Marina Bennati

Dynamic nuclear polarization at high magnetic fields in liquids.

MPI for Biophysical Chemistry Gottingen, Am Fassberg 11, 37077 Gottingen, Germany b Free University Berlin, Inst. of Experimental Physics, Arnimallee 14, 14195 Berlin, Germany Magnetic Resonance Center (CERM) and Department of Chemistry, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Italy Bruker Biospin GmbH, Silberstreifen 4, 76287 Rheinstetten, Germany e Technische Universitat Munchen, Department of Chemistry, Lichtenbergstr. 4, 85747 Garching, Germany Goethe University Frankfurt, Max von Laue Strasse 7, 60438 Frankfurt, Germany

Field dependent dynamic nuclear polarization with radicals in aqueous solution.

The dynamic nuclear polarization (DNP) effect between the polarizers TEMPOL and trityl and the 1H protons of water solution was investigated at two different magnetic fields corresponding to electron pumping frequencies of 9 and 94 GHz. For TEMPOL, large DNP enhancements up to about −100 (9 GHz) and −20 (94 GHz) were observed by continuous microwave irradiation on one of the three nitroxide hyperfine lines. These enhancements are considerably larger than those ever reported for this system and also larger than the ones we obtained with the second proposed polarizer agent, the trityl radical, under the same conditions. 1H Overhauser coupling factors were extracted from NMRD relaxation dispersion experiments and used to estimate the degree of effective saturation of the TEMPOL EPR line. Our results show that nitroxide radicals are well-suited polarizers for DNP experiments in aqueous solutions at variable fields. The large enhancements open up attractive perspectives for the application potential of DNP in ...

High-field pulsed electron–electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y·) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2′-deoxynucleoside diphosphates. It has been proposed that the function of the tyrosyl radical in R2 is to generate a transient thiyl radical (C439·) in R1 over a distance of 35 Å, which in turn initiates the reduction process. EPR distance measurements provide a tool with which to study the mechanism of radical initiation in class I RNRs. These types of experiments at low magnetic fields and frequencies (0.3 T, 9 GHz) give insight into interradical distances and populations. We present a pulsed electron–electron double resonance (PELDOR) experiment at high EPR frequency (180-GHz electron Larmor frequency) that detects the dipolar interaction between the Y·s in each protomer of RNR R2 from Escherichia coli. We observe a correlation between the orientation-dependent dipolar interaction and their resolved g-tensors. This information has allowed us to define the relative orientation of two radicals embedded in the active homodimeric protein in solution. This experiment demonstrates that high-field PELDOR spectroscopy is a powerful tool with which to study the assembly of proteins that contain multiple paramagnetic centers.

New developments in high field electron paramagnetic resonance with applications in structural biology

Recent developments in microwave technologies have led to a renaissance of electron paramagnetic resonance (EPR) due to the implementation of new spectrometers operating at frequencies ?90?GHz. EPR at high fields and high frequencies (HF-EPR) has been established up to THz (very high frequency (VHF) EPR) in continuous wave (cw) operation and up to about 300?GHz in pulsed operation. To date, its most prominent application field is structural biology. This review article first gives an overview of the theoretical basics and the technical aspects of HF-EPR methodologies, such as cw and pulsed HF-EPR, as well as electron nuclear double resonance at high fields (HF-ENDOR). In the second part, the article illustrates different application areas of HF-EPR in studies of protein structure and function. In particular, HF-EPR has delivered essential contributions to disentangling complex spectra of radical cofactors or reaction intermediates in photosynthetic reaction centres, radical enzymes (such as ribonucleotide reductase) and in metalloproteins. Furthermore, HF-EPR combined with site-directed spin labelling in membranes and soluble proteins provides new methods of investigating complex molecular dynamics and intermolecular distances.

Heterodisulfide reductase from methanogenic archaea : a new catalytic role for an iron-sulfur cluster

Abstract Heterodisulfide reductase (HDR) from methanogenic archaea is an iron-sulfur protein that catalyzes reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol-coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH). Via the characterization of a paramagnetic reaction intermediate generated upon oxidation of the enzyme in the presence of coenzyme M, the enzyme was shown to contain a [4Fe-4S] cluster in its active site that catalyzes reduction of the disulfide substrate in two one-electron reduction steps. The formal thiyl radical generated by the initial one-electron reduction of the disulfide is stabilized via reduction and coordination of the resultant thiol to the [4Fe-4S] cluster.

Optimization of dynamic nuclear polarization experiments in aqueous solution at 15 MHz/9.7 GHz: a comparative study with DNP at 140 MHz/94 GHz.

Dynamic nuclear polarization is emerging as a potential tool to increase the sensitivity of NMR aiming at the detection of macromolecules in liquid solution. One possibility for such an experimental design is to perform the polarization step between electrons and nuclei at low magnetic fields and then transfer the sample to a higher field for NMR detection. In this case, an independent optimization of the polarizer and detection set ups is required. In the present paper we describe the optimization of a polarizer set up at 15 MHz (1)H NMR/9.7 GHz EPR frequencies based on commercial hardware. The sample consists of the nitroxide radical TEMPONE-D,(15)N in water, for which the dimensions were systematically decreased to fit the homogeneous B(1) region of a dielectric ENDOR resonator. With an available B(1) microwave field up to 13 G we observe a maximum DNP enhancement of -170 at room temperature by irradiating on either one of the EPR lines. The DNP enhancement was saturated at all polarizer concentrations. Pulsed ELDOR experiments revealed that the saturation level of the two hyperfine lines is such that the DNP enhancements are well consistent with the coupling factors derived from NMRD data. By raising the polarizing field and frequencies 10-fold, i.e. to 140 MHz (1)H/94 GHz EPR, we reach an enhancement of -43 at microwave field strengths (B(1) approximately 5 G). The results are discussed in view of an application for a DNP spectrometer.

Probing secondary structures of spin-labeled RNA by pulsed EPR spectroscopy.

The ability of RNA to interconvert between multiple conformational states is essential for the diversity of biological functions that have been discovered in the recent past. For example, the correct operation of regulatory RNA elements, such as riboswitches, is based on the precise interplay of alternative RNA conformations. Studying the molecular mechanisms of RNA function entails probing RNA-folding intermediates on the energy landscape. EPR spectroscopy, in particular, has been increasingly applied to obtain structural information on nucleic acids, including local conformational changes in RNA and the identification of metal-ion binding sites. Pulsed EPR techniques (PELDOR/ DEER) have been used to determine distances between paramagnetic centers in specifically modified RNA. PELDOR should therefore be suitable for the detection of alternative RNA conformations that involve distinct changes in base-pairing patterns. The accessibility of spin-labeled RNA still poses the major challenge for the widespread applicability of powerful EPR techniques. Nitroxide radicals are the most commonly used type of paramagnetic labels for nucleic acids. Several methods have been reported for attaching nitroxide groups at internal positions at the ribose, the phosphate backbone, or at nucleobases, often by means of multiatom linkers that provide several unwanted degrees of rotational freedom. Rigid nitroxide spin labels conjugated to the nucleobase or to nucleobase analogues have been reported for DNA. Our RNA spin-labeling approach addresses the direct attachment of nitroxide labels onto RNA nucleobases, such that conformational changes can be directly detected by PELDOR (i.e., by the change in distance between two labeled nucleotides). The nucleobase spin labels used in this study are also designed to preserve the Watson–Crick base-pairing capability of labeled nucleotides and not to interfere with alternative base-pairing patterns in different RNA conformations. Here, we describe the installation of nitroxide spin labels on exocyclic amino groups of the RNA nucleobases guanine, adenine, and cytosine (Figure 1) with unprecedented effi-

1H and 13C dynamic nuclear polarization in aqueous solution with a two-field (0.35 T/14 T) shuttle DNP spectrometer.

Dynamic nuclear polarization (DNP) permits increasing the NMR signal of nuclei by pumping the electronic spin transitions of paramagnetic centers nearby. This method is emerging as a powerful tool to increase the inherent sensitivity of NMR in structural biology aiming at detection of macromolecules. In aqueous solution, additional technical issues associated with the penetration of microwaves in water and heating effects aggravate the performance of the experiment. To examine the feasibility of low-field (9.7 GHz/0.35 T) DNP in high resolution NMR, we have constructed the prototype of a two-field shuttle DNP spectrometer that polarizes nuclei at 9.7 GHz/0.35 T and detects the NMR spectrum at 14 T. We report our first (1)H and (13)C DNP enhancements with this spectrometer. Effective enhancements up to 15 were observed for small molecules at (1)H 600 MHz/14 T as compared to the Boltzmann signal. The results provide a proof of principle for the feasibility of a shuttle DNP experiment and open up perspectives for the application potential of this method in solution NMR.

Saturation factor of nitroxide radicals in liquid DNP by pulsed ELDOR experiments.

We propose the use of the pulse electron double resonance (ELDOR) method to determine the effective saturation factor of nitroxide radicals for dynamic nuclear polarization (DNP) experiments in liquids. The obtained values for the nitroxide radical TEMPONE-D,(15)N at different concentrations are rationalized in terms of spin relaxation and are shown to fulfil the Overhauser theory.

W-band orientation selective DEER measurements on a Gd3+/nitroxide mixed-labeled protein dimer with a dual mode cavity.

Double electron-electron resonance (DEER) at W-band (95 GHz) was applied to measure the distance between a pair of nitroxide and Gd(3+) chelate spin labels, about 6 nm apart, in a homodimer of the protein ERp29. While high-field DEER measurements on systems with such mixed labels can be highly attractive in terms of sensitivity and the potential to access long distances, a major difficulty arises from the large frequency spacing (about 700 MHz) between the narrow, intense signal of the Gd(3+) central transition and the nitroxide signal. This is particularly problematic when using standard single-mode cavities. Here we show that a novel dual-mode cavity that matches this large frequency separation dramatically increases the sensitivity of DEER measurements, allowing evolution times as long as 12 μs in a protein. This opens the possibility of accessing distances of 8 nm and longer. In addition, orientation selection can be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd(3+)-nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide spin labels.