Olav Schiemann

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.

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.

MtsslWizard: In Silico Spin-Labeling and Generation of Distance Distributions in PyMOL

MtsslWizard is a computer program, which operates as a plugin for the PyMOL molecular graphics system. MtsslWizard estimates distances between spin labels on proteins quickly with user-configurable options through a simple graphical interface. In default mode, the program searches for ensembles of possible MTSSL conformations that do not clash with a static model of the protein. Once conformations are assigned, distance distributions between two or more ensembles are calculated, displayed, and can be exported to other software. The program’s use is evaluated in a number of challenging test cases and its strengths and weaknesses evaluated. The benefits of the program are its accuracy and simplicity.

PELDOR Measurements on a Nitroxide-Labeled Cu(II) Porphyrin : Orientation Selection, Spin-Density Distribution, and Conformational Flexibility

Metal ions are functionally or structurally important centers in metalloproteins or RNAs, which makes them interesting targets for spectroscopic investigations. In combination with site-directed spin labeling, pulsed electron-electron double resonance (PELDOR or DEER) could be a well-suited method to characterize and localize them. Here, we report on the synthesis, full characterization, and PELDOR study of a copper(II) porphyrin/nitroxide model system. The X-band PELDOR time traces contain besides the distance information a convolution of orientational selectivity, conformational flexibility, exchange coupling, and spin density distribution, which can be deconvoluted by experiments with different frequency offsets and simulations. The simulations are based on the known experimental and spin Hamiltonian parameters and make use of a geometric model as employed for structurally similar bis-nitroxides and spin density parameters as obtained from density functional theory calculations. It is found that orientation selection with respect to dipolar angles is only weakly resolvable at X-band frequencies due to the large nitrogen hyperfine coupling of the copper porphyrin. On the other hand, the PELDOR time traces reveal a much faster oscillation damping than observed for structurally similar bis-nitroxides, which is mainly assigned to a small distribution in exchange couplings J. Taking the effects of orientation selectivity, distribution in J, and spin density distribution into account leads finally to a narrow distance distribution caused solely by the flexibility of the structure, which is in agreement with distributions from known bis-nitroxides of similar structure. Thus, X-band PELDOR measurements at different frequency offsets in combination with explicit time trace simulations allow for distinguishing between structural models and quantitative interpretation of copper-nitroxide PELDOR data gives access to localization of copper(II) ions.

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.

Conformational flexibility of nitroxide biradicals determined by X-band PELDOR experiments

PELDOR (pulsed electron–electron double resonance) experiments have been performed at X-band (9 GHz) frequencies on a linear and a bent nitroxide biradical. All PELDOR time traces were recorded with the pump frequency νB set at the center of the nitroxide spectra to achieve maximum pumping efficiency, while the probe frequency νA was stepped between a frequency offset ΔνAB = νA − νB of +40 to +80 MHz. The modulation frequencies and the damping of the oscillations change as a function ΔνAB, whereas the modulation depth λ for our investigated systems was only very slightly altered. This can be explained by the selection of different orientations of nitroxide radicals with respect to the external magnetic field as a function of frequency offset. Quantitative simulations of the PELDOR time traces could be achieved for both molecules and for all offset frequencies using a simple geometric model, described by a free rotation of the nitroxide radical around its acetylene bond and a single bending mode of the interconnecting molecular bridge. The results show that the distribution function for the relative orientations of the nitroxides with respect to each other and with respect to the dipolar vector R deviates from a random distribution and thus has to be taken into account to quantitatively simulate the PELDOR traces. Vice versa, a quantitative simulation of PELDOR time traces with variable offset frequencies allows the determination of the conformational freedom of such molecules.

Trityl radicals: spin labels for nanometer-distance measurements.

Spin labelling with trityls: to gather information about the structure and dynamics of trityl radicals, spin-labelled polymers were measured with pulsed electron-electron double resonance (PELDOR) and double-quantum coherence (DQC). This study demonstrates that trityl radicals have great potential as spin labels that eliminate some limitations of nitroxide spin labels.

Pulsed electron–electron double resonance: beyond nanometre distance measurements on biomacromolecules

PELDOR (or DEER; pulsed electron-electron double resonance) is an EPR (electron paramagnetic resonance) method that measures via the dipolar electron-electron coupling distances in the nanometre range, currently 1.5-8 nm, with high precision and reliability. Depending on the quality of the data, the error can be as small as 0.1 nm. Beyond mere mean distances, PELDOR yields distance distributions, which provide access to conformational distributions and dynamics. It can also be used to count the number of monomers in a complex and allows determination of the orientations of spin centres with respect to each other. If, in addition to the dipolar through-space coupling, a through-bond exchange coupling mechanism contributes to the overall coupling both mechanisms can be separated and quantified. Over the last 10 years PELDOR has emerged as a powerful new biophysical method without size restriction to the biomolecule to be studied, and has been applied to a large variety of nucleic acids as well as proteins and protein complexes in solution or within membranes. Small nitroxide spin labels, paramagnetic metal ions, amino acid radicals or intrinsic clusters and cofactor radicals have been used as spin centres.