Akiva Feintuch

Theoretical aspects of dynamic nuclear polarization in the solid state – The solid effect

Dynamic nuclear polarization has gained high popularity in recent years, due to advances in the experimental aspects of this methodology for increasing the NMR and MRI signals of relevant chemical and biological compounds. The DNP mechanism relies on the microwave (MW) irradiation induced polarization transfer from unpaired electrons to the nuclei in a sample. In this publication we present nuclear polarization enhancements of model systems in the solid state at high magnetic fields. These results were obtained by numerical calculations based on the spin density operator formalism. Here we restrict ourselves to samples with low electron concentrations, where the dipolar electron-electron interactions can be ignored. Thus the DNP enhancement of the polarizations of the nuclei close to the electrons is described by the Solid Effect mechanism. Our numerical results demonstrate the dependence of the polarization enhancement on the MW irradiation power and frequency, the hyperfine and nuclear dipole-dipole spin interactions, and the relaxation parameters of the system. The largest spin system considered in this study contains one electron and eight nuclei. In particular, we discuss the influence of the nuclear concentration and relaxation on the polarization of the core nuclei, which are coupled to an electron, and are responsible for the transfer of polarization to the bulk nuclei in the sample via spin diffusion.

Fast passage dynamic nuclear polarization on rotating solids

Magic Angle Spinning (MAS) Dynamic Nuclear Polarization (DNP) has proven to be a very powerful way to improve the signal to noise ratio of NMR experiments on solids. The experiments have in general been interpreted considering the Solid-Effect (SE) and Cross-Effect (CE) DNP mechanisms while ignoring the influence of sample spinning. In this paper, we show experimental data of MAS-DNP enhancements of (1)H and (13)C in proline and SH3 protein in glass forming water/glycerol solvent containing TOTAPOL. We also introduce a theoretical model that aims at explaining how the nuclear polarization is built in MAS-DNP experiments. By using Liouville space based simulations to include relaxation on two simple spin models, {electron-nucleus} and {electron-electron-nucleus}, we explain how the basic MAS-SE-DNP and MAS-CE-DNP processes work. The importance of fast energy passages and short level anti-crossing is emphasized and the differences between static DNP and MAS-DNP is explained. During a single rotor cycle the enhancement in the {electron-electron-nucleus} system arises from MAS-CE-DNP involving at least three kinds of two-level fast passages: an electron-electron dipolar anti-crossing, a single quantum electron MW encounter and an anti-crossing at the CE condition inducing nuclear polarization in- or decrements. Numerical, powder-averaged, simulations were performed in order to check the influence of the experimental parameters on the enhancement efficiencies. In particular we show that the spinning frequency dependence of the theoretical MAS-CE-DNP enhancement compares favorably with the experimental (1)H and (13)C MAS-DNP enhancements of proline and SH3.

Probing Protein Conformation in Cells by EPR Distance Measurements using Gd3+ Spin Labeling

Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electron-electron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd(3+) chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells.

Dynamic nuclear polarization assisted spin diffusion for the solid effect case.

The dynamic nuclear polarization (DNP) process in solids depends on the magnitudes of hyperfine interactions between unpaired electrons and their neighboring (core) nuclei, and on the dipole-dipole interactions between all nuclei in the sample. The polarization enhancement of the bulk nuclei has been typically described in terms of a hyperfine-assisted polarization of a core nucleus by microwave irradiation followed by a dipolar-assisted spin diffusion process in the core-bulk nuclear system. This work presents a theoretical approach for the study of this combined process using a density matrix formalism. In particular, solid effect DNP on a single electron coupled to a nuclear spin system is considered, taking into account the interactions between the spins as well as the main relaxation mechanisms introduced via the electron, nuclear, and cross-relaxation rates. The basic principles of the DNP-assisted spin diffusion mechanism, polarizing the bulk nuclei, are presented, and it is shown that the polarization of the core nuclei and the spin diffusion process should not be treated separately. To emphasize this observation the coherent mechanism driving the pure spin diffusion process is also discussed. In order to demonstrate the effects of the interactions and relaxation mechanisms on the enhancement of the nuclear polarization, model systems of up to ten spins are considered and polarization buildup curves are simulated. A linear chain of spins consisting of a single electron coupled to a core nucleus, which in turn is dipolar coupled to a chain of bulk nuclei, is considered. The interaction and relaxation parameters of this model system were chosen in a way to enable a critical analysis of the polarization enhancement of all nuclei, and are not far from the values of (13)C nuclei in frozen (glassy) organic solutions containing radicals, typically used in DNP at high fields. Results from the simulations are shown, demonstrating the complex dependences of the DNP-assisted spin diffusion process on variations of the relevant parameters. In particular, the effect of the spin lattice relaxation times on the polarization buildup times and the resulting end polarization are discussed, and the quenching of the polarizations by the hyperfine interaction is demonstrated.

Spectroscopic selection of distance measurements in a protein dimer with mixed nitroxide and Gd3+ spin labels

The pulse DEER (Double Electron-Electron Resonance) technique is frequently applied for measuring nanometer distances between specific sites in biological macromolecules. In this work we extend the applicability of this method to high field distance measurements in a protein assembly with mixed spin labels, i.e. a nitroxide spin label and a Gd(3+) tag. We demonstrate the possibility of spectroscopic selection of distance distributions between two nitroxide spin labels, a nitroxide spin label and a Gd(3+) ion, and two Gd(3+) ions. Gd(3+)-nitroxide DEER measurements possess high potential for W-band long range distance measurements (6 nm) by combining high sensitivity with ease of data analysis, subject to some instrumental improvements.

Gadolinium(III) spin labels for high-sensitivity distance measurements in transmembrane helices.

Distance determination, by pulse EPR techniques, between two spin labels attached to biomolecules has become an attractive methodology to probe conformations and assemblies of biomolecules in frozen solutions. Among these techniques, double electron-electron resonance (DEER or PELDOR), which can access distances in the range of 1.7 to 8 nm, is highly popular, and the most widely used spin labels are nitroxide radicals. Membrane proteins in their natural environment are of particular interest for DEER applications, since those pose a considerable challenge for Xray crystallography and NMR spectroscopy. DEER studies of peptides and proteins in either reconstituted or model membranes are considerably more challenging than those in solution, because the high local concentration of the spins in the membrane decreases the phase memory time and, therefore, sensitivity. Most DEER measurements on nitroxide-labeled biomolecules are carried out at X-band frequencies (9.5 GHz, 0.35 T), and recently such measurements were demonstrated in frozen cells. A major difficulty of such measurements is the reduction of nitroxides in the cell, which severely limits the scope of such exciting developments. Recently, Gd (S= 7/2) spin labels have been suggested as an alternative to nitroxide spin labels for W-band and Qband DEER distance measurements. Gd tags can be attached to proteins, similar to nitroxides, by site-directed spin labeling (SDSL). Gd features high sensitivity at high frequencies and the DEER measurements are free of orientation selection effects so that the distance distribution can readily be extracted from a single DEER measurement. Moreover, in the context of future development of in-cell DEER measurements, Gd chelates are stable under in vivo conditions as known from their applications as contrast agents for magnetic resonance imaging (MRI). Gd-Gd DEER has been demonstrated on model systems, proteins, peptides, and DNA, all in isotropic membrane-free solutions. Distance measurements between a Gd label and a nitroxide label have also been shown to yield attractive sensitivity. In this work, we continue to develop the approach of Gd–Gd DEER distance measurements and demonstrate for the first time such measurements in a model membrane. The model system we chose consists of the well-studied transmembrane helical WALP peptides in 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) vesicles. We demonstrate the sensitivity of W-band Gd–Gd DEER to small distance variations in a membrane. Using WALP peptides of different lengths we show that such measurements pick up, in addition to the helix extension, also subtle “cis–trans” effects arising from different positions of the labels with respect to the helix axes. In addition, we report the effect of the spin label interaction with the membranes on the measured distance distribution. We compared W-band DEER on WALP23 labeled with two different Gd tags with X-band DEER onWALP23 labeled with nitroxide tags. Here we used X-band, rather than W-band, to avoid complications owing to orientation selection. The differences observed are important and suggest that by employing different spin labels such effects can be isolated. Finally, we show that the effect of hydrophobic mismatch between peptide and membrane can be explored by Gd–Gd DEER. WALP23 was labeled at the N and C termini (see Table 1) with two nitroxides (WAL23-NO) using (l-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSSL) and two different Gd-DOTA derivatives, shown in Figure 1. WALP23-DOTA is labeled with a DOTA chelate and WALP23-C1 with DOTA with phenylethylamine substituents. The bulky substituents were designed to restrict the flexibility of the tag. The hydrophobic length of WALP23 (ca. 2.6 nm) is very close to that of the hydrophobic thickness of DOPC bilayers (ca. 2.7 nm). The sample composition was 50 mm WALP23 in DOPC multilamellar vesicles (MLV; 1:1000 peptide/lipid molar ratio). Details of the sample preparation are given in the Supporting Information. [*] Dr. E. Matalon, Dr. A. Feintuch, Prof. D. Goldfarb Department of Chemical Physics, Weizmann Institute of Science Rehovot, 76100 (Israel) E-mail: Daniella.goldfarb@weizmann.ac.il

The interplay between the solid effect and the cross effect mechanisms in solid state ¹³C DNP at 95 GHz using trityl radicals.

The (13)C solid state Dynamic Nuclear Polarization (DNP) mechanism using trityl radicals (OX63) as polarizers was investigated in the temperature range of 10-60K. The solutions used were 6M (13)C urea in DMSO/H2O (50% v/v) with 15 mM and 30 mM OX63. The measurements were carried out at ∼3.5 T, which corresponds to Larmor frequencies of 95 GHz and 36 MHz for the OX63 and the (13)C nuclei, respectively. Measurements of the (13)C signal intensity as a function of the microwave (MW) irradiation frequency yielded (13)C DNP spectra with temperature dependent lineshapes for both samples. The maximum enhancement for the 30 mM sample was reached at 40K, while that of the 15 mM sample at 20-30K. Furthermore, the lineshapes observed showed that both the cross effect (CE) and the solid effect (SE) DNP mechanisms are active in this temperature range and that their relative contribution is temperature dependent. Simulations of the spectra with the relative contributions of the CE and SE mechanisms as a fit parameter revealed that for both samples the CE contribution decreases with decreasing temperature while the SE contribution increases. In addition, for the 15 mM sample the contributions of the two mechanisms are comparable from 20K to 60K while for the 30 mM the CE dominates in this range, as expected from the higher concentration. The steep decrease of the CE contribution towards low temperatures is however unexpected. The temperature dependence of the OX63 longitudinal relaxation, DNP buildup times and (13)C spin lattice relaxation times did not reveal any obvious correlation with the DNP temperature dependence. A similar behavior of the CE and SE mechanism was observed for (1)H DNP with the nitroxide radical TEMPOL as a polarizer. This suggests that this effect is a general phenomenon involving a temperature dependent competition between the CE and SE mechanisms, the source of which is, however, still unknown.

A Dynamic Nuclear Polarization spectrometer at 95 GHz/144 MHz with EPR and NMR excitation and detection capabilities

A spectrometer specifically designed for systematic studies of the spin dynamics underlying Dynamic Nuclear Polarization (DNP) in solids at low temperatures is described. The spectrometer functions as a fully operational NMR spectrometer (144 MHz) and pulse EPR spectrometer (95 GHz) with a microwave (MW) power of up to 300 mW at the sample position, generating a MW B(1) field as high as 800 KHz. The combined NMR/EPR probe comprises of an open-structure horn-reflector configuration that functions as a low Q EPR cavity and an RF coil that can accommodate a 30-50 μl sample tube. The performance of the spectrometer is demonstrated through some basic pulsed EPR experiments, such as echo-detected EPR, saturation recovery and nutation measurements, that enable quantification of the actual intensity of MW irradiation at the position of the sample. In addition, DNP enhanced NMR signals of samples containing TEMPO and trityl are followed as a function of the MW frequency. Buildup curves of the nuclear polarization are recorded as a function of the microwave irradiation time period at different temperatures and for different MW powers.

Theoretical aspects of Magic Angle Spinning - Dynamic Nuclear Polarization

Magic Angle Spinning (MAS) combined with Dynamic Nuclear Polarization (DNP) has been proven in recent years to be a very powerful method for increasing solid-state NMR signals. Since the advent of biradicals such as TOTAPOL to increase the nuclear polarization new classes of radicals, with larger molecular weight and/or different spin properties have been developed. These have led to unprecedented signal gain, with varying results for different experimental parameters, in particular the microwave irradiation strength, the static field, and the spinning frequency. Recently it has been demonstrated that sample spinning imposes DNP enhancement processes that differ from the active DNP mechanism in static samples as upon sample spinning the DNP enhancements are the results of energy level anticrossings occurring periodically during each rotor cycle. In this work we present experimental results with regards to the MAS frequency dependence of the DNP enhancement profiles of four nitroxide-based radicals at two different sets of temperature, 110 and 160K. In fact, different magnitudes of reduction in enhancement are observed with increasing spinning frequency. Our simulation code for calculating MAS-DNP powder enhancements of small model spin systems has been improved to extend our studies of the influence of the interaction and relaxation parameters on powder enhancements. To achieve a better understanding we simulated the spin dynamics of a single three-spin system {ea-eb-n} during its steady state rotor periods and used the Landau-Zener formula to characterize the influence of the different anti-crossings on the polarizations of the system and their necessary action for reaching steady state conditions together with spin relaxation processes. Based on these model calculations we demonstrate that the maximum steady state nuclear polarization cannot become larger than the maximum polarization difference between the two electrons during the steady state rotor cycle. This study also shows the complexity of the MAS-DNP process and therefore the necessity to rely on numerical simulations for understanding parametric dependencies of the enhancements. Finally an extension of the spin system up to five spins allowed us to probe the first steps of the transfer of polarization from the nuclei coupled to the electrons to further away nuclei, demonstrating a decrease in the spin-diffusion barrier under MAS conditions.

Dynamic nuclear polarization using frequency modulation at 3.34 T

During dynamic nuclear polarization (DNP) experiments polarization is transferred from unpaired electrons to their neighboring nuclear spins, resulting in dramatic enhancement of the NMR signals. While in most cases this is achieved by continuous wave (cw) irradiation applied to samples in fixed external magnetic fields, here we show that DNP enhancement of static samples can improve by modulating the microwave (MW) frequency at a constant field of 3.34 T. The efficiency of triangular shaped modulation is explored by monitoring the (1)H signal enhancement in frozen solutions containing different TEMPOL radical concentrations at different temperatures. The optimal modulation parameters are examined experimentally and under the most favorable conditions a threefold enhancement is obtained with respect to constant frequency DNP in samples with low radical concentrations. The results are interpreted using numerical simulations on small spin systems. In particular, it is shown experimentally and explained theoretically that: (i) The optimal modulation frequency is higher than the electron spin-lattice relaxation rate. (ii) The optimal modulation amplitude must be smaller than the nuclear Larmor frequency and the EPR line-width, as expected. (iii) The MW frequencies corresponding to the enhancement maxima and minima are shifted away from one another when using frequency modulation, relative to the constant frequency experiments.