Gunnar Jeschke

DeerAnalysis2006 - a comprehensive software package for analyzing pulsed ELDOR data

Pulsed electron-electron double resonance techniques such as the four-pulse double electron-electron resonance experiment measure a dipolar evolution function of the sample. For a sample consisting of spin-carrying nanoobjects, this function is the product of a form factor, corresponding to the internal structure of the nanoobject, and a background factor, corresponding to the distribution of nanoobjects in space. The form factor contains information on the spin-to-spin distance distribution within the nanoobject and on the average number of spins per nanoobject, while the background factor depends on constraints, such as a confinement of the nanoobjects to a two-dimensional layer. Separation of the dipolar evolution function into these two contributions and extraction of the spin-to-spin distance distribution require numerically stable mathematical algorithms that can handle data for different classes of samples, e.g., spin-labelled biomacromolecules and synthetic materials. Furthermore, experimental imperfections such as the limited excitation bandwidth of microwave pulses need to be considered. The software package DeerAnalysis2006 provides access to a comprehensive set of tools for such data analysis within a common user interface. This interface allows for several tests of the reliability and precision of the extracted information. User-supplied models for the spin-to-spin distance distribution within a certain class of nanoobjects can be added to an existing library and be fitted with a universal algorithm.

DEER Distance Measurements on Proteins

Distance distributions between paramagnetic centers in the range of 1.8 to 6 nm in membrane proteins and up to 10 nm in deuterated soluble proteins can be measured by the DEER technique. The number of paramagnetic centers and their relative orientation can be characterized. DEER does not require crystallization and is not limited with respect to the size of the protein or protein complex. Diamagnetic proteins are accessible by site-directed spin labeling. To characterize structure or structural changes, experimental protocols were optimized and techniques for artifact suppression were introduced. Data analysis programs were developed, and it was realized that interpretation of the distance distributions must take into account the conformational distribution of spin labels. First methods have appeared for deriving structural models from a small number of distance constraints. The present scope and limitations of the technique are illustrated.

Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonance

The biological function of protein, DNA, and RNA molecules often depends on relative movements of domains with dimensions of a few nanometers. This length scale can be accessed by distance measurements between spin labels if pulsed electron paramagnetic resonance (EPR) techniques such as electron-electron double resonance (ELDOR) and double-quantum EPR are used. The approach does not require crystalline samples and is well suited to biomacromolecules with an intrinsic flexibility as distributions of distances can be measured. Furthermore, oligomerization or complexation of biomacromolecules can also be studied, even if it is incomplete. The sensitivity of the technique and the reliability of the measured distance distribution depend on careful optimization of the experimental conditions and procedures for data analysis. Interpretation of spin-to-spin distance distributions in terms of the structure of the biomacromolecules furthermore requires a model for the conformational distribution of the spin labels.

Distance Measurements in the Nanometer Range by Pulse EPR

Distances of 1.5 to 8 nm in macromolecules and supramolecular assemblies can be measured by pulse electron paramagnetic resonance (EPR) experiments. In conjunction with site-directed spin labeling or the use of paramagnetic tracers this opens up a new method for the structure determination of complex systems such as biomacromolecules or nanostructured polymer materials. The extent of information obtained from such measurements crucially depends on the proper choice of the pulse sequence, on optimization of temperature, concentration, and matrix and on the procedure used for data analysis.

Large Molecular Weight Nitroxide Biradicals Providing Efficient Dynamic Nuclear Polarization at Temperatures up to 200 K

A series of seven functionalized nitroxide biradicals (the bTbK biradical and six derivatives) are investigated as exogenous polarization sources for dynamic nuclear polarization (DNP) solid-state NMR at 9.4 T and with ca. 100 K sample temperatures. The impact of electron relaxation times on the DNP enhancement (ε) is examined, and we observe that longer inversion recovery and phase memory relaxation times provide larger ε. All radicals are tested in both bulk 1,1,2,2-tetrachloroethane solutions and in mesoporous materials, and the difference in ε between the two cases is discussed. The impact of the sample temperature and magic angle spinning frequency on ε is investigated for several radicals each characterized by a range of electron relaxation times. In particular, TEKPol, a bulky derivative of bTbK with a molecular weight of 905 g·mol(-1), is presented. Its high-saturation factor makes it a very efficient polarizing agent for DNP, yielding unprecedented proton enhancements of over 200 in both bulk and materials samples at 9.4 T and 100 K. TEKPol also yields encouraging enhancements of 33 at 180 K and 12 at 200 K, suggesting that with the continued improvement of radicals large ε may be obtained at higher temperatures.

Data analysis procedures for pulse ELDOR measurements of broad distance distributions

The reliability of procedures for extracting the distance distribution between spins from the dipolar evolution function is studied with particular emphasis on broad distributions. A new numerically stable procedure for fitting distance distributions with polynomial interpolation between sampling points is introduced and compared to Tikhonov regularization in the dipolar frequency and distance domains and to approximate Pake transformation. Distance distribution with only narrow peaks are most reliably extracted by distance-domain Tikhonov regularization, while frequency-domain Tikhonov regularization is favorable for distributions with only broad peaks. For the quantification of distributions by their mean distance and variance, Hermite polynomial interpolation provides the best results. Distributions that contain both broad and narrow peaks are most difficult to analyze. In this case a high signal-to-noise ratio is strictly required and approximate Pake transformation should be applied. A procedure is given for renormalizing primary experimental data from protein preparations with slightly different degrees of spin labelling, so that they can be compared directly. Performance of all the data analysis procedures is demonstrated on experimental data for a shape-persistent biradical with a label-to-label distance of 5 nm, for a [2]catenane with a broad distance distribution, and for a doubly spin-labelled double mutant of plant light harvesting complex II

Determination of the nanostructure of polymer materials by electron paramagnetic resonance spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy is one the few methods that can characterize structural features in the range between 0.5 and 5 nm in systems that lack long-range order. Approaches based on EPR spectroscopy provide good structural contrast even in complex materials, as the sites of interest can be selectively labeled or addressed by suitably functionalized spin probes using well established techniques. This article assesses the EPR experiments available for distance measurements on nanoscales in terms of the accessible distance range, precision, and sensitivity. Recommendations are derived for the proper choice of experiment for a given problem. Both simple and sophisticated methods for data analysis are described and their limitations are evaluated. It is discussed which assumptions must be made to extract a pair correlation function from EPR data. Finally, applications to the study of polymer chain conformation and the structure of ionically functionalized diblock copolymers are highlighted. Cartoon of the structure of an ionically end-functionalized diblock copolymer as derived from SAXS data (long period of 20 nm of the lamellar morphology) and EPR data (ion cluster sizes of 2 nm and intercluster distances of 5-7 nm).

High sensitivity and versatility of the DEER experiment on nitroxide radical pairs at Q-band frequencies.

Measurement of distances with the Double Electron-Electron Resonance (DEER) experiment at X-band frequencies using a pair of nitroxides as spin labels is a popular biophysical tool for studying function-related conformational dynamics of proteins. The technique is intrinsically highly precise and can potentially access the range from 1.5 to 6-10 nm. However, DEER performance drops strongly when relaxation rates of the nitroxide spin labels are high and available material quantities are low, which is usually the case for membrane proteins reconstituted into liposomes. This leads to elevated noise levels, very long measurement times, reduced precision, and a decrease of the longest accessible distances. Here we quantify the performance improvement that can be achieved at Q-band frequencies (34.5 GHz) using a high-power spectrometer. More than an order of magnitude gain in sensitivity is obtained with a homebuilt setup equipped with a 150 W TWT amplifier by using oversized samples. The broadband excitation enabled by the high power ensures that orientation selection can be suppressed in most cases, which facilitates extraction of distance distributions. By varying pulse lengths, Q-band DEER can be switched between orientationally non-selective and selective regimes. Because of suppression of nuclear modulations from matrix protons and deuterons, analysis of the Q-band data is greatly simplified, particularly in cases of very small DEER modulation depth due to low binding affinity between proteins forming a complex or low labelling efficiency. Finally, we demonstrate that a commercial Q-band spectrometer can be readily adjusted to the high-power operation.

Distance measurements in the borderline region of applicability of CW EPR and DEER: A model study on a homologous series of spin-labelled peptides

Inter-spin distances between 1 nm and 4.5 nm are measured by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) methods for a series of nitroxide-spin-labelled peptides. The upper distance limit for measuring dipolar coupling by the broadening of the CW spectrum and the lower distance limit for the present optimally-adjusted double electron electron resonance (DEER) set-up are determined and found to be both around 1.6-1.9 nm. The methods for determining distances and corresponding distributions from CW spectral line broadening are reviewed and further developed. Also, the work shows that a correction factor is required for the analysis of inter-spin distances below approximately 2 nm for DEER measurements and this is calculated using the density matrix formalism.

Dipolar Spectroscopy and Spin Alignment in Electron Paramagnetic Resonance

Abstract Two single-frequency techniques for refocusing (SIFTER) dipolar couplings between electron spins are introduced. The experiments are based on the solid-echo and Jeener–Broekaert sequences, well established in dipolar NMR spectroscopy of solids, and open up new routes to high-resolution two-dimensional EPR spectroscopy with only moderate requirements on the spectrometer. For distances between paramagnetic centres larger than 3 nm, SIFTER provides better resolution than double electron–electron resonance (DEER). Good agreement between distances from SIFTER measurements and force-field computations is found for shape-persistent biradicals with distances up to 5.1 nm corresponding to a dipolar frequency of 390 kHz.