Burkhard Endeward

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.

Pulsed Electron-Electron Double Resonance Determination of Spin Label Distances and Orientations on the Tetrameric Potassium Ion Channel KcsA

Pulsed electron-electron double-resonance (PELDOR) measurements are presented from the potassium ion channel KcsA both solubilized in detergent and reconstituted in lipids. Site-directed spin-labeling using (1-oxyl-2,2,5,5-tetramethyl-3-pyrrolin-3-yl)methyl methanethiosulfonate was performed with a R64C mutant of the protein. The orientations of the spin-labels in the tetramer were determined by PELDOR experiments performed at two magnetic field strengths (0.3 T/X-band and 1.2 T/Q-band) and variable probe frequency. Quantitative simulation of the PELDOR data supports a strongly restricted nitroxide, oriented at an angle of 65 degrees relative to the central channel axis. In general, poorer quality PELDOR data were obtained from membrane-reconstituted preparations compared to soluble proteins or detergent-solubilized samples. One reason for this is the reduced transverse spin relaxation time T(2) of nitroxides due to crowding of tetramers within the membrane that occurs even at low protein to lipid ratios. This reduced T(2) can be overcome by reconstituting mixtures of unlabeled and labeled proteins, yielding high-quality PELDOR data. Identical PELDOR oscillation frequencies and their dependencies on the probe frequency were observed in the detergent and membrane-reconstituted preparations, indicating that the position and orientation of the spin-labels are the same in both environments.

Shaped optimal control pulses for increased excitation bandwidth in EPR

A 1 ns resolution pulse shaping unit has been developed for pulsed EPR spectroscopy to enable 14-bit amplitude and phase modulation. Shaped broadband excitation pulses designed using optimal control theory (OCT) have been tested with this device at X-band frequency (9 GHz). FT-EPR experiments on organic radicals in solution have been performed with the new pulses, designed for uniform excitation over a significantly increased bandwidth compared to a classical rectangular π/2 pulse of the same B(1) amplitude. The concept of a dead-time compensated prefocused pulse has been introduced to EPR with a self-refocusing of 200 ns after the end of the pulse. Echo-like refocused signals have been recorded and compared to the performance of a classical Hahn-echo sequence. The impulse response function of the microwave setup has been measured and incorporated into the algorithm for designing OCT pulses, resulting in further significant improvements in performance. Experimental limitations and potential new applications of OCT pulses in EPR spectroscopy will be discussed.

Confirmed by X‐ray Crystallography: The B⋅B One‐Electron σ Bond

Is one electron sufficient to bring about significant σ bonding between two atoms? The chemists view on the chemical bond is usually tied to the concept of shared electron pairs, and not too much experimental evidence exists to challenge this firm belief. Whilst species with the unusual one-electron σ-bonding motif between homonuclear atoms have so far been identified mainly by spectroscopic evidence, we present herein the first crystallographic characterization, augmented by a detailed quantum-chemical validation, for a radical anion featuring a B⋅B one-electron-two-center σ bond.

PELDOR spectroscopy reveals preorganization of the neomycin-responsive riboswitch tertiary structure.

Pulsed electron double resonance (PELDOR) spectroscopy reveals a prearranged tertiary structure of the 27 nucleotides long engineered neomycin-responsive riboswitch. Measured distances between spin labels at positions U4-U14, U4-U15, U14-U26, and U15-U26 were unchanged upon neomycin binding which implies that the global stem-loop architecture is preserved in the absence and presence of the ligand. On the basis of our results, we infer that low-temperature PELDOR data unambiguously demonstrate the existence of an enthalpically favorable set of RNA conformations ready to bind the ligand without major global rearrangement.

A preorganized ditopic borane as highly efficient one- or two-electron trap.

Reduction of the bis(9-borafluorenyl)methane 1 with excess lithium furnishes the red dianion salt Li2[1]. The corresponding dark green monoanion radical Li[1] is accessible through the comproportionation reaction between 1 and Li2[1]. EPR spectroscopy on Li[1] reveals hyperfine coupling of the unpaired electron to two magnetically equivalent boron nuclei (a((11)B) = 5.1 ± 0.1 G, a((10)B) = 1.7 ± 0.2 G). Further coupling is observed to the unique B-CH-B bridgehead proton (a((1)H) = 7.2 ± 0.2 G) and to eight aromatic protons (a((1)H) = 1.4 ± 0.1 G). According to X-ray crystallography, the B···B distances continuously decrease along the sequence 1 → [1](•-) → [1](2-) with values of 2.534(2), 2.166(4), and 1.906(3) Å, respectively. Protonation of Li2[1] leads to the cyclic borohydride species Li[1H] featuring a B-H-B two-electron-three-center bond. This result strongly indicates a nucleophilic character of the boron atoms; the reaction can also be viewed as rare example of the protonation of an element-element σ bond. According to NMR spectroscopy, EPR spectroscopy, and quantum-chemical calculations, [1](2-) represents a closed-shell singlet without any spin contamination. Detailed wave function analyses of [1](•-) and [1](2-) reveal strongly localized interactions of the two boron pz-type orbitals, with small delocalized contributions of the 9-borafluorenyl π systems. Overall, our results provide evidence for a direct B-B one-electron and two-electron bonding interaction in [1](•-) and [1](2-), respectively.

Structure and dynamics of nucleic acids.

In this chapter we describe the application of CW and pulsed EPR methods for the investigation of structural and dynamical properties of RNA and DNA molecules and their interaction with small molecules and proteins. Special emphasis will be given to recent applications of dipolar spectroscopy on nucleic acids.

Carr–Purcell Pulsed Electron Double Resonance with Shaped Inversion Pulses

Pulsed electron paramagnetic resonance (EPR) spectroscopy allows the determination of distances, in the range of 1.5-8 nm, between two spin-labels attached to macromolecules containing protons. Unfortunately, for hydrophobic lipid-bound or detergent-solubilized membrane proteins, the maximum distance accessible is much lower, because of a strongly reduced coherence time of the electron spins. Here we introduce a pulse sequence, based on a Carr-Purcell decoupling scheme on the observer spin, where each π-pulse is accompanied by a shaped sech/tanh inversion pulse applied to the second spin, to overcome this limitation. This pump/probe excitation scheme efficiently recouples the dipolar interaction, allowing a substantially longer observation time window to be achieved. This increases the upper limit and accuracy of distances that can be determined in membrane protein complexes. We validated the method on a bis-nitroxide model compound and applied this technique to the trimeric betaine transporter BetP. Interprotomer distances as long as 6 nm could be reliably determined, which is impossible with the existing methods.

Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (QD) located in the vicinity of the distal heme bD. Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. 14N and 15N ESEEM and HYSCORE measurements carried out at X-band (∼9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with 15N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant Aiso(14N) ∼0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using 14N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, ∼3.4 GHz), the 14N nuclear quadrupolar parameters of the interacting nitrogen nucleus (κ = 0.49, η = 0.50) were determined and correspond to those of a histidine Nδ, assigned to the heme bD ligand His-66 residue. Moreover S-band 15N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T(15N) = 0.16 MHz). A distance of ∼2.2 Åbetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the QD site of NarGHI.

Hydrogen Bonds between Nitrogen Donors and the Semiquinone in the Qi-site of the bc1 Complex

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (∼9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nϵ of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Nδ in other quinone-processing sites. The experiments with 15N showed that the Nϵ of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nϵ of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38 ± 0.13Å. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.