Andriy Marko

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.

Molecular orientation studies by pulsed electron-electron double resonance experiments

Pulsed electron-electron double resonance (PELDOR) has proven to be a valuable tool to measure the distribution of long range distances in noncrystalline macromolecules. These experiments commonly use nitroxide spin labels as paramagnetic markers that are covalently attached to the macromolecule at specific positions. Unless these spin labels are flexible in such a manner that they exhibit an almost random orientation, the PELDOR signals will-apart from the interspin distance-also depend on the orientation of the spin labels. This effect needs to be considered in the analysis of PELDOR signals and can, moreover, be used to obtain additional information on the structure of the molecule under investigation. In this work, we demonstrate that the PELDOR signal can be represented as a convolution of a kernel function containing the distance distribution function and an orientation intensity function. The following strategy is proposed to obtain both functions from the experimental data. In a first step, the distance distribution function is estimated by the Tikhonov regularization, using the average over all PELDOR time traces with different frequency offsets and neglecting angular correlations of the spin labels. Second, the convolution relation is employed to determine the orientation intensity function, using again the Tikhonov regularization. Adopting small nitroxide biradical molecules as simple examples, it is shown that the approach works well and is internally consistent. Furthermore, independent molecular dynamics simulations are performed and used to calculate PELDOR signals, distance distributions, and orientational intensity functions. The calculated and experimental results are found to be in excellent overall agreement.

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.

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.

Conformational dynamics of nucleic acid molecules studied by PELDOR spectroscopy with rigid spin labels

Nucleic acid molecules can adopt a variety of structures and exhibit a large degree of conformational flexibility to fulfill their various functions in cells. Here we describe the use of Pulsed Electron-Electron Double Resonance (PELDOR or DEER) to investigate nucleic acid molecules where two cytosine analogs have been incorporated as spin probes. Because these new types of spin labels are rigid and incorporated into double stranded DNA and RNA molecules, there is no additional flexibility of the spin label itself present. Therefore the magnetic dipole-dipole interaction between both spin labels encodes for the distance as well as for the mutual orientation between the spin labels. All of this information can be extracted by multi-frequency/multi-field PELDOR experiments, which gives very precise and valuable information about the structure and conformational flexibility of the nucleic acid molecules. We describe in detail our procedure to obtain the conformational ensembles and show the accuracy and limitations with test examples and application to double-stranded DNA.

Broadband spin echoes and broadband SIFTER in EPR

Applications of broadband pulses for EPR have been reported for FID, echo detection and inversion pulses recently. Here we present a broadband Hahn, stimulated and refocused echo sequence derived from adiabatic pulses. The formation of echoes is accomplished by using variable chirp rates and pulse lengths. In all three broadband echo experiments the complete spectral shape of a nitroxide (about 70 Gauss at X-band frequency) could be recovered by Fourier transformation of the quadrature detected echo signals. Such broadband echoes provide an exciting opportunity to optimize pulse sequences where a full excitation of the spectrum is mandatory for an optimum performance. We applied our pulses to the SIFTER (single frequency technique for refocusing dipolar couplings) experiment, a solid echo based pulse sequence to measure the dipolar coupling between two unpaired electron spins. By employing our broadband Hahn echo sequence on a nitroxide biradical we could achieve an artifact free dipolar evolution time trace in the SIFTER experiment with 95% modulation depth at X-band frequency and of 10% modulation depth at Q-band frequency.

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. Thus, not only the distance but additionally the three Euler angles between both the nitroxide axis systems and the two polar angles of the interconnecting vector with respect to the nitroxide axis systems can be extracted from a single pair of spin labels. To extract all these parameters independently and unambiguously, a set of multifrequency/multifield pulsed EPR experiments have to be performed. We will describe the experimental procedure as well as newly developed spin labels, which are helpful to disentangle all these parameters, and tools which we have developed to analyze such data sets. The procedures and analyses will be illustrated by examples from our laboratory.

Out-of-phase PELDOR

Pulsed electron–electron double resonance (PELDOR) is a method frequently used to determine the structure of bio-macromolecule on a nanometre scale. Usually PELDOR experiments are carried out in the high-temperature limit, when the Boltzmann population of spins oriented parallel and antiparallel to the external magnetic field are almost equal. Also the well-developed theories describing PELDOR apply to this case. However, the high-temperature conditions are no more fulfilled for experiments done in a high magnetic field (above 6 T) and at low temperatures (below 5 K), when the Zeeman interaction energy of an electron spin becomes comparable with thermal energy . In this work we demonstrate that PELDOR signals measured at these conditions differ from the usual PELDOR signals. Additional to the standard in-phase component the PELDOR signal at low temperature and high magnetic field also contains an out-of-phase component that disappears in the high-temperature limit. This means that we observe not only the modulation of the refocused transverse magnetisation along a single axis in the rotating coordinate system but rather its precession in the x-y plane with a dipolar frequency. Here, we provide a quantitative explanation as well as a detailed analysis of the spin magnetisation dynamics under such conditions based on density matrix formalism. Understanding the PELDOR phenomena in high field and at low temperatures offers a tool to separate intra from intermolecular interactions, which might be extremely helpful and important for applications to biomolecules with a high degree of conformational flexibility.

Separation of intra- and intermolecular contributions to the PELDOR signal

Pulsed Electron-electron Double Resonance (PELDOR) is commonly used to measure distances between native paramagnetic centers or spin labels attached to complex biological macromolecules. In PELDOR the energies of electron magnetic dipolar interactions are measured by analyzing the oscillation frequencies of the recorded time resolved signal. Since PELDOR is an ensemble method, the detected signal contains contributions from intramolecular, as well as intermolecular electron spin interactions. The intramolecular part of the signal contains the information about the structure of the studied molecules, thus it is very important to accurately separate intra- and intermolecular contributions to the total signal. This separation can become ambiguous, when the length of the PELDOR signal is not much longer than twice the oscillation period of the signal. In this work we suggest a modulation depth scaling method, which can use short PELDOR signals in order to extract the intermolecular contribution. Using synthetic data we demonstrate the advantages of the new approach and analyze its stability with regard to signal noise. The method was also successfully tested on experimental data of three systems measured at Q-Band frequencies, two model compounds in deuterated and protonated solvents and one biological sample, namely BetP. The application of the new method with an assigned value of the signal modulation depth enables us to determine the interspin distances in all cases. This is especially interesting for the model compound with an interspin distance of 5.2nm in the protonated solvent and the biological sample, since an accurate separation of the intra- and intermolecular PELDOR signal contributions would be difficult with the standard approach in those cases.