A. D. Milov

Electron-electron double resonance in electron spin echo: Model biradical systems and the sensitized photolysis of decalin

Abstract Model systems, comprising frozen glassy solutions of stabilized radicals and biradicals of the nitroxyl type, have been used to test the applicability of electron-electron double resonance in electron spin echo (ELDOR ESE) in studies of the spatial distributions of free radicals arranged in groups in solids. The method was used to investigate the spatial distribution of alkyl radicals generated by the sensitized photolysis of glassy naphthalene solutions in decalin at 77 K. and detected radical pairs.

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%.

The effect of microwave pulse duration on the distance distribution function between spin labels obtained by PELDOR data analysis

A method is proposed for obtaining a distance distribution function between spin labels in spin-labeled molecules on the basis of the data of pulsed electron-electron double resonance with regard to the finite duration of microwave (mw) pulses. It is shown that taking into account the finite duration of mw pulses makes it possible to extend the range of the studied distances between spin labels to the region of short distance in which the magnitude of the dipole-dipole interaction becomes comparable to or exceeds the spectrum widths of exciting mw pulses. With frozen glassy solutions of biradicals as model systems with a length between spins of less than 2 nm, the shape for the distance distribution function between labels was obtained, and the value and sign of the exchange interaction were estimated. It is demonstrated that the analysis of the dipole-dipole interaction neglecting the duration of mw pulses can lead to substantial distortions in the shape of the distance distribution function.

Double electron-electron resonance in electron spin echo: Conformations of spin-labeled poly-4-vinilpyridine in glassy solutions

Double electron-electron resonance in electron spin echo has been used to study the glassy solutions of poly-4-vinylpyridine doped by nitroxyl radicals frozen in liquid nitrogen. The phase relaxation of spin labels due to spin-spin interaction of unpaired electrons has been studied. The intramolecular and intermolecular contributions of the dipole-dipole interaction of spin labels into relaxation process have been separated. It has been established that both the intramolecular and intermolecular spin-spin interaction of spin labels lead to the dependence of echo signal on timeT of the exp (−aTq) type. It is shown that for the intramolecular interaction the experimentalq value is 0.3, for the intermolecular one it is 2. The assumption has been made of the linear structure of polymeric molecules due to the presence of a sufficiently high density of an electric charge on polymeric molecules.

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.

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.

Electron spin echo as a method of ESR tomography

Abstract The electron spin echo method combined with pulsed gradients of an external magnetic field has been proposed for studies on the spatial distribution of paramagnetic centres. The relationship has been found between the distribution function of paramagnetic centres and the dependence of the spin echo signal on the parameters of the applied gradients. The resolution of the method depends on the ratio of the inverse phase relaxation time ( T 2 −1 ) to the magnetic field-gradient and may be much higher than the resolution of standard ESR tomography. The method has been applied to a model system.

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.

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 α-helix with a 2.8 nm distance between labels and the 310-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.