Macroscopic sample shape effect on pulse electron double resonance (PELDOR) signal.

Pulse electron double resonance (PELDOR), also called double electron-electron resonance (DEER), is a technique capable of measuring the strength of electron spin dipolar interactions, revealing spin-spin distance distributions in ordered and disordered solid materials. Previous work has shown that PELDOR signals acquire an out-of-phase component under conditions of high electron spin polarization, such as at low temperatures and high fields. In this paper, we show theoretically and experimentally that the size and sign of this effect depends on the macroscopic shape of the sample and its orientation in the external magnetic field. This effect is caused by dipolar interactions between distant spins and provides new insights into the fundamental physics of PELDOR.

Simulation of nitrogen nuclear spin magnetization of liquid solved nitroxides.

Nitroxide radicals are widely used in electron paramagnetic resonance (EPR) applications. Nitroxides are stable organic radicals containing the N-O˙ group with hyperfine coupled unpaired electron and nitrogen nuclear spins. In the past, much attention was devoted to studying nitroxide EPR spectra and electron spin magnetization evolution under various experimental conditions. However, the dynamics of nitrogen nuclear spin has not been investigated in detail so far. In this work, we performed quantitative prediction and simulation of nitrogen nuclear spin magnetization evolution in several magnetic resonance experiments. Our research was focused on fast rotating nitroxide radicals in liquid solutions. We used a general approach allowing us to compute electron and nitrogen nuclear spin magnetization from the same time-dependent spin density matrix obtained by solving the Liouville/von Neumann equation. We investigated the nitrogen nuclear spin dynamics subjected to various radiofrequency magnetic fields. Furthermore, we predicted a large dynamic nuclear polarization of nitrogen upon nitroxide irradiation with microwaves and analyzed its effect on the nitroxide EPR saturation factor.

Separation of intra- and intermolecular contributions to the PELDOR signal.

Pulsed Electron-electron Double Resonance (PELDOR) is commonly used to measure distances between native paramagnetic centers or spin labels attached to complex biological macromolecules. In PELDOR the energies of electron magnetic dipolar interactions are measured by analyzing the oscillation frequencies of the recorded time resolved signal. Since PELDOR is an ensemble method, the detected signal contains contributions from intramolecular, as well as intermolecular electron spin interactions. The intramolecular part of the signal contains the information about the structure of the studied molecules, thus it is very important to accurately separate intra- and intermolecular contributions to the total signal. This separation can become ambiguous, when the length of the PELDOR signal is not much longer than twice the oscillation period of the signal. In this work we suggest a modulation depth scaling method, which can use short PELDOR signals in order to extract the intermolecular contribution. Using synthetic data we demonstrate the advantages of the new approach and analyze its stability with regard to signal noise. The method was also successfully tested on experimental data of three systems measured at Q-Band frequencies, two model compounds in deuterated and protonated solvents and one biological sample, namely BetP. The application of the new method with an assigned value of the signal modulation depth enables us to determine the interspin distances in all cases. This is especially interesting for the model compound with an interspin distance of 5.2nm in the protonated solvent and the biological sample, since an accurate separation of the intra- and intermolecular PELDOR signal contributions would be difficult with the standard approach in those cases.

Broadband spin echoes and broadband SIFTER in EPR.

Applications of broadband pulses for EPR have been reported for FID, echo detection and inversion pulses recently. Here we present a broadband Hahn, stimulated and refocused echo sequence derived from adiabatic pulses. The formation of echoes is accomplished by using variable chirp rates and pulse lengths. In all three broadband echo experiments the complete spectral shape of a nitroxide (about 70 Gauss at X-band frequency) could be recovered by Fourier transformation of the quadrature detected echo signals. Such broadband echoes provide an exciting opportunity to optimize pulse sequences where a full excitation of the spectrum is mandatory for an optimum performance. We applied our pulses to the SIFTER (single frequency technique for refocusing dipolar couplings) experiment, a solid echo based pulse sequence to measure the dipolar coupling between two unpaired electron spins. By employing our broadband Hahn echo sequence on a nitroxide biradical we could achieve an artifact free dipolar evolution time trace in the SIFTER experiment with 95% modulation depth at X-band frequency and of 10% modulation depth at Q-band frequency.

An algorithm to analyze PELDOR data of rigid spin label pairs.

Pulsed Electron-electron Double Resonance (PELDOR) is a method frequently used to determine the distances between paramagnetic centers in biomacromolecules on the nanometer scale. A standard algorithm for determination of distances from the experimental data assumes that all possible mutual orientations of the spin labels are equally probable. However, in many applications the mobility of the spin labels attached to large molecules can be significantly restricted. In order to determine the total PELDOR signal in this case, the individual contributions of each rigid biradical should be explicitly calculated for the given frequencies of the probe and pump pulses. The solution of the inverse problem of determination of the ensemble of molecular structures that fit the experimental PELDOR data acquired at multiple microwave frequencies and magnetic fields has proven to be a non-trivial task, especially, when no information about the molecular structure under study is available. In this work we present a fitting algorithm that reconstructs experimental data by searching for an optimal combination of presimulated PELDOR time traces for nitroxide biradicals with all relative orientations and with inter-spin distances in the experimentally accessible range. The generated library of PELDOR time traces has been employed to excellently fit experimental data containing orientation selection effects gathered on model biradical systems.

Structure and dynamics of nucleic acids.

In this chapter we describe the application of CW and pulsed EPR methods for the investigation of structural and dynamical properties of RNA and DNA molecules and their interaction with small molecules and proteins. Special emphasis will be given to recent applications of dipolar spectroscopy on nucleic acids.

Conformational flexibility of DNA.

Pulsed Electron-Electron Double Resonance (PELDOR) on double-stranded DNA (ds-DNA) was used to investigate the conformational flexibility of helical DNA. Stretching, twisting, and bending flexibility of ds-DNA was determined by incorporation of two rigid nitroxide spin labels into a series of 20 base pair (bp) DNA duplexes. Orientation-selective PELDOR experiments performed at both X-band (9 GHz/0.3 T) and G-band (180 GHz/6.4 T) with spin label distances in the range of 2-4 nm allowed us to differentiate between different simple models of DNA dynamics existing in the literature. All of our experimental results are in full agreement with a dynamic model for ds-DNA molecules, where stretching of the molecule leads to a slightly reduced radius of the helix induced by a cooperative twist-stretch coupling.

Optimization of transversal relaxation of nitroxides for pulsed electron-electron double resonance spectroscopy in phospholipid membranes.

Pulsed electron-electron double resonance (PELDOR) spectroscopy is increasingly applied to spin-labeled membrane proteins. However, after reconstitution into liposomes, spin labels often exhibit a much faster transversal relaxation (T(m)) than in detergent micelles, thus limiting application of the method in lipid bilayers. In this study, the main reasons for enhanced transversal relaxation in phospholipid membranes were investigated systematically by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin clustering and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples of low local concentrations does deuteration of acyl chains and buffer significantly prolong T(m). In these cases, values of up to 7 µs have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect T(m), either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (<50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions.

Analytical method to determine the orientation of rigid spin labels in DNA.

We demonstrate the ability of pulsed electron double resonance (PELDOR) experiments to determine the orientation of spin labels in biological macromolecules. Thus, the distance information usually obtained from PELDOR data can be complemented by the mutual orientation of macromolecular domains. A method to determine the angle beta between the spin label normal and the interspin axis is proposed and analyzed mathematically. The obtained analytical expression allows extraction of angles beta without a fitting procedure if these angles are equal for both nitroxide of biradical. The method was applied to the experimental data gathered on ten spin-labeled DNA samples. The angles estimated from the PELDOR data are in excellent agreement with literature values.

Application of a portable nuclear magnetic resonance surface probe to porous media.

A portable nuclear magnetic resonance (NMR) surface probe was used to determine the time-dependent self-diffusion coefficient D(t) of water molecules in two fluid-filled porous media. The measuring equipment and the inhomogeneous magnetic fields in the sensitive volume of the probe are described. It is discussed how to evaluate D(t) using a surface probe from the primary and stimulated echoes generated in three-pulse experiments. Furthermore, the evaluation of D(t) allows one to determine the geometrical structure of porous materials.

Diffusion studies in porous media with the "inside-out" technique.

Diffusion measurements of water in a glass beads porous media were carried out by the "One-Sided Access" sensor. An approach to determine the long time mean square displacement of hydrogen molecules by measuring the ratio of stimulated echo to primary echo in a three pulse sequence in the presence of permanent static field gradient is analysed. The difference between the apparent diffusion coefficient in a pure water sample and in water/glass beads mixtures becomes visible.