Vasyl Denysenkov

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

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.

High-field dynamic nuclear polarization in aqueous solutions.

Unexpected high DNP enhancements of more than 10 have been achieved in liquid water samples at room temperature and magnetic fields of 9.2 T (corresponding to 400 MHz (1)H NMR frequency and 260 GHz EPR frequency). The liquid samples were polarized in situ using a double-resonance structure, which allows simultaneous excitation of NMR and EPR transitions and achieves significant DNP enhancements at very low incident microwave power of only 45 mW. These results demonstrate the first important step toward the application of DNP to high-resolution NMR, increasing the sensitivity on biomolecules with small sample volumes and at physiologically low concentrations.

Conformational Flexibility of DNA

Pulsed Electron-Electron Double Resonance (PELDOR) on double-stranded DNA (ds-DNA) was used to investigate the conformational flexibility of helical DNA. Stretching, twisting, and bending flexibility of ds-DNA was determined by incorporation of two rigid nitroxide spin labels into a series of 20 base pair (bp) DNA duplexes. Orientation-selective PELDOR experiments performed at both X-band (9 GHz/0.3 T) and G-band (180 GHz/6.4 T) with spin label distances in the range of 2-4 nm allowed us to differentiate between different simple models of DNA dynamics existing in the literature. All of our experimental results are in full agreement with a dynamic model for ds-DNA molecules, where stretching of the molecule leads to a slightly reduced radius of the helix induced by a cooperative twist-stretch coupling.

High-frequency 180 GHz PELDOR

For aromatic organic radicals, pulsed electron-electron double resonance (PELDOR) experiments at high magnetic fields offer the possibility to achieve orientation-selective pumping and detection that could allow one not only to determine the distance between paramagnetic species but also their relative orientation with respect to the interconnecting dipolar axis. We present a PELDOR two-frequency setup that was introduced into our homebuilt 180 GHz pulsed electron paramagnetic resonance (EPR) spectrometer and we discuss its technical and experimental features. The capability of 180 GHz PELDOR has been tested using the three-pulse ELDOR sequence on the protein RNR-R2 (ribonucleotide reductase) fromEscherichia coli, which contains two tyrosyl radicals at a distance of 3.3 nm. At 180 GHz, orientation selectivity is observed and the modulation frequency was found in good agreement with theoretical predictions, which take into account the relative orientation of the radicals from X-ray data.

Host–Guest Complexes as Water-Soluble High-Performance DNP Polarizing Agents

Dynamic nuclear polarization (DNP) enhances the sensitivity of solid-state NMR (SSNMR) spectroscopy by orders of magnitude and, therefore, opens possibilities for novel applications from biology to materials science. This multitude of opportunities implicates a need for high-performance polarizing agents, which integrate specific physical and chemical features tailored for various applications. Here, we demonstrate that for the biradical bTbK in complex with captisol (CAP), a β-cyclodextrin derivative, host-guest assembling offers a new and easily accessible approach for the development of new polarizing agents. In contrast to bTbK, the CAP-bTbK complex is water-soluble and shows significantly improved DNP performance compared to the commonly used DNP agent TOTAPOL. Furthermore, NMR and EPR data reveal improved electron and nuclear spin relaxation properties for bTbK within the host molecule. The numerous possibilities to functionalize host molecules will permit designing novel radical complexes targeting diverse applications.

Liquid state DNP at high magnetic fields: Instrumentation, experimental results and atomistic modelling by molecular dynamics simulations

Dynamic nuclear polarization (DNP) at high magnetic fields has recently become one of the major research areas in magnetic resonance spectroscopy and imaging. Whereas much work has been devoted to experiments where the polarization transfer from the electron spin to the nuclear spin is performed in the solid state, only a few examples exist of experiments where the polarization transfer is performed in the liquid state. Here we describe such experiments at a magnetic field of 9.2 T, corresponding to a nuclear Larmor frequency of 400 MHz for proton spins and an excitation frequency of 263 GHz for the electron spins. The technical requirements to perform such experiments are discussed in the context of the double resonance structures that we have implemented. The experimental steps that allowed access to the enhancement factors for proton spins of several organic solvents with nitroxide radicals as polarizing agents are described. A computational scheme for calculating the coupling factors from molecular dynamics (MD) simulations is outlined and used to highlight the limitations of the classical models based on translational and rotational motion that are typically employed to quantify the observed coupling factors. The ability of MD simulations to predict enhancements for a variety of radicals and solvent molecules at any magnetic field strength should prove useful in optimizing experimental conditions for DNP in the liquid state.

Liquid state Dynamic Nuclear Polarization probe with Fabry–Perot resonator at 9.2 T

Recent achievements in liquid state DNP at high magnetic fields showing significant enhancements on aqueous solutions have initiated strong interest in possible applications of this method to biomolecular research. However, in situ DNP of biomolecules at ambient temperatures is a challenging task due to high microwave losses leading to excessive sample heating. To avoid such heating the sample volume has to be reduced strongly to keep it away from the electric component of the microwave field. A helical double resonance structure, used for the first demonstrations of the applicability of Overhauser DNP to aqueous solutions at high magnetic fields (9.2 T), restricted the sample size to a very small volume of 2 nl. Together with a poor spectral resolution this resulted in small overall signal amplitude, hampering observations of biomolecules. Here we present a new type of the double resonance structure for liquid-state DNP which consists of a Fabry-Perot resonator for the microwave excitation and a stripline resonator for the NMR detection. This new double resonance structure (260 GHz/400 MHz) offers a 30-fold increase in aqueous sample volume (80 nl) with respect to the helical probe and exhibits improved NMR sensitivity and linewidth.

Multifrequency electron paramagnetic resonance characterization of PpoA, a CYP450 fusion protein that catalyzes fatty acid dioxygenation.

PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans . It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = (5)/(2) high-spin (HS) and Fe(III) S = (1)/(2) low-spin (LS) hemes. (14)N ENDOR spectroscopy on the S = (5)/(2) signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = (5)/(2) heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T(1)-spin-lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.

Advanced EPR Methods for Studying Conformational Dynamics of Nucleic Acids.

Pulsed electron paramagnetic resonance (EPR) spectroscopy has become an important tool for structural characterization of biomolecules allowing measurement of the distances between two paramagnetic spin labels attached to a biomolecule in the 2-8 nm range. In this chapter, we will focus on applications of this approach to investigate tertiary structure elements as well as conformational dynamics of nucleic acid molecules. Both aspects take advantage of using specific spin labels that are rigidly attached to the nucleobases, as they allow obtaining not only the distance but also the relative orientation between both nitroxide moieties with high accuracy. Thus, not only the distance but additionally the three Euler angles between both the nitroxide axis systems and the two polar angles of the interconnecting vector with respect to the nitroxide axis systems can be extracted from a single pair of spin labels. To extract all these parameters independently and unambiguously, a set of multifrequency/multifield pulsed EPR experiments have to be performed. We will describe the experimental procedure as well as newly developed spin labels, which are helpful to disentangle all these parameters, and tools which we have developed to analyze such data sets. The procedures and analyses will be illustrated by examples from our laboratory.