Bela E. Bode

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.

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 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.

Optimization of Transversal Relaxation of Nitroxides for Pulsed Electron—Electron Double Resonance Spectroscopy in Phospholipid Membranes

Pulsed electron-electron double resonance (PELDOR) spectroscopy is increasingly applied to spin-labeled membrane proteins. However, after reconstitution into liposomes, spin labels often exhibit a much faster transversal relaxation (T(m)) than in detergent micelles, thus limiting application of the method in lipid bilayers. In this study, the main reasons for enhanced transversal relaxation in phospholipid membranes were investigated systematically by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin clustering and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples of low local concentrations does deuteration of acyl chains and buffer significantly prolong T(m). In these cases, values of up to 7 μs have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect T(m), either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (<50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions.

PELDOR in rotationally symmetric homo-oligomers

Nanometre distance measurements by pulsed electron−electron double resonance (PELDOR) spectroscopy have become an increasingly important tool in structural biology. The theoretical underpinning of the experiment is well defined for systems containing two nitroxide spin-labels (spin pairs); however, recently experiments have been reported on homo-oligomeric membrane proteins consisting of up to eight spin-labelled monomers. We have explored the theory behind these systems by examining model systems based on multiple spins arranged in rotationally symmetric polygons. The results demonstrate that with a rising number of spins within the test molecule, increasingly strong distortions appear in distance distributions obtained from an analysis based on the simple spin pair approach. These distortions are significant over a range of system sizes and remain so even when random errors are introduced into the symmetry of the model. We present an alternative approach to the extraction of distances on such systems based on a minimisation that properly treats multi-spin correlations. We demonstrate the utility of this approach on a spin-labelled mutant of the heptameric Mechanosensitive Channel of Small Conductance of E. coli.

Electron spin density distribution in the special pair triplet of Rhodobacter sphaeroides R26 revealed by magnetic field dependence of the solid-state photo-CIDNP effect.

Photo-CIDNP (photochemically induced dynamic nuclear polarization) can be observed in frozen and quinone-blocked photosynthetic reaction centers (RCs) as modification of magic-angle spinning (MAS) NMR signal intensity under illumination. Studying the carotenoidless mutant strain R26 of Rhodobacter sphaeroides, we demonstrate by experiment and theory that contributions to the nuclear spin polarization from the three-spin mixing and differential decay mechanism can be separated from polarization generated by the radical pair mechanism, which is partially maintained due to differential relaxation (DR) in the singlet and triplet branch. At a magnetic field of 1.4 T, the latter contribution leads to dramatic signal enhancement of about 80,000 and dominates over the two other mechanisms. The DR mechanism encodes information on the spin density distribution in the donor triplet state. Relative peak intensities in the photo-CIDNP spectra provide a critical test for triplet spin densities computed for different model chemistries and conformations. The unpaired electrons are distributed almost evenly over the two moieties of the special pair of bacteriochlorophylls, with only slight excess in the L branch.

Pulsed electron–electron double resonance (PELDOR) distance measurements in detergent micelles

Pulsed electron-electron double resonance (PELDOR) spectroscopy is a powerful tool for measuring nanometer distances in spin-labeled systems. A common approach is doubly covalent spin-labeling of a macromolecule and measurement of the inter-spin distance, or to use singly-labeled components of a system that forms aggregates or oligomers. This situation has been described as a spin-cluster. The PELDOR signal, however, does not only contain the desired dipolar coupling between the spin-labels of the molecule or cluster under study. In samples of finite concentration the dipolar coupling between the spin-labels of the randomly distributed molecules or spin-clusters also contributes significantly. In homogeneous frozen solutions or lipid vesicle membranes this second contribution can be considered to be an exponential or stretched exponential decay, respectively. In this study, we show that this assumption is not valid in detergent micelles. Spin-labeled fatty acids that are randomly partitioned into different detergent micelles give rise to PELDOR time traces which clearly deviate from stretched exponential decays. The obtained signals can be modeled quantitatively based on the size of the micelles, their aggregation number, the spin-label concentration and the degree of spin-labeling. As a main conclusion a PELDOR signal deviating from a stretched exponential decay does not necessarily prove the observation of specific distance information on the molecule or cluster. These results are important for the interpretation of PELDOR experiments on membrane proteins or lipophilic peptides solubilized in detergent micelles or small vesicles, which often do not show pronounced dipolar oscillations in their time traces.

Accurate extraction of nanometer distances in multimers by pulse EPR

Abstract Pulse electron paramagnetic resonance (EPR) is gaining increasing importance in structural biology. The PELDOR (pulsed electron–electron double resonance) method allows extracting distance information on the nanometer scale. Here, we demonstrate the efficient extraction of distances from multimeric systems such as membrane‐embedded ion channels where data analysis is commonly hindered by multi‐spin effects.

Theory of solid-state photo-CIDNP in the earth's magnetic field.

To date, solid-state photo-CIDNP experiments have been performed only using magic angle spinning NMR in a high-field regime, which is not associated with physiologically relevant spin dynamics. Here, we predict that nuclear spin polarization up to 10%, almost 9 orders of magnitude larger than thermal equilibrium polarization, can arise in cyclic photoreactions at the earth field due to a coherent three-spin mixing mechanism in the S-T(-) or S-T(+) manifold. The effect is maximal at a distance of about 30 Å between the two radicals, which nearly coincides with the separation between the donor and secondary acceptor in natural photosynthetic reaction centers. Analytical expressions are given for a simple limiting case. Numerical computations for photosynthetic reaction centers show that many nuclei in the chromophores and their vicinity are likely to become polarized. The theory predicts that only modest hyperfine couplings of a few hundred kilohertz are required to generate polarization of more than 1% for radical-radical distances between 20 and 50 Å, that is, for a large number of radical pairs in electron-transfer proteins.

Binding dynamics of a monomeric SSB protein to DNA: a single-molecule multi-process approach

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single-monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between high-density binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.