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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

HYSCORE and DEER with an upgraded 95GHz pulse EPR spectrometer

The set-up of a new microwave bridge for a 95 GHz pulse EPR spectrometer is described. The virtues of the bridge are its simple and flexible design and its relatively high output power (0.7 W) that generates pi pulses of 25 ns and a microwave field, B(1)=0.71 mT. Such a high B(1) enhances considerably the sensitivity of high field double electron-electron resonance (DEER) measurements for distance determination, as we demonstrate on a nitroxide biradical with an interspin distance of 3.6 nm. Moreover, it allowed us to carry out HYSCORE (hyperfine sublevel-correlation) experiments at 95 GHz, observing nuclear modulation frequencies of 14N and 17O as high as 40 MHz. This opens a new window for the observation of relatively large hyperfine couplings, yet not resolved in the EPR spectrum, that are difficult to observe with HYSCORE carried out at conventional X-band frequencies. The correlations provided by the HYSCORE spectra are most important for signal assignment, and the improved resolution due to the two dimensional character of the experiment provides 14N quadrupolar splittings.     

Binding of MRI contrast agents to albumin: a high-field EPR study

High-field electron paramagnetic resonance (EPR) experiments to monitor binding of lipophilic Gd(III) complexes to human serum albumin (HSA) are described. It was observed that magnetic interactions between the nitroxide moiety ofn-doxyl-stearic acids bound to HSA and Gd(III) complexes resulted in broadening of nitroxide continuous-wave EPR spectra. The broadening effect can be well described by a one-parameter model of additional Lorentzian broadening At 95 GHz, continuous-wave EPR spectra from Gd(III) complexes are fully resolved from the nitroxide signal allowing for simultaneous and independent line shape analysis. Analysis of the line width broadening effects for spectra from a series ofn-doxyl-stearic acids bound to HSA indicated a progressive decrease of spin label-Gd(III) magnetic interactions along the fatty acid (FA) binding channel, consistent with binding of Gd-DOTAP complex in the vicinity of the main FA binding site. The substantial difference in spin label-metal interactions along the FA binding channel for lipophilic Gd(III) complexes with different chelates is indicative of binding to different sites. We also report measurements of dissociation constant for noncovalent binding of Gd(III) complexes to HSA on the basis of analyses of 95 GHz Gd(III) EPR line shapes.     

一种新型的红外成像材料

基于超大磁阻(CMR)材料在绝缘体—金属转变(I—M)点附近的巨大电阻变化，本研究了La位Gd掺杂对La0.7-xGdxSr0.3MnO3(x=0.20、0.30、0.40、0.50)电阻温度系数(TCR)的影响。实验结果表明：Gd掺杂将引起电阻率曲线的急剧变化，导致出现大的TCR；而且随Gd掺杂的增加，TCRR在x＝0．30出现峰值，然后随掺杂量增加逐步降低，大的TCR行为将成为新型的红外成像材料。     

三氟乙酰丙酮—Gd（Ⅲ）—CTMAB配合物体系荧光法研究及应用

本文研究了Ｇｄ－三氟乙酰丙酮－ＣＴＭＡＢ的三元配合物荧光体系，并用钇的测定，最低检测限为：０．００３μｇ／ｍＬ，Ｇｄ在０．００５－０．４μｇ／ｍＬ范围内呈线性关系，回归方程Ｆ＝３１．６５ｃ＋０．２１，相关系数为ｒ＝０．９９９６，将此法用于稀土矿的痕量Ｇｄ的测定，与ＩＣＰ－ＡＥＳ法相比，结果令人满意。     

One-dimensional metallofullerene crystal generated inside single-walled carbon nanotubes

Electron microscope imaging for gadolinium metallofullerenes encapsulating in single-wall carbon nanotubes [(Gd@C82)n@SWNTs] identifies the single Gd atom encaged in each. The intermolecular distance between Gd@C82 is extremely regular, regarding the chains of Gd@C82 as novel one-dimensional crystals. Chemical state analysis of Gd atoms suggests evidence for charge transfer from Gd to either a fullerene cage or a nanotube. The slopes of the temperature dependence of electric resistance for the mat-like films of (Gd@C82)n@SWNTs and (C60)n@SWNTs are much steeper than that for empty SWNTs, suggesting the electron scattering due to the electrostatic potential from inside fullerenes playing an important role.     

The influence of the solution preparation method on the co-luminescence intensity of chelates of Eu(III) ions and estimation of the size of arising nanostructures

The luminescence intensity of Eu(III) ions (I ^Eu) in 3: 1 aqueous solutions of thenoyltrifluoroacetone, n-methoxybenzoylacetone, and dibenzoylmethane with 1,10-phenanthroline is studied in the presence and the absence of La(III), Gd(III), and Nd(III) ions. It is found that, in binary solutions of Eu(III) with La and Gd, the intensity I ^Eu, as well as the influence of chelates of La and Gd on I ^Eu, is considerably greater if these ions are introduced into the solutions of β-diketones and 1,10-phenanthroline simultaneously and homogeneously than when they are introduced into these solutions successively. This result is explained by the difference in the distribution of different Ln chelates over nanostructures. The average size of the structures arising is estimated.     

紫外差光谱测定Gd(Ⅲ), Yb(Ⅲ)与HBED配合物的条件稳定常数

在0.01 mol·L-1 N-2-羟乙基哌嗪-N'-2-乙磺酸(Hepes), pH 7.4, 室温条件下, 应用紫外差光谱滴定观察了Gd(Ⅲ), Yb(Ⅲ)与N, N'-二(2-羟苄基)乙二胺-N, N'-二乙酸(HBED)的结合. 结果表明 Gd(Ⅲ), Yb(Ⅲ)与HBED均形成1∶1的配合物, 其紫外差光谱均于237和291 nm处出现吸收峰, 在237 nm处配合物Gd-HBED与Yb-HBED的摩尔吸光系数分别为 ΔεGd=(22.52±0.20)×103 cm-1·mol-1·L, ΔεYb=(27.15±0.11)×103 cm-1·mol-1·L; 配合物Gd-HBED与Yb-HBED的条件稳定常数分别为 lgKGd-HBED=13.56±0.28, lgKYb-HBED=16.06±0.03, 符合线性自由能关系.     

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.