Pulsed Dipolar EPR for Self-Limited Complexes of Oligonucleotides Studies.

Pulsed electron-electron double resonance (PELDOR) spectroscopy is a powerful method for determining nucleic acid (NA) structure and conformational dynamics. PELDOR with molecular dynamics (MD) simulations opens up unique possibilities for defining the conformational ensembles of flexible, three-dimensional, self-assembled complexes of NA. Understanding the diversity and structure of these complexes is vital for uncovering matrix and regulative biological processes in the human body and artificially influencing them for therapeutic purposes.

Implementation and applications of shaped pulses in EPR.

In this review, we describe the application of shaped pulses for EPR spectroscopy. Pulses generated by fast arbitrary waveform generators are mostly used in the field of EPR spectroscopy for broadband (200 MHz-1 GHz) excitation of paramagnetic species. The implementation and optimization of such broadband pulses in existing EPR spectrometers, often designed and optimized for short rectangular microwave pulses, is demanding.

Spin-Labeled Riboswitch Synthesized from a Protected TPA Phosphoramidite Building Block.

The nitroxide TPA (2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene) is an excellent spin label for EPR studies of RNA. Previous synthetic methods, however, are complicated and require special equipment. Herein, we describe a uridine derived phosphoramidite with a photocaged TPA unit attached.

Long-range distance determination in fully deuterated RNA with pulsed EPR spectroscopy.

Pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy is powerful in structure and dynamics study of biological macromolecules by providing distance distribution information ranging from 1.8 to 6 nm, providing that the biomolecules are site-specifically labeled with paramagnetic tags. However, long distances up to 16 nm have been measured on perdeuterated and spin-labeled proteins in deuterated solvent by PELDOR.

Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results.

The power of trichlorosilylation: isolable trisilylated allyl anions, allyl radicals, and allenyl anions.

Treatment of hexachloropropene (ClC[double bond, length as m-dash]C(Cl)-CCl) with SiCl and [BuN]Cl (1 : 4 : 1) in CHCl results in a quantitative conversion to the trisilylated, dichlorinated allyl anion salt [BuN][ClC[double bond, length as m-dash]C(SiCl)-C(SiCl)] ([BuN][]). Tetrachloroallene ClC[double bond, length as m-dash]C[double bond, length as m-dash]CCl was identified as the first intermediate of the reaction cascade. In the solid state, [] adopts approximate symmetry with a dihedral angle between the planes running through the olefinic and carbanionic fragments of [] of C[double bond, length as m-dash]C-Si//Si-C-Si = 78.

Multiquantum Counting of Trityl Radicals.

We demonstrate a series of multitrityl radical compounds where accurate spin-counting by pulsed electron paramagnetic resonance (EPR) can be achieved at X-band (9 GHz) frequencies, even for molecules with very short and flexible linkers. Multiquantum filter experiments, well-known from NMR, were used to count the number of coupled electron spins in these compounds. The six pulse double quantum filter sequence used in EPR for distance determinations in biradicals was used.

Interpretation of spectroscopic data using molecular simulations for the secondary active transporter BetP.

Mechanistic understanding of dynamic membrane proteins such as transporters, receptors, and channels requires accurate depictions of conformational ensembles, and the manner in which they interchange as a function of environmental factors including substrates, lipids, and inhibitors. Spectroscopic techniques such as electron spin resonance (ESR) pulsed electron-electron double resonance (PELDOR), also known as double electron-electron resonance (DEER), provide a complement to atomistic structures obtained from x-ray crystallography or cryo-EM, since spectroscopic data reflect an ensemble and can be measured in more native solvents, unperturbed by a crystal lattice. However, attempts to interpret DEER data are frequently stymied by discrepancies with the structural data, which may arise due to differences in conditions, the dynamics of the protein, or the flexibility of the attached paramagnetic spin labels.

Exhaustively Trichlorosilylated C and C Building Blocks: Beyond the Müller-Rochow Direct Process.

The Cl-induced heterolysis of the Si-Si bond in SiCl generates an [SiCl] ion as reactive intermediate. When carried out in the presence of CCl or ClC═CCl (CHCl solutions, room temperature or below), the reaction furnishes the monocarbanion [C(SiCl)] ([A]; 92%) or the vicinal dianion [(ClSi)C-C(SiCl)] ([B]; 85%) in excellent yields. Starting from [B], the tetrasilylethane (ClSi)(H)C-C(H)(SiCl) (HB) and the tetrasilylethene (ClSi)C═C(SiCl) (B; 96%) are readily available through protonation (CFSOH) or oxidation (CuCl), respectively.

A semi-rigid isoindoline-derived nitroxide spin label for RNA.

A new isoindoline-derived benzimidazole nitroxide spin label, ImUm, was synthesized and incorporated into RNA oligoribonucleotides. ImUm is the first example of a conformationally unambiguous spin label for RNA, in which the nitroxide N-O bond lies on the same axis as the single bond used to attach the rigid isoindoline-based spin label to a uridine base. This results in minimal displacement of the nitroxide upon rotation of this single bond, which is a useful property for a label to be used for distance measurements.

Flexibilities of isoindoline-derived spin labels for nucleic acids by orientation selective PELDOR.

Pulsed electron electron double resonance experiments with rigid spin labels can reveal very detailed information about the structure and conformational flexibility of nucleic acid molecules. On the other hand, the analysis of such data is more involved the distance and orientation information encoded in the time domain data need to be extracted and separated. In this respect studies with different spin labels with variable internal mobility are interesting and can help to unambiguously interpret the EPR data.

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.

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.

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.

Pulsed electron-electron double resonance spectroscopy between a high-spin Mn(2+) ion and a nitroxide spin label.

Pulsed Electron-Electron Double Resonance (PELDOR) has attracted considerable attention for biomolecular applications, as it affords precise measurements of distances between pairs of spin labels in the range of 1.5-8 nm. Usually nitroxide moieties incorporated by site-directed spin labelling with cysteine residues are used as spin probes in protein systems.

Room temperature quantum coherence in a potential molecular qubit.

The successful development of a quantum computer would change the world, and current internet encryption methods would cease to function. However, no working quantum computer that even begins to rival conventional computers has been developed yet, which is due to the lack of suitable quantum bits. A key characteristic of a quantum bit is the coherence time.

Conformationally restricted isoindoline-derived spin labels in duplex DNA: distances and rotational flexibility by pulsed electron-electron double resonance spectroscopy.

Three structurally related isoindoline-derived spin labels that have different mobilities were incorporated into duplex DNA to systematically study the effect of motion on orientation-dependent pulsed electron-electron double resonance (PELDOR) measurements. To that end, a new nitroxide spin label, (ExIm)U, was synthesized and incorporated into DNA oligonucleotides. (ExIm)U is the first example of a conformationally unambiguous spin label for nucleic acids, in which the nitroxide N-O bond lies on the same axis as the three single bonds used to attach the otherwise rigid isoindoline-based spin label to a uridine base.

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 chemist's 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.

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.

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR.

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons.

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.

The RNA-DNA hybrid structure determined by EPR, CD and RNase H1.

A or B: RNA-DNA hybrids, key intermediates in gene regulation, were classified by pulsed electron-electron double resonance (PELDOR) in combination with CD spectroscopy into two classes, interpreted as A- and B-like structures. RNase H1 cleavage of these hybrids is in full agreement with these assignments, cleaving the hybrids with A-like geometry preferentially. This combined analytical approach allows the interpretation and eventually the design of more easily cleavable hybrids as needed for the antisense technology.

Coherence times and Rabi oscillations in CaWO₄:Cr(5+) crystal.

The coherence times of dopant pentavalent chromium ions in CaWO₄ single crystal (0.0006at.% Cr(5+)) were investigated both theoretically and experimentally.

Liquid state DNP using a 260 GHz high power gyrotron.

Dynamic nuclear polarization (DNP) at high magnetic fields (9.2 T, 400 MHz (1)H NMR frequency) requires high microwave power sources to achieve saturation of the EPR transitions. Here we describe the first high-field liquid-state DNP results using a high-power gyrotron microwave source (20 W at 260 GHz).

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.