Thomas F. Prisner

Long-range distance determinations in biomacromolecules by EPR spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy provides a variety of tools to study structures and structural changes of large biomolecules or complexes thereof. In order to unravel secondary structure elements, domain arrangements or complex formation, continuous wave and pulsed EPR methods capable of measuring the magnetic dipole coupling between two unpaired electrons can be used to obtain long-range distance constraints on the nanometer scale. Such methods yield reliably and precisely distances of up to 80 A, can be applied to biomolecules in aqueous buffer solutions or membranes, and are not size limited. They can be applied either at cryogenic or physiological temperatures and down to amounts of a few nanomoles. Spin centers may be metal ions, metal clusters, cofactor radicals, amino acid radicals, or spin labels. In this review, we discuss the advantages and limitations of the different EPR spectroscopic methods, briefly describe their theoretical background, and summarize important biological applications. The main focus of this article will be on pulsed EPR methods like pulsed electron-electron double resonance (PELDOR) and their applications to spin-labeled biosystems.

Dynamic Nuclear Polarization with a Rigid Biradical

A new polarizing agent with superior performance in dynamic nuclear polarization experiments is introduced, and utilizes two TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) moieties connected through a rigid spiro tether (see structure). The observed NMR signal intensities were enhanced by a factor of 1.4 compared to those of TOTAPOL, a previously described TEMPO-based biradical with a flexible tether.

High field dynamic nuclear polarization-the renaissance

Sensitivity is a critical issue in NMR spectroscopy, microscopy and imaging, and the factor that often limits the success of various applications. The origin of low sensitivity in NMR is well known to be due to the small magnetic moment of nuclear spins, which yields small Boltzmann polarizations and weak absorption signals. Historically, each advance in technology and methodology that has increased the signal-to-noise in NMR has shifted the boundary of what is achievable, often opening new areas of application and directions of research. The archetypal example of this phenomenon was the introduction of Fourier transform spectroscopy which led to increases of ∼ 102-fold in signal-to-noise, revolutionizing NMR and many other forms of spectroscopy.1 More recent technological developments of note include the continuing development of higher field superconducting magnets which increases polarization, and cryoprobes in which the excitation/detection coil is maintained at low temperatures increasing sensitivity through a higher probe Q and decreasing receiver noise.2 In addition, innovations in NMR methodology have improved sensitivity, classic examples being Hartmann–Hahn cross polarization,3,4 and J-coupling meditated5 transfer methods, and the introduction of 1H detection of 13C/15N resonances.6 Furthermore, techniques for non-inductive detection of resonance, such as the AFM-based technique of magnetic resonance force microscopy (MRFM), have recently allowed observation of a single electron spin,7 and ∼ 100 nuclear spins/√Hz.8

Relative Orientation of Rigid Nitroxides by PELDOR: Beyond Distance Measurements in Nucleic Acids

Show me your angle: Incorporation of the rigid spin label C allows determination of both distance and orientation of two nitroxide spin labels in DNA by PELDOR experiments at common X-band frequencies. The orientational information is obtained by varying the position of the detection pulses over the nitroxide spectrum. Simulation of the set of time traces yields very precise distances and angles.

Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances

In this protocol, we describe the facile synthesis of the nitroxide spin-label 2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene (TPA) and then its coupling to DNA/RNA through Sonogashira cross-coupling during automated solid-phase synthesis. Subsequently, we explain how to perform distance measurements between two such spin-labels on RNA/DNA using the pulsed electron paramagnetic resonance method pulsed electron double resonance (PELDOR). This combination of methods can be used to study global structure elements of oligonucleotides in frozen solution at RNA/DNA amounts of ∼10 nmol. We especially focus on the Sonogashira cross-coupling step, the advantages of the ACE chemistry together with the appropriate parameters for the RNA synthesizer and on the PELDOR data analysis. This procedure is applicable to RNA/DNA strands of up to ∼80 bases in length and PELDOR yields reliably spin–spin distances up to ∼6.5 nm. The synthesis of TPA takes ∼5 days and spin labeling together with purification ∼4 days. The PELDOR measurements usually take ∼16 h and data analysis from an hour up to several days depending on the extent of analysis.

Base-specific spin-labeling of RNA for structure determination

To facilitate the measurement of intramolecular distances in solvated RNA systems, a combination of spin-labeling, electron paramagnetic resonance (EPR), and molecular dynamics (MD) simulation is presented. The fairly rigid spin label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA) was base and site specifically introduced into RNA through a Sonogashira palladium catalyzed cross-coupling on column. For this purpose 5-iodo-uridine, 5-iodo-cytidine and 2-iodo-adenosine phosphoramidites were synthesized and incorporated into RNA-sequences. Application of the recently developed ACE® chemistry presented the main advantage to limit the reduction of the nitroxide to an amine during the oligonucleotide automated synthesis and thus to increase substantially the reliability of the synthesis and the yield of labeled oligonucleotides. 4-Pulse Electron Double Resonance (PELDOR) was then successfully used to measure the intramolecular spin–spin distances in six doubly labeled RNA-duplexes. Comparison of these results with our previous work on DNA showed that A- and B-Form can be differentiated. Using an all-atom force field with explicit solvent, MD simulations gave results in good agreement with the measured distances and indicated that the RNA A-Form was conserved despite a local destabilization effect of the nitroxide label. The applicability of the method to more complex biological systems is discussed.

Long‐Range Distance Measurements on Nucleic Acids in Cells by Pulsed EPR Spectroscopy

There-fore, it is important to investigate whether the in vitrodetermined NA structure reflects the intracellular (in vivo)conformation.BecauseofthehighsensitivityofEPRspectroscopy,itcanalso be applied to in vivo systems. In the past, nitroxide labelshave been used for in vivo EPR applications to determine theoxygen concentration, pH value, redox state, molecularmobility, and polarity of the local environment, and forspatial mapping of the free-radical metabolism.

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

Facing and Overcoming Sensitivity Challenges in Biomolecular NMR Spectroscopy

In the Spring of 2013, NMR spectroscopists convened at the Weizmann Institute in Israel to brainstorm on approaches to improve the sensitivity of NMR experiments, particularly when applied in biomolecular settings. This multi-author interdisciplinary Review presents a state-of-the-art description of the primary approaches that were considered. Topics discussed included the future of ultrahigh-field NMR systems, emerging NMR detection technologies, new approaches to nuclear hyperpolarization, and progress in sample preparation. All of these are orthogonal efforts, whose gains could multiply and thereby enhance the sensitivity of solid- and liquid-state experiments. While substantial advances have been made in all these areas, numerous challenges remain in the quest of endowing NMR spectroscopy with the sensitivity that has characterized forms of spectroscopies based on electrical or optical measurements. These challenges, and the ways by which scientists and engineers are striving to solve them, are also addressed.

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.