Distance between a native cofactor and a spin label in the reaction centre of Rhodobacter sphaeroides by a two-frequency pulsed electron paramagnetic resonance method and molecular dynamics simulations

The distance between the paramagnetic state of a native cofactor and a spin label is measured in the photosynthetic reaction centre from the bacterium Rhodobacter sphaeroides R26. A two-frequency pulsed electron paramagnetic resonance method [double-electron-electron spin resonance (DEER)] is used. A distance of 3.05 nm between the semiquinone anion state of the primary acceptor (Q(A)) and the spin label at the native cysteine at position 156 in the H-subunit is found. Molecular-dynamics (MD) simulations are performed to interpret the distance. A 6 ns run comprising the entire RC protein yields a distance distribution that is close to the experimental one. The average distance found by the MD simulation is smaller than the distance obtained by DEER by at least 0.2 nm. To better represent the experiments performed at low temperature (60K), a MD method to mimic the freezing-in of the room-temperature conformations is introduced. Both MD methods yield similar distances, but the second method has a trend towards a wider distance distribution.     

Measuring Cu2+-Nitroxide Distances Using Double Electron–Electron Resonance and Saturation Recovery

Distance measurements were obtained between a bound Cu²⁺ and a spin label on two polypeptides of differing length using the double electron–electron resonance (DEER) and saturation recovery experiments. Distance distributions obtained from the DEER results resolved differences between the average distance and distribution of distances for each peptide. An average distance was also obtained for each peptide using the relaxation-based saturation recovery experiment. Predicted average distances for the relaxation-based method, <r_ESR>, were calculated using the distance distributions from the DEER experiment. The predicted <r_ESR> values were similar to those measured by saturation recovery; both were biased to shorter values compared with the DEER results. The breadth of the distance distributions had a significant effect on the average distance measured by saturation recovery. This work highlights the advantage of using DEER to measure metal-nitroxide distances in that the average distances measured are less biased than in relaxation-based techniques.     

DEER in biological multispin-systems: a case study on the fatty acid binding to human serum albumin

In this study, self-assembled systems of human serum albumin (HSA) and spin-labeled fatty acids are characterized by double electron-electron resonance (DEER). HSA, being the most important transport protein of the human blood, is capable to host up to seven paramagnetic fatty acid derivatives. DEER measurements of these self-assembled multispin clusters are strongly affected by correlations of more than two spins, the evaluation of the latter constituting the central topic of this paper. While the DEER modulation depth can be used to obtain qualitative information of the number of coupled spins, the quantitative analysis is hampered by the occurrence of cluster mixtures with different numbers of coupled spins and contributions from unbound spin-labeled material. Applying flip angle dependent DEER measurements, unwanted multispin correlations were found to lead not only to a broadening of the distance peaks but also to cause small distances to be overestimated and large distances to be suppressed. It is thus favorable to use spin-diluted systems with an average of two paramagnetic molecules per spin cluster when a quantitative analysis of the distance distribution is sought.     

Observer-selective double electron-electron-spin resonance, a pulse sequence to improve orientation selection

By pulsed double electron-electron resonance (DEER), distances between spin labels in disordered systems up to 8 nm can be measured. In addition, the relative orientation of the interacting radicals can be determined, provided that the bandwidth of the pulses is sufficiently small. On the other hand, the bandwidth has to exceed the dipolar interaction considerably, because otherwise the DEER modulations become distorted and the modulation depth decreases, making distance determination impossible. Therefore, small bandwidths, i.e. long pulses, place a lower limit on the distance that can be determined. Two new pulse sequences, observer-selective DEER (os-DEER) and dead-time free os-DEER, are introduced that make it possible to use long observer pulses with bandwidths that are smaller than the dipolar interaction. The new pulse sequences do not suffer from the distortions caused by the limited bandwidth of the observer pulses, as demonstrated by measurements on a nitroxide biradical. With observer pulses of 140 ns, i.e., significantly longer than the 32 ns used in the conventional DEER sequence, a dipolar interaction of 7.8 MHz has been measured.     

Visualization of distance distribution from pulsed double electron-electron resonance data

Double electron-electron resonance (DEER), also known as pulsed electron-electron double resonance (PELDOR), is a time-domain electron paramagnetic resonance method that can measure the weak dipole-dipole interactions between unpaired electrons. DEER has been applied to discrete pairs of free radicals in biological macromolecules and to clusters containing small numbers of free radicals in polymers and irradiated materials. The goal of such work is to determine the distance or distribution of distances between radicals, which is an underdetermined problem. That is, the spectrum of dipolar interactions can be readily calculated for any distribution of free radicals, but there are many, quite different distributions of radicals that could produce the same experimental dipolar spectrum. This paper describes two methods that are useful for approximating the distance distributions for the large subset of cases in which the mutual orientations of the free radicals are uncorrelated and the width of the distribution is more than a few percent of its mean. The first method relies on a coordinate transformation and is parameter-free, while the second is based on iterative least-squares with Tikhonov regularization. Both methods are useful in DEER studies of spin-labeled biomolecules containing more than two labels.     

Dead-time free measurement of dipole-dipole interactions between electron spins. 2000

A four-pulse version of the pulse double electron–electron resonance (DEER) experiment is presented, which is designed for the determination of interradical distances on a nanoscopic lengthscale. With the new pulse sequence electron–electron couplings can be studied without dead-time artifacts, so that even broad distributions of electron–electron distances can be characterized. A version of the experiment that uses a pulse train in the detection period exhibits improved signal-to-noise ratio. Tests on two nitroxide biradicals with known length indicate that the accessible range of distances extends from about 1.5 to 8 nm. The four-pulse DEER spectra of an ionic spin probe in an ionomer exhibit features due to probe molecules situated both on the same and on different ion clusters. The former feature provides information on the cluster size and is inaccessible with previous methods.     

Distance determination in human ubiquitin by pulsed double electron-electron resonance and double quantum coherence ESR methods

Recently, distance measurements by pulsed ESR (electron spin resonance) have been obtained using pulsed DEER (double electron-electron resonance) and DQC (double quantum coherence) in SDSL (site directed spin labeling) proteins. These methods can observe long range dipole interactions (15-80A). We applied these methods to human ubiquitin proteins. The distance between the 20th and the 35th cysteine was estimated in doubly spin labeled human ubiquitin. Pulsed DEER requires two microwave sources. However, a phase cycle is not usually required in this method. On the other hand, DQC-ESR at X-band ( approximately 9GHz) can acquire a large echo signal by using pulses of short duration and high power, but this method has an ESEEM (electron spin echo envelope modulation) problem. We used a commercial pulsed ESR spectrometer and compared these two methods.     

Dead-time free measurement of dipole-dipole interactions between electron spins

A four-pulse version of the pulse double electron-electron resonance (DEER) experiment is presented, which is designed for the determination of interradical distances on a nanoscopic length-scale. With the new pulse sequence electron-electron couplings can be studied without dead-time artifacts, so that even broad distributions of electron-electron distances can be characterized. A version of the experiment that uses a pulse train in the detection period exhibits improved signal-to-noise ratio. Tests on two nitroxide biradicals with known length indicate that the accessible range of distances extends from about 1.5 to 8 nm. The four-pulse DEER spectra of an ionic spin probe in an ionomer exhibit features due to probe molecules situated both on the same and on different ion clusters. The former feature provides information on the cluster size and is inaccessible with previous methods.     

Maltose Binding Protein Is Partially Structured in Its Molten Globule State

Seven double cysteine mutants of maltose binding protein (MBP) were generated with one each in the active cleft at position 298 and the second cysteine distributed over both domains of the protein. These cysteines were spin labeled and distances between the labels in biradical pairs determined by pulsed double electron–electron resonance (DEER) measurements. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structure of MBP without maltose and were found to be in excellent agreement. MBP is in a molten globule state at pH 3.3 and is known to still bind its substrate maltose. The nitroxide spin label was sufficiently stable under these conditions. In preliminary experiments, DEER measurements were carried out with one of the mutants yielding a broad distance distribution as was to be expected if there is no explicit tertiary structure and the individual helices pointing into all possible directions.     

Two-Dimensional Distance Correlation Maps from Pulsed Triple Electron Resonance (TRIER) on Proteins with Three Paramagnetic Centers

Recently, we introduced the pulsed triple electron resonance (TRIER) experiment, which correlates dipolar frequencies of molecules with three electron spins. These correlation patterns contain valuable information: in combination with double electron–electron resonance (DEER) they allow to interpret distance distributions of biological systems that exist in more than one conformation. Together with an improved sequence and new data processing, we were now for the first time able to obtain two-dimensional distance correlation maps of the previously investigated model compounds as well as of spin-labeled proteins. For this we applied two-dimensional approximate Pake transformation to TRIER data. This enabled us to get distance correlation plots from two triple-labeled protein samples that were in good agreement with DEER data and simulations. With such information it should then be possible to assign peaks in DEER distance distributions for macromolecules that can exist in more than one conformation.     

Double Electron–Electron Resonance Between Trapped Electron and Hole in a Semiconductor

We demonstrate the spin interactions between dispersedly trapped electrons and holes in a semiconductor using the double electron–electron resonance (DEER) method of the pulsed electron paramagnetic resonance (EPR) techniques. An aluminum-doped titanium dioxide crystal is adopted as a spin system, in which optically generated electrons and holes are trapped, to reveal EPR signals that appear close to each other at a selected crystal orientation under an external magnetic field. We used the four-pulse DEER method by applying two microwave frequencies to a microwave cavity for pumping electrons and probing holes at the optimum temperature of 32 K. The dipolar modulation in the probed signal by pumping interacting spins was successfully detected. The observed non-oscillating decay shape indicates that the detected interaction is caused by widely distributed trapped electron and hole spins over long distances. We were able to extract a spin-pair distribution function by the first derivative of a background-corrected curve, referring to a previously reported method.     

A short note on orientation selection in the DEER experiments on a native cofactor and a spin label in the reaction center ofRhodobacter sphaeroides

The analysis of the two-frequency pulsed electron paramagnetic resonance (EPR) (double electron-electron spin resonance, DEER) investigation on the coupling between the semiquinone anion state of the primary acceptor (Q_A) and the spin label at the cysteine 156 in the H-subunit in the photosynthetic reaction center (RC) fromRhodobacter sphaerodes (R26) (I. V. Borovykh, S. Ceola, P. Gajula, P. Gast, H. J. Steinhoff, M. Huber: J. Magn. Reson. 180, 178–185, 2006) is reinvestigated to include orientation selection. The combination of the EPR properties of the two radicals and the pump and observer frequencies suggests that such an effect could play a role even at the X-band (9 GHz) EPR fields and frequencies employed. The magnitude of the effect is estimated from the structures obtained from the molecular-dynamics (MD) simulations from the previous study: the distance change is small (around 2%) and the distance of 3.05 nm obtained then is possibly underestimated by 0.06 nm. Thus, the difference of at least 0.2 nm between the measured distance and the average distance of 2.8 nm found by the MD simulation remains, suggesting a significant difference between the measurement and the X-ray structure of the RC, as discussed previously.     

Conformational Properties of the Spin-Labeled Tylopeptin B and Heptaibin Peptaibiotics Based on PELDOR Spectroscopy Data

X-Band pulsed electron–electron double resonance (PELDOR) spectroscopy was used to investigate for the first time the magnetic dipole–dipole interaction between spin labels for frozen glassy methanol solutions at 77 K of double spin-labeled, medium-length peptaibiotics, namely, tylopeptin B and heptaibin. This study was conducted on tylopeptin labeled at positions 3 and 13 (T313) and heptaibin labeled at positions 2 and 14 (H214). PELDOR data analysis was carried out using the theory developed for short inter-spin distances. The distance distribution functions between spin labels for T313 (maximum at 1.76 nm, half-width of 0.07 nm) and H214 (maximum at 2.30 nm, half-width of 0.065 nm) were determined. It is found that the distance distribution function for peptide T313 has the Gaussian shape. The main part of the distance spectrum for H214 has Gaussian shape and additional less intensive broad lines are shifted to high distances range 2.5–3.5 nm. The upper limit of distance spectrum in this case corresponds approximately to the length of extended peptide molecule and the number of such configurations is low. Intramolecular distances between the labels at maxima observed allowed us to assign α-helical conformation to T313 and 3₁₀-helical structure to H214 in methanol solution.     

Geometric model-based fitting algorithm for orientation-selective PELDOR data

Pulsed electron–electron double resonance (PELDOR or DEER) spectroscopy is frequently used to determine distances between spin centres in biomacromolecular systems. Experiments where mutual orientations of the spin pair are selectively excited provide the so-called orientation-selective PELDOR data. This data is characterised by the orientation dependence of the modulation depth parameter and of the dipolar frequencies. This dependence has to be taken into account in the data analysis in order to extract distance distributions accurately from the experimental time traces. In this work, a fitting algorithm for such data analysis is discussed. The approach is tested on PELDOR data-sets from the literature and is compared with the previous results.     

Optimization of Pulsed-DEER Measurements for Gd-Based Labels: Choice of Operational Frequencies, Pulse Durations and Positions, and Temperature

In this work, the experimental conditions and parameters necessary to optimize the long-distance (≥60 Å) double electron–electron resonance (DEER) measurements of biomacromolecules labeled with Gd(III) tags are analyzed. The specific parameters discussed are the temperature, microwave band, the separation between the pumping and observation frequencies, pulse train repetition rate, pulse durations and pulse positioning in the electron paramagnetic resonance spectrum. It was found that: (1) in optimized DEER measurements, the observation pulses have to be applied at the maximum of the electron paramagnetic resonance spectrum; (2) the optimal temperature range for K_a-band measurements is 14–17 K, while in W-band the optimal temperatures are between 6 and 9 K; (iv) W-band is preferable to K_a-band for DEER measurements. Recent achievements and the conditions necessary for short-distance measurements (<15 Å) are also briefly discussed.     

Nucleotide Spin Labeling for ESR Spectroscopy of ATP-Binding Proteins

Site-directed spin labeling of proteins by chemical modification of engineered cysteine residues with the molecule MTSSL (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate) has been an invaluable tool for conducting double electron electron resonance (DEER) spectroscopy experiments. However, this method is generally limited to recombinant proteins with a limited number of reactive Cys residues that when modified will not impair protein function. Here, we present a method that allows for spin labeling of protein-nucleotide-binding sites by adenosine diphosphate (ADP) modified with a nitroxide moiety on the β-phosphate (ADP-β-S-SL). The synthesis of this ADP analog is straightforward and isolation of pure product is readily achieved on a standard reverse-phase high-performance liquid chromatography (HPLC) system. Furthermore, analyses of isolated ADP-β-S-SL by LC–mass spectrometry confirm that the molecule is very stable under ambient conditions. The crystal structure of ADP-β-S-SL bound to the ATP pocket of the histidine kinase CheA reveals specific targeting of the probe, whose nitroxide moiety is mobile on the protein surface. Continuous wave and pulsed-ESR measurements demonstrate the capability of ADP-β-S-SL to report on active site environment and provide reliable DEER distance constraints.     

Relaxation-based distance measurements between a nitroxide and a lanthanide spin label

Distance measurements by electron paramagnetic resonance techniques between labels attached to biomacromolecules provide structural information on systems that cannot be crystallized or are too large to be characterized by NMR methods. However, existing techniques are limited in their distance range and sensitivity. It is anticipated by theoretical considerations that these limits could be extended by measuring the enhancement of longitudinal relaxation of a nitroxide label due to a lanthanide complex label at cryogenic temperatures. The relaxivity of the dysprosium complex with the macrocyclic ligand DOTA can be determined without direct measurements of longitudinal relaxation rates of the lanthanide and without recourse to model compounds with well defined distance by analyzing the dependence of relaxation enhancement on either temperature or concentration in homogeneous glassy frozen solutions. Relaxivities determined by the two calibration techniques are in satisfying agreement with each other. Error sources for both techniques are examined. A distance of about 2.7 nm is measured in a model compound of the type nitroxide–spacer–lanthanide complex and is found in good agreement with the distance in a modeled structure. Theoretical considerations suggest that an increase of the upper distance limit requires measurements at lower fields and temperatures.     

The determination of pair distance distributions by pulsed ESR using Tikhonov regularization

Pulsed ESR techniques with the aid of site-directed spin labeling have proven useful in providing unique structural information about proteins. The determination of distance distributions in electron spin pairs directly from the dipolar time evolution of the pulsed ESR signals by means of the Tikhonov regularization method is reported. The difficulties connected with numerically inverting this ill-posed mathematical problem are clearly illustrated. The Tikhonov regularization with the regularization parameter determined by the L-curve criterion is then described and tested to confirm its accuracy and reliability. The method is applied to recent experimental results on doubly labeled proteins that have been studied using two pulsed ESR techniques, double quantum coherence (DQC) ESR and double electron-electron resonance (DEER). The extracted distance distributions are able to provide valuable information about the conformational constraints in various partially folded states of proteins. This study supplies a mathematically reliable method for extracting pair distributions from pulsed ESR experimental data and has extended the use of pulsed ESR to provide results of greater value for structural biology.     

Membrane-peptide interaction studied by PELDOR and CW ESR: Peptide conformations and cholesterol effect on the spatial peptide distribution in the membrane

Pulsed electron-electron double resonance (PELDOR) combined with continuous-wave electron paramagnetic resonance was used to study inter- and intramolecular dipole-dipole interactions between spin labels for spin-labeled analogs of trichogin GA IV bound to multilamellar membranes of egg L-α-phosphatidylcholine (ePC) and in ePC membranes containing cholesterol. All samples were frozen to 77 K. For mono-labeled peptide concentrations in lipid over the range between 0.5 to 2.2 mol%, it is shown that in these membranes trichogin molecules are distributed homogeneously and are likely to be located on or near the inner and outer membrane surfaces. Addition of cholesterol to a final concentration of 16.5 mol% leads to an increase of the local concentration of trichogin molecules in the membranes. For the double-labeled trichogin, a distribution of the intramolecular distance between the two spin labels was observed. The distribution function is characterized by two main maxima located at distances of 1.3 and 1.8 nm. The distance of 1.3 nm is close to that expected for the α-helix structure of the peptide chain. The distance of 1.8 nm corresponds to a mixed structure in which a 3₁₀-helix is combined with a set of even more elongated conformations.