Publications by authors named "Akiva Feintuch"

55 Publications

Substrate binding in the multidrug transporter MdfA in detergent solution and in lipid nanodiscs.

Biophys J 2021 May 24;120(10):1984-1993. Epub 2021 Mar 24.

Departments of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

MdfA from Escherichia coli is a prototypical secondary multi-drug (Mdr) transporter that exchanges drugs for protons. MdfA-mediated drug efflux is driven by the proton gradient and enabled by conformational changes that accompany the recruitment of drugs and their release. In this work, we applied distance measurements by W-band double electron-electron resonance (DEER) spectroscopy to explore the binding of mito-TEMPO, a nitroxide-labeled substrate analog, to Gd(III)-labeled MdfA. The choice of Gd(III)-nitroxide DEER enabled measurements in the presence of excess of mito-TEMPO, which has a relatively low affinity to MdfA. Distance measurements between mito-TEMPO and MdfA labeled at the periplasmic edges of either of three selected transmembrane helices (TM3, TM5, and TM9) revealed rather similar distance distributions in detergent micelles (n-dodecyl-β-d-maltopyranoside, DDM)) and in lipid nanodiscs (ND). By grafting the predicted positions of the Gd(III) tag on the inward-facing (I) crystal structure, we looked for binding positions that reproduced the maxima of the distance distributions. The results show that the location of the mito-TEMPO nitroxide in DDM-solubilized or ND-reconstituted MdfA is similar (only 0.4 nm apart). In both cases, we located the nitroxide moiety near the ligand binding pocket in the I structure. However, according to the DEER-derived position, the substrate clashes with TM11, suggesting that for mito-TEMPO-bound MdfA, TM11 should move relative to the I structure. Additional DEER studies with MdfA labeled with Gd(III) at two sites revealed that TM9 also dislocates upon substrate binding. Together with our previous reports, this study demonstrates the utility of Gd(III)-Gd(III) and Gd(III)-nitroxide DEER measurements for studying the conformational behavior of transporters.
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http://dx.doi.org/10.1016/j.bpj.2021.03.014DOI Listing
May 2021

Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme.

Nat Commun 2020 11 23;11(1):5945. Epub 2020 Nov 23.

Research School of Chemistry, The Australian National University, Canberra, ACT, 2601, Australia.

Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron-electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational sampling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively samples catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.
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http://dx.doi.org/10.1038/s41467-020-19695-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683729PMC
November 2020

In-cell destabilization of a homodimeric protein complex detected by DEER spectroscopy.

Proc Natl Acad Sci U S A 2020 08 11;117(34):20566-20575. Epub 2020 Aug 11.

Department of Chemical and Biological Physics, Weizmann Institute of Science, 76100 Rehovot, Israel;

The complexity of the cellular medium can affect proteins' properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer-monomer dissociation constant was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.
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http://dx.doi.org/10.1073/pnas.2005779117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456071PMC
August 2020

Two closed ATP- and ADP-dependent conformations in yeast Hsp90 chaperone detected by Mn(II) EPR spectroscopic techniques.

Proc Natl Acad Sci U S A 2020 01 20;117(1):395-404. Epub 2019 Dec 20.

Department of Chemical and Biological Physics, Weizmann Institute of Science, 76100 Rehovot, Israel;

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.
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http://dx.doi.org/10.1073/pnas.1916030116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6955315PMC
January 2020

Proton polarization enhancement of up to 150 with dynamic nuclear polarization of plasma-treated glucose powder.

Solid State Nucl Magn Reson 2019 08 16;100:26-35. Epub 2019 Mar 16.

Schulich Faculty of Chemistry, Technion - Israel Institute of Technology, Haifa, 32000, Israel. Electronic address:

Dynamic nuclear polarization (DNP) for the enhancement of the NMR signals of specific metabolites has recently found applications in the context of magnetic resonance imaging (MRI). Currently, DNP signal enhancement is implemented in clinical systems through the use of exogenous stable organic free radicals, known as polarization agents (PAs), mixed in a solution with the metabolite of interest. These PAs are medically undesirable and thus must be filtered out prior to patient injection - a task that involves considerable technical complexity and consumes valuable time during which the polarization decays. Here, we aim to demonstrate DNP enhancements large enough for clinical relevance using a process free of exogenous PAs. This is achieved by processing (soft grinding) the metabolite in its solid form and subsequently exposing it to plasma in a dilute atmosphere to produce chemically-unstable free radicals (herein referred to as electrical-discharge-induced radicals - EDIRs) within the powder. These samples are then subjected to the normal DNP procedure of microwave irradiation while placed under a high static magnetic field, and their NMR signal is measured to quantify the enhancement of the protons' signal in the solid. Proton signal enhancements (measured as the ratio of the NMR signal with microwave irradiation to the NMR signal without microwave irradiation) of up to 150 are demonstrated in glucose. Upon fast dissolution, the free radicals are annihilated, leaving the sample in its original chemical composition (which is safe for clinical use) without any need for filtration and cumbersome quality control procedures. We thus conclude that EDIRs are found to be highly efficient in providing DNP enhancement levels that are on par with those achieved with the exogenous PAs, while being safe for clinical use. This opens up the possibility of applying our method to clinical scenarios with minimal risks and lower costs per procedure.
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http://dx.doi.org/10.1016/j.ssnmr.2019.03.003DOI Listing
August 2019

Theoretical Aspects of the Cross Effect Enhancement of Nuclear Polarization under Static Dynamic Nuclear Polarization Conditions.

J Phys Chem Lett 2019 Apr 1;10(8):1769-1778. Epub 2019 Apr 1.

Department of Chemical and Biological Physics , Weizmann Institute of Science , Rehovot 76100 , Israel.

In this study, we perform quantum calculations of the spin dynamics of a small spin system that includes nine coupled electrons and one nucleus placed in an external magnetic field and exposed to microwave irradiation. This is an extension of a previous work in which we have demonstrated on a system of 11 coupled electron spins the dynamics of the electron polarizations composing the electron paramagnetic resonance (EPR) line during static dynamic nuclear polarization (DNP) experiments. There we have shown that the electron polarizations are determined by a spectral diffusion process, induced by the dipolar interaction and cross-relaxation. Additionally, we showed that a distinction had to be made between strong and weak dipolar-coupled systems relative to the inhomogeneity of the EPR line with only the first behaving according to the thermal mixing DNP (with two electron spin temperatures) description. The EPR spectra in the weak and strong dipolar interaction cases show different types of spectral features. In the extended spin system, we again make a distinction between weak and strong electron-electron interactions and show that the DNP spectra for the two cases are different in nature but that the DNP spectra can be derived in all cases from the EPR line shapes using the indirect cross effect.
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http://dx.doi.org/10.1021/acs.jpclett.8b03615DOI Listing
April 2019

DEER distance measurements on trityl/trityl and Gd(iii)/trityl labelled proteins.

Phys Chem Chem Phys 2019 May;21(20):10217-10227

Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot 76100, Israel.

Triarylmethyl (TAM or trityl) radicals are becoming important for measuring distances in proteins and nucleic acids. Here, we report on a new trityl spin label CT02MA, which conjugates to a protein via a redox stable thioether bond. The performance of the new spin label was demonstrated in W-band double electron-electron resonance (DEER) distance measurements on doubly trityl-labelled mutants of immunoglobulin G-binding protein 1 (GB1) and ubiquitin. For both doubly CT02MA-labelled proteins we measured, by applying chirped pump pulse(s), relatively narrow distance distributions, comparable to those obtained with the same protein mutants doubly labelled with BrPy-DO3MA-Gd(iii). We noticed, however, that the sample contained some free CT02MA that was difficult to remove at the purification step. Dual labelling of ubiquitin with one CT02MA tag and one BrPy-DO3MA-Gd(iii) tag was achieved as well and the trityl-Gd(iii) distance distribution was measured, facilitated by the use of a dual mode cavity in combination with a chirped pump pulse. We also measured the Gd(iii)-Gd(iii) distance distribution in this sample, showing that the labelling procedure was not fully selective. Nevertheless, these measurements demonstrate the potential of the high sensitivity Gd(iii)-trityl W-band DEER distance measurements in proteins, which can be further exploited by designing orthogonal Gd(iii)/trityl labelling schemes.
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http://dx.doi.org/10.1039/c8cp07249cDOI Listing
May 2019

The effects of sample conductivity on the efficacy of dynamic nuclear polarization for sensitivity enhancement in solid state NMR spectroscopy.

Solid State Nucl Magn Reson 2019 Jul 20;99:7-14. Epub 2019 Feb 20.

Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, 7610001, Israel. Electronic address:

In recent years dynamic nuclear polarization (DNP) has greatly expanded the range of materials systems that can be studied by solid state NMR spectroscopy. To date, the majority of systems studied by DNP were insulating materials including organic and inorganic solids. However, many technologically-relevant materials used in energy conversion and storage systems are electrically conductive to some extent or are employed as composites containing conductive additives. Such materials introduce challenges in their study by DNP-NMR which include microwave absorption and sample heating that were not thoroughly investigated so far. Here we examine several commercial carbon allotropes, commonly employed as electrodes or conductive additives, and consider their effect on the extent of solvent polarization achieved in DNP from nitroxide biradicals. We then address the effect of sample conductivity systematically by studying a series of carbons with increasing electrical conductivity prepared via glucose carbonization. THz spectroscopy measurements are used to determine the extent of μw absorption. Our results show that while the DNP performance significantly drops in samples containing the highly conductive carbons, sufficient signal enhancement can still be achieved with some compromise on conductivity. Furthermore, we show that the deleterious effect of conductive additives on DNP enhancements can be partially overcome through pulse-DNP experiments.
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http://dx.doi.org/10.1016/j.ssnmr.2019.02.003DOI Listing
July 2019

Experimental quantification of electron spectral-diffusion under static DNP conditions.

Phys Chem Chem Phys 2018 Dec;21(1):478-489

Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot 7610001, Israel.

Dynamic Nuclear Polarization (DNP) is an efficient technique for enhancing NMR signals by utilizing the large polarization of electron spins to polarize nuclei. The mechanistic details of the polarization transfer process involve the depolarization of the electrons resulting from microwave (MW) irradiation (saturation), as well as electron-electron cross-relaxation occurring during the DNP experiment. Recently, electron-electron double resonance (ELDOR) experiments have been performed under DNP conditions to map the depolarization profile along the EPR spectrum as a consequence of spectral diffusion. A phenomenological model referred to as the eSD model was developed earlier to describe the spectral diffusion process and thus reproduce the experimental results of electron depolarization. This model has recently been supported by quantum mechanical calculations on a small dipolar coupled electron spin system, experiencing dipolar interaction based cross-relaxation. In the present study, we performed a series of ELDOR measurements on a solid glassy solution of TEMPOL radicals in an effort to substantiate the eSD model and test its predictability in terms of electron depolarization profiles, in the steady-state and under non-equilibrium conditions. The crucial empirical parameter in this model is ΛeSD, which reflects the polarization exchange rate among the electron spins. Here, we explore further the physical basis of this parameter by analyzing the ELDOR spectra measured in the temperature range of 3-20 K and radical concentrations of 20-40 mM. Simulations using the eSD model were carried out to determine the dependence of ΛeSD on temperature and concentration. We found that for the samples studied, ΛeSD is temperature independent. It, however, increases with a power of ∼2.6 of the concentration of TEMPOL, which is proportional to the average electron-electron dipolar interaction strength in the sample.
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http://dx.doi.org/10.1039/c8cp05930fDOI Listing
December 2018

High Sensitivity In-Cell EPR Distance Measurements on Proteins using an Optimized Gd(III) Spin Label.

J Phys Chem Lett 2018 Oct 9;9(20):6119-6123. Epub 2018 Oct 9.

Department of Chemical and Biological Physics , Weizmann Institute of Science , Rehovot 76100 , Israel.

Distance measurements by electron-electron double resonance (DEER) carried out on spin-labeled proteins delivered into cells provide new insights into the conformational states of proteins in their native environment. Such measurements depend on spin labels that exhibit high redox stability and high DEER sensitivity. Here we present a new Gd(III)-based spin label, BrPSPy-DO3A-Gd(III), which was derived from an earlier label, BrPSPy-DO3MA-Gd(III), by removing the methyl group from the methyl acetate pending arms. The small chemical modification led to a reduction in the zero-field splitting and to a significant increase in the phase memory time, which together culminated in a remarkable improvement of in-cell DEER sensitivity, while maintaining the high distance resolution. The excellent performance of BrPSPy-DO3A-Gd(III) in in-cell DEER measurements was demonstrated on doubly labeled ubiquitin and GB1 delivered into HeLa cells by electroporation.
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http://dx.doi.org/10.1021/acs.jpclett.8b02663DOI Listing
October 2018

Small neutral Gd(iii) tags for distance measurements in proteins by double electron-electron resonance experiments.

Phys Chem Chem Phys 2018 Sep;20(36):23535-23545

Research School of Chemistry, The Australian National University, Canberra, ACT 2601, Australia.

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.
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http://dx.doi.org/10.1039/c8cp03532fDOI Listing
September 2018

Triple resonance EPR spectroscopy determines the Mn coordination to ATP.

J Magn Reson 2018 09 24;294:143-152. Epub 2018 Jul 24.

Department of Chemical and Biological Physics, Weizmann Institute of Science, Israel. Electronic address:

Mn often serves as a paramagnetic substitute to Mg, providing means for exploring the close environment of Mg in many biological systems where it serves as an essential co-factor. This applies to proteins with ATPase activity, where the ATP hydrolysis requires the binding of Mg-ATP to the ATPase active site. In this context, it is important to distinguish between the Mn coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band P electron-nuclear double resonance (ENDOR) we obtained information about the coordination to the phosphates, whereas from electron-electron double resonance (ELDOR) - detected NMR (EDNMR) we determined the coordination to an adenosine nitrogen. The coordination to these ligands has been reported earlier, but whether the nitrogen and phosphate coordination is within the same nucleotide molecules or different ones is still under debate. By applying the correlation technique, THYCOS (triple hyperfine correlation spectroscopy), and measuring N-P correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn ion. We also carried out DFT calculations to substantiate this finding. In addition, we expanded the understanding of the THYCOS experiment by comparing it to 2D-EDNMR for Mn-P correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with N-P correlations.
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http://dx.doi.org/10.1016/j.jmr.2018.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230374PMC
September 2018

Paramagnetic Metal-Ion Dopants as Polarization Agents for Dynamic Nuclear Polarization NMR Spectroscopy in Inorganic Solids.

Chemphyschem 2018 09 12;19(17):2139-2142. Epub 2018 Jun 12.

Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, 761000, Israel.

Dynamic nuclear polarization (DNP), a technique in which the high electron spin polarization is transferred to surrounding nuclei via microwave irradiation, equips solid-state NMR spectroscopy with unprecedented sensitivity. The most commonly used polarization agents for DNP are nitroxide radicals. However, their applicability to inorganic materials is mostly limited to surface detection. Paramagnetic metal ions were recently introduced as alternatives for nitroxides. Doping inorganic solids with paramagnetic ions can be used to tune material properties and introduces endogenous DNP agents that can potentially provide sensitivity in the particles' bulk and surface. Here we demonstrate the approach by doping Li Ti O (LTO), an anode material for lithium ion batteries, with paramagnetic ions. By incorporating Gd(III) and Mn(II) in LTO we gain up to 14 fold increase in signal intensity in static Li DNP-NMR experiments. These results suggest that doping with paramagnetic ions provides an efficient route for sensitivity enhancement in the bulk of micron size particles.
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http://dx.doi.org/10.1002/cphc.201800462DOI Listing
September 2018

Small Gd(III) Tags for Gd(III)-Gd(III) Distance Measurements in Proteins by EPR Spectroscopy.

Inorg Chem 2018 May 9;57(9):5048-5059. Epub 2018 Apr 9.

Department of Chemical Physics , Weizmann Institute of Science , Rehovot 76100 , Israel.

The C7-Gd and C8-Gd tags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of the C7-Gd ( S configuration) and C8-Gd ( R configuration) tags loaded with Gd(III) on two mutants of the homodimeric ERp29 protein. The W-band EPR spectra were found to differ between the tags in the free state and after conjugation to the protein. In addition, the spectra were sensitive to the labeling position, which may originate from an environment-dependent charge density on the Gd(III)-coordinating oxygens. This is in agreement with previous NMR experiments with different lanthanide ions, which suggested sensitivity to H-bonding. W-band H-ENDOR (electron-electron double resonance) experiments detected effects from orientation selection in the central transition, due to a relatively narrow distribution in the ZFS parameters as indicated by simulations. In contrast, the distance distributions derived from DEER (double electron-electron resonance) measurements were insensitive to the R or S configuration of the tags and did not exhibit any orientation selection effects. The DEER measurements faithfully reflected the different widths of the distance distributions at the different protein sites in agreement with previous DEER measurements using other Gd(III) tags. Due to their small size, short tether to the protein, and a broad central EPR transition, the C7-Gd and C8-Gd tags are attractive Gd(III) tags for measurements of relatively short (<4 nm) distances by EPR spectroscopy.
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http://dx.doi.org/10.1021/acs.inorgchem.8b00133DOI Listing
May 2018

Electron-Electron Cross-Relaxation and Spectral Diffusion during Dynamic Nuclear Polarization Experiments on Solids.

J Phys Chem Lett 2018 Apr 27;9(7):1793-1802. Epub 2018 Mar 27.

Department of Chemical and Biological Physics , Weizmann Institute of Science , Rehovot - 76100 , Israel.

Recently it has been shown that experimental electron-electron double resonance (ELDOR) spectra of amorphous glasses containing free radicals with inhomogeneously broadened electron paramagnetic resonance (EPR) spectra can be analyzed using a set of coupled rate equations for the electron polarizations of frequency bins composing these spectra, named the eSD (electron spectral diffusion) model. The rate matrix defining these equations has elements depending on the microwave, the spin-lattice relaxation rates and on eSD rate constants responsible for polarization exchange. In this study, we show that in addition to the static dipolar flip-flop terms in the Hamiltonian a zero-quantum electron cross-relaxation mechanism can be responsible for the polarization exchange process in our samples. This conclusion was reached by calculating the EPR lineshapes of a system of 11 coupled electrons exposed to microwave irradiation using an eigenstate population rate equation derived from the spin density vector rate equation in Liouville space. These equations involve all terms of the Hamiltonian and in addition rate constants representing longitudinal relaxation and cross-relaxation mechanisms as well as MW irradiation. The results of these calculations are compared with the results obtained from the eSD model.
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http://dx.doi.org/10.1021/acs.jpclett.8b00090DOI Listing
April 2018

Surface loop-gap resonators for electron spin resonance at W-band.

Rev Sci Instrum 2017 Dec;88(12):123901

Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa 32000, Israel.

Electron spin resonance (ESR) is a spectroscopic method used to detect paramagnetic materials, reveal their structure, and also image their position in a sample. ESR makes use of a large static magnetic field to split the energy levels of the electron magnetic moment of the paramagnetic species. A strong microwave magnetic field is applied to excite the spins, and subsequently the ESR system detects their faint microwave signal response. The sensitivity of an ESR system is greatly influenced by the magnitude of the static field and the properties of the microwave resonator used to detect the spin signal. In general terms, the higher the static field (microwave frequency) and the smaller the resonator, the more sensitive the system will be. Previous work aimed at high-sensitivity ESR was focused on the development and testing of very small resonators operating at moderate magnetic fields in the range of ∼0.1-1.2 T (maximum frequency of ∼35 GHz). Here, we describe the design, construction, and testing of recently developed miniature surface loop-gap resonators used in ESR and operating at a much higher frequency of ∼95 GHz (W-band, corresponding to a field of ∼3.4 T). Such resonators can greatly enhance the sensitivity of ESR and also improve the resulting spectral resolution due to the higher static field employed. A detailed description of the resonator's design and coupling mechanism, as well as the supporting probe head, is provided. We also discuss the production method of the resonators and probe head and, in the end, provide preliminary experimental results that show the setup's high spin sensitivity and compare it to theoretical predictions.
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http://dx.doi.org/10.1063/1.5000946DOI Listing
December 2017

Selective Distance Measurements Using Triple Spin Labeling with Gd, Mn, and a Nitroxide.

J Phys Chem Lett 2017 Nov 13;8(21):5277-5282. Epub 2017 Oct 13.

Department of Chemical Physics, Weizmann Institute of Science , Rehovot 76100, Israel.

Distance measurements by pulse electron paramagnetic resonance techniques, such as double electron-electron resonance (DEER, also called PELDOR), have become an established tool to explore structural properties of biomacromolecules and their assemblies. In such measurements a pair of spin labels provides a single distance constraint. Here we show that by employing three different types of spin labels that differ in their spectroscopic and spin dynamics properties it is possible to extract three independent distances from a single sample. We demonstrate this using the Antennapedia homeodomain orthogonally labeled with Gd and Mn tags in complex with its cognate DNA binding site labeled with a nitroxide.
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http://dx.doi.org/10.1021/acs.jpclett.7b01739DOI Listing
November 2017

Improved sensitivity for W-band Gd(III)-Gd(III) and nitroxide-nitroxide DEER measurements with shaped pulses.

J Magn Reson 2017 10 10;283:1-13. Epub 2017 Aug 10.

Department of Chemical Physics, Weizmann Institute of Science, 76100 Rehovot, Israel. Electronic address:

Chirp and shaped pulses have been recently shown to be highly advantageous for improving sensitivity in DEER (double electron-electron resonance, also called PELDOR) measurements due to their large excitation bandwidth. The implementation of such pulses for pulse EPR has become feasible due to the availability of arbitrary waveform generators (AWG) with high sampling rates to support pulse shaping for pulses with tens of nanoseconds duration. Here we present a setup for obtaining chirp pulses on our home-built W-band (95GHz) spectrometer and demonstrate its performance on Gd(III)-Gd(III) and nitroxide-nitroxide DEER measurements. We carried out an extensive optimization procedure on two model systems, Gd(III)-PyMTA-spacer-Gd(III)-PyMTA (Gd-PyMTA ruler; zero-field splitting parameter (ZFS) D∼1150MHz) as well as nitroxide-spacer-nitroxide (nitroxide ruler) to evaluate the applicability of shaped pulses to Gd(III) complexes and nitroxides, which are two important classes of spin labels used in modern DEER/EPR experiments. We applied our findings to ubiquitin, doubly labeled with Gd-DOTA-monoamide (D∼550MHz) asa model for a system with a small ZFS. Our experiments were focused on the questions (i) what are the best conditions for positioning of the detection frequency, (ii) which pump pulse parameters (bandwidth, positioning in the spectrum, length) yield the best signal-to-noise ratio (SNR) improvements when compared to classical DEER, and (iii) how do the sample's spectral parameters influence the experiment. For the nitroxide ruler, we report an improvement of up to 1.9 in total SNR, while for the Gd-PyMTA ruler the improvement was 3.1-3.4 and for Gd-DOTA-monoamide labeled ubiquitin it was a factor of 1.8. Whereas for the Gd-PyMTA ruler the two setups pump on maximum and observe on maximum gave about the same improvement, for Gd-DOTA-monoamide a significant difference was found. In general the choice of the best set of parameters depends on the D parameter of the Gd(III) complex.
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http://dx.doi.org/10.1016/j.jmr.2017.08.003DOI Listing
October 2017

Time domain simulation of Gd-Gd distance measurements by EPR.

J Chem Phys 2017 Jul;147(4):044201

Department of Chemical Physics, Weizmann Institute of Science, Rehovot 7610001, Israel.

Gd-based spin labels are useful as an alternative to nitroxides for intramolecular distance measurements at high fields in biological systems. However, double electron-electron resonance (DEER) measurements using model Gd complexes featured a low modulation depth and an unexpected broadening of the distance distribution for short Gd-Gd distances, when analysed using the software designed for S = 1/2 pairs. It appears that these effects result from the different spectroscopic characteristics of Gd-the high spin, the zero field splitting (ZFS), and the flip-flop terms in the dipolar Hamiltonian that are often ignored for spin-1/2 systems. An understanding of the factors affecting the modulation frequency and amplitude is essential for the correct analysis of Gd-Gd DEER data and for the educated choice of experimental settings, such as Gd spin label type and the pulse parameters. This work uses time-domain simulations of Gd-Gd DEER by explicit density matrix propagation to elucidate the factors shaping Gd DEER traces. The simulations show that mixing between the |+½, -½〉 and |-½, +½〉 states of the two spins, caused by the flip-flop term in the dipolar Hamiltonian, leads to dampening of the dipolar modulation. This effect may be mitigated by a large ZFS or by pulse frequency settings allowing for a decreased contribution of the central transition and the one adjacent to it. The simulations reproduce both the experimental line shapes of the Fourier-transforms of the DEER time domain traces and the trends in the behaviour of the modulation depth, thus enabling a more systematic design and analysis of Gd DEER experiments.
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http://dx.doi.org/10.1063/1.4994084DOI Listing
July 2017

Thiolate Spin Population of Type I Copper in Azurin Derived from S Hyperfine Coupling.

Inorg Chem 2017 Jun 16;56(11):6163-6174. Epub 2017 May 16.

Department of Chemical Physics, Weizmann Institute of Science , Rehovot 76100, Israel.

The electron transfer mediating properties of type I copper proteins stem from the intricate ligand coordination sphere of the Cu ion in their active site. These redox properties are in part due to unusual cysteine thiol coordination, which forms a highly covalent copper-sulfur (Cu-S) bond. The structure and electronic properties of type I copper have been the subject of many experimental and theoretical studies. The measurement of spin delocalization of the Cu(II) unpaired electron to neighboring ligands provides an elegant experimental way to probe the fine details of the electronic structure of type I copper. To date, the crucial parameter of electron delocalization to the sulfur atom of the cysteine ligand has not been directly determined experimentally. We have prepared S-enriched azurin and carried out W-band (95 GHz) electron paramagnetic resonance (EPR) and electron-electron double resonance detected NMR (EDNMR) measurements and, for the first time, recorded the S nuclear frequencies, from which the hyperfine coupling and the spin population on the sulfur of the thiolate ligand were derived. The overlapping S and N EDNMR signals were resolved using a recently introduced two-dimensional correlation technique, 2D-EDNMR. The S hyperfine tensor was determined by simulations of the EDNMR spectra using S hyperfine and quadrupolar tensors predicted by QM/MM DFT calculations as starting points for a manual spectral fit procedure. To reach a reasonable agreement with the experimental spectra, the S hyperfine principal value, A, and one of the corresponding Euler angles had to be modified. The final values obtained gave an experimentally determined sulfur spin population of 29.8 ± 0.7%, significantly improving the wide range of 29-62% reported in the literature. Our direct, experimentally derived value now provides an important constraint for further theoretical work aimed at unravelling the unique electronic properties of this site.
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http://dx.doi.org/10.1021/acs.inorgchem.7b00167DOI Listing
June 2017

Effect of electron spectral diffusion on static dynamic nuclear polarization at 7 Tesla.

Phys Chem Chem Phys 2017 Feb;19(5):3596-3605

Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA 93106, USA. and Department of Chemical Engineering, University of California Santa Barbara, Santa Barbara, CA 93106, USA.

Here, we present an integrated experimental and theoretical study of H dynamic nuclear polarization (DNP) of a frozen aqueous glass containing free radicals at 7 T, under static conditions and at temperatures ranging between 4 and 20 K. The DNP studies were performed with a home-built 200 GHz quasi-optics microwave bridge, powered by a tunable solid-state diode source. DNP using monochromatic and continuous wave (cw) irradiation applied to the electron paramagnetic resonance (EPR) spectrum of the radicals induces the transfer of polarization from the electron spins to the surrounding nuclei of the solvent and solutes in the frozen aqueous glass. In our systematic experimental study, the DNP enhanced H signals are monitored as a function of microwave frequency, microwave power, radical concentration, and temperature, and are interpreted with the help of electron spin-lattice relaxation times, experimental MW irradiation parameters, and the electron spectral diffusion (eSD) model introduced previously. This comprehensive experimental DNP study with mono-nitroxide radical spin probes was accompanied with theoretical calculations. Our results consistently demonstrate that eSD effects can be significant at 7 T under static DNP conditions, and can be systematically modulated by experimental conditions.
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http://dx.doi.org/10.1039/c6cp06893fDOI Listing
February 2017

On The Potential of Dynamic Nuclear Polarization Enhanced Diamonds in Solid-State and Dissolution (13) C NMR Spectroscopy.

Chemphyschem 2016 Sep 15;17(17):2691-701. Epub 2016 Jul 15.

Chemical Physics Department, Weizmann Institute of Science, Rehovot, Israel.

Dynamic nuclear polarization (DNP) is a versatile option to improve the sensitivity of NMR and MRI. This versatility has elicited interest for overcoming potential limitations of these techniques, including the achievement of solid-state polarization enhancement at ambient conditions, and the maximization of (13) C signal lifetimes for performing in vivo MRI scans. This study explores whether diamond's (13) C behavior in nano- and micro-particles could be used to achieve these ends. The characteristics of diamond's DNP enhancement were analyzed for different magnetic fields, grain sizes, and sample environments ranging from cryogenic to ambient temperatures, in both solution and solid-state experiments. It was found that (13) C NMR signals could be boosted by orders of magnitude in either low- or room-temperature solid-state DNP experiments by utilizing naturally occurring paramagnetic P1 substitutional nitrogen defects. We attribute this behavior to the unusually long electronic/nuclear spin-lattice relaxation times characteristic of diamond, coupled with a time-independent cross-effect-like polarization transfer mechanism facilitated by a matching of the nitrogen-related hyperfine coupling and the (13) C Zeeman splitting. The efficiency of this solid-state polarization process, however, is harder to exploit in dissolution DNP-enhanced MRI contexts. The prospects for utilizing polarized diamond approaching nanoscale dimensions for both solid and solution applications are briefly discussed.
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http://dx.doi.org/10.1002/cphc.201600301DOI Listing
September 2016

Correction: Gd(iii)-Gd(iii) EPR distance measurements - the range of accessible distances and the impact of zero field splitting.

Phys Chem Chem Phys 2016 Jul 21;18(27):18614. Epub 2016 Jun 21.

Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 76100, Israel.

Correction for 'Gd(iii)-Gd(iii) EPR distance measurements - the range of accessible distances and the impact of zero field splitting' by Arina Dalaloyan et al., Phys. Chem. Chem. Phys., 2015, 17, 18464-18476.
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http://dx.doi.org/10.1039/c6cp90156eDOI Listing
July 2016

Overcoming artificial broadening in Gd(3+)-Gd(3+) distance distributions arising from dipolar pseudo-secular terms in DEER experiments.

Phys Chem Chem Phys 2016 05 22;18(18):12847-59. Epub 2016 Apr 22.

Department of Chemical Physics, Weizmann Institute of Science, Rehovot 7610001, Israel.

By providing accurate distance measurements between spin labels site-specifically attached to bio-macromolecules, double electron-electron resonance (DEER) spectroscopy provides a unique tool to probe the structural and conformational changes in these molecules. Gd(3+)-tags present an important family of spin-labels for such purposes, as they feature high chemical stability and high sensitivity in high-field DEER measurements. The high sensitivity of the Gd(3+) ion is associated with its high spin (S = 7/2) and small zero field splitting (ZFS), resulting in a narrow spectral width of its central transition at high fields. However, under the conditions of short distances and exceptionally small ZFS, the weak coupling approximation, which is essential for straightforward DEER data analysis, becomes invalid and the pseudo-secular terms of the dipolar Hamiltonian can no longer be ignored. This work further explores the effects of pseudo-secular terms on Gd(3+)-Gd(3+) DEER measurements using a specifically designed ruler molecule; a rigid bis-Gd(3+)-DOTA model compound with an expected Gd(3+)-Gd(3+) distance of 2.35 nm and a very narrow central transition at the W-band (95 GHz). We show that the DEER dipolar modulations are damped under the standard W-band DEER measurement conditions with a frequency separation, Δν, of 100 MHz between the pump and observe pulses. Consequently, the DEER spectrum deviates considerably from the expected Pake pattern. We show that the Pake pattern and the associated dipolar modulations can be restored with the aid of a dual mode cavity by increasing Δν from 100 MHz to 1.09 GHz, allowing for a straightforward measurement of a Gd(3+)-Gd(3+) distance of 2.35 nm. The increase in Δν increases the contribution of the |-5/2〉→|-3/2〉 and |-7/2〉→|-5/2〉 transitions to the signal at the expense of the |-3/2 〉→|-1/2〉 transition, thus minimizing the effect of dipolar pseudo-secular terms and restoring the validity of the weak coupling approximation. We apply this approach to the A93C/N140C mutant of T4 lysozyme labeled with two different Gd(3+) tags that have narrow central transitions and show that even for a distance of 4 nm there is still a significant (about two-fold) broadening that is removed by increasing Δν to 636 MHz and 898 MHz.
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http://dx.doi.org/10.1039/c6cp00829aDOI Listing
May 2016

A New Gd(3+) Spin Label for Gd(3+)-Gd(3+) Distance Measurements in Proteins Produces Narrow Distance Distributions.

J Phys Chem Lett 2015 Dec 7;6(24):5016-21. Epub 2015 Dec 7.

Department of Chemical Physics, Weizmann Institute of Science , Rehovot 76100, Israel.

Gd(3+) tags have been shown to be useful for performing distance measurements in biomolecules via the double electron-electron resonance (DEER) technique at Q- and W-band frequencies. We introduce a new cyclen-based Gd(3+) tag that exhibits a relatively narrow electron paramagnetic resonance (EPR) spectrum, affording high sensitivity, and which yields exceptionally narrow Gd(3+)-Gd(3+) distance distributions in doubly tagged proteins owing to a very short tether. Both the maxima and widths of distance distributions measured for tagged mutants of the proteins ERp29 and T4 lysozyme, featuring Gd(3+)-Gd(3+) distances of ca. 6 and 4 nm, respectively, were well reproduced by simulated distance distributions based on available crystal structures and sterically allowed rotamers of the tag. The precision of the position of the Gd(3+) ion is comparable to that of the nitroxide radical in an MTSL-tagged protein and thus the new tag represents an attractive tool for performing accurate distance measurements and potentially probing protein conformational equilibria.
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http://dx.doi.org/10.1021/acs.jpclett.5b02451DOI Listing
December 2015

Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags.

J Biomol NMR 2016 Jan 23;64(1):39-51. Epub 2015 Nov 23.

Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.

Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd(3+)-Gd(3+) distances measured by double electron-electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm(3+) and C3-Tb(3+) tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb(3+) and Tm(3+) rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.
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http://dx.doi.org/10.1007/s10858-015-0003-zDOI Listing
January 2016

Gd³⁺ Spin Labeling for Measuring Distances in Biomacromolecules: Why and How?

Methods Enzymol 2015 1;563:415-57. Epub 2015 Sep 1.

Department of Chemical Physics, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

Applications of distance measurements by pulse dipolar electron-paramagnetic resonance (PD-EPR) spectroscopy to structural biology are based on introducing spin labels (SLs) at well-defined locations in the biomacromolecule. The most commonly used SLs are nitroxyl radicals, but recently SLs based on high-spin Gd(3+) (S=7/2) complexes have been shown to be an attractive alternative for PD-EPR, particularly double electron-electron resonance (DEER), at spectrometer frequencies higher than 30 GHz. In this chapter, we describe the advantage of using this new family of SLs in terms of sensitivity, stability, and chemical diversity. We present current labeling strategies for proteins, discuss the approximations under which DEER data analysis of a pair of Gd(3+) SLs (GdSLs) is equivalent to that of a pair of S=1/2 SLs, and discuss the reduction in multispin effects in a cluster of GdSLs, as opposed to a cluster of nitroxide labels, which can be found in oligomeric systems. In addition, we provide a brief overview of the current, rather limited, knowledge of Gd(3+) phase relaxation behavior and describe experimental strategies in terms of optimizing sensitivity. The possibility of using several types of SLs in a system allows one to isolate effects due to the chemical nature of the SL itself; several such examples are presented, focusing on comparing nitroxide and GdSLs. Finally, we will discuss the initial results on in-cell DEER with GdSLs.
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http://dx.doi.org/10.1016/bs.mie.2015.07.006DOI Listing
July 2016

The effect of Gd on trityl-based dynamic nuclear polarisation in solids.

Phys Chem Chem Phys 2015 Oct;17(40):26969-78

Magnetic Resonance Center (CERM) and Department of Chemistry "Ugo Schiff", University of Florence, Italy.

In dynamic nuclear polarisation (DNP) experiments performed under static conditions at 1.4 K we show that the presence of 1 mM Gd(iii)-DOTAREM increases the (13)C polarisation and decreases the (13)C polarisation buildup time of (13)C-urea dissolved in samples containing water/DMSO mixtures with trityl radical (OX063) concentrations of 10 mM or higher. To account for these observations further measurements were carried out at 6.5 K, using a combined EPR and NMR spectrometer. At this temperature, frequency swept DNP spectra of samples with 5 or 10 mM OX063 were measured, with and without 1 mM Gd-DOTA, and again a (13)C enhancement gain was observed due to the presence of Gd-DOTA. These measurements were complemented by electron-electron double resonance (ELDOR) measurements to quantitate the effect of electron spectral diffusion (eSD) on the DNP enhancements and lineshapes. Simulations of the ELDOR spectra were done using the following parameters: (i) a parameter defining the rate of the eSD process, (ii) an "effective electron-proton anisotropic hyperfine interaction parameter", and (iii) the transverse electron spin relaxation time of OX063. These parameters, together with the longitudinal electron spin relaxation time, measured by EPR, were used to calculate the frequency profile of electron polarisation. This, in turn, was used to calculate two basic solid effect (SE) and indirect cross effect (iCE) DNP spectra. A properly weighted combination of these two normalized DNP spectra provided a very good fit of the experimental DNP spectra. The best fit simulation parameters reveal that the addition of Gd(iii)-DOTA causes an increase in both the SE and the iCE contributions by similar amounts, and that the increase in the overall DNP enhancements is a result of narrowing of the ELDOR spectra (increased electron polarisation gradient across the EPR line). These changes in the electron depolarisation profile are a combined result of shortening of the longitudinal and transverse electron spin relaxation times, as well as an increase in the eSD rate and in the effective electron-proton anisotropic hyperfine interaction parameter.
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http://dx.doi.org/10.1039/c5cp04138dDOI Listing
October 2015

Nuclear depolarization and absolute sensitivity in magic-angle spinning cross effect dynamic nuclear polarization.

Phys Chem Chem Phys 2015 Sep 3;17(34):21824-36. Epub 2015 Aug 3.

Univ. Grenoble Alpes, INAC, SCIB, F-38000 Grenoble, France.

Over the last two decades solid state Nuclear Magnetic Resonance has witnessed a breakthrough in increasing the nuclear polarization, and thus experimental sensitivity, with the advent of Magic Angle Spinning Dynamic Nuclear Polarization (MAS-DNP). To enhance the nuclear polarization of protons, exogenous nitroxide biradicals such as TOTAPOL or AMUPOL are routinely used. Their efficiency is usually assessed as the ratio between the NMR signal intensity in the presence and the absence of microwave irradiation εon/off. While TOTAPOL delivers an enhancement εon/off of about 60 on a model sample, the more recent AMUPOL is more efficient: >200 at 100 K. Such a comparison is valid as long as the signal measured in the absence of microwaves is merely the Boltzmann polarization and is not affected by the spinning of the sample. However, recent MAS-DNP studies at 25 K by Thurber and Tycko (2014) have demonstrated that the presence of nitroxide biradicals combined with sample spinning can lead to a depolarized nuclear state, below the Boltzmann polarization. In this work we demonstrate that TOTAPOL and AMUPOL both lead to observable depolarization at ≈110 K, and that the magnitude of this depolarization is radical dependent. Compared to the static sample, TOTAPOL and AMUPOL lead, respectively, to nuclear polarization losses of up to 20% and 60% at a 10 kHz MAS frequency, while Trityl OX63 does not depolarize at all. This experimental work is analyzed using a theoretical model that explains how the depolarization process works under MAS and gives new insights into the DNP mechanism and into the spin parameters, which are relevant for the efficiency of a biradical. In light of these results, the outstanding performance of AMUPOL must be revised and we propose a new method to assess the polarization gain for future radicals.
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http://dx.doi.org/10.1039/c5cp03457dDOI Listing
September 2015