular endowed with a large population of α‐helical conformers. We also observed a significant quenching effect from the TOAC3 nitroxyl radical on the fluorescence of Trp1, compatible with that expected when both residues are closely located on the same helix segment. Combined continuous wave and pulsed electron‐electron double resonance electron paramagnetic resonance methodologies converge on the conclusion obtained from the other aforementioned spectroscopies, namely, that the [TOAC3,13] ampullosporin A analog is mostly folded in the α‐helical conformation. A liposome leakage assay demonstrated that the membrane‐modifying properties of this bis‐labeled analog are remarkable and even slightly superior to those of the natural peptaibiotic itself.

Peptide Aggregation and Conformation Properties as Studied by Pulsed Electron-Electron Double Resonance

Pulsed ELDOR is described as a technique for structural studies of spin labeled peptides in frozen solutions and for determining crystallographic distances on the order of 10 A between spins by measuring magnetic dipole interactions. Results from studies of double labeled trichogin GA IV analogues demonstrate conformational variations with solvent polarity, and aggregates of four peptides are detected in weakly polar (frozen) solvents. An aggregate model of four 310-helical trichogin molecules has been proposed.