Laser-Induced Magnetic Dipole Spectroscopy - ACS Publications

Letter pubs.acs.org/JPCL

Laser-Induced Magnetic Dipole Spectroscopy Christian Hintze,†,¶ Dennis Bücker,†,¶ Silvia Domingo Köhler,† Gunnar Jeschke,‡ and Malte Drescher*,† †

Department of Chemistry, University of Konstanz, 78464 Konstanz, Germany Laboratory of Physical Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zürich, Switzerland

‡

S Supporting Information *

ABSTRACT: Pulse electron paramagnetic resonance measurements of nanometer scale distance distributions have proven highly effective in structural studies. They exploit the magnetic dipole−dipole coupling between spin labels site-specifically attached to macromolecules. The most commonly applied technique is double electron−electron resonance (DEER, also called pulsed electron double resonance (PELDOR)). Here we present the new technique of laser-induced magnetic dipole (LaserIMD) spectroscopy based on optical switching of the dipole−dipole coupling. In a proof of concept experiment on a model peptide, we find, already at a low quantum yield of triplet excitation, the same sensitivity for measuring the distance between a porphyrin and a nitroxide label as in a DEER measurement between two nitroxide labels. On the heme protein cytochrome C, we demonstrate that LaserIMD allows for distance measurements between a heme prosthetic group and a nitroxide label, although the heme triplet state is not directly observable by an electron spin echo.

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n the past 15 years, pulsed EPR distance measurements have become an important tool to obtain structural information on the nanometer scale about macromolecules, may it be in biological1−4 or synthetic systems.5−10 Especially the pulsed EPR techniques double quantum coherence (DQC)11,12 and double electron electron resonance (DEER, also called pulsed electron double resonance, PELDOR)4 have become widely used tools for this purpose.13 To obtain distance information, pairs of paramagnetic spin labels are attached to the nanostructure of interest via site-directed spin labeling.14−19 Mainly nitroxides14,18,20 but also other types of persistent paramagnetic molecules21−28 have been used as spin labels. Especially the recent finding of the porphyrin triplet state being a potential spin label for DEER distance measurements29 is very important for this work. DEER/PELDOR and DQC determine the electron−electron dipolar interaction between the spin labels by separating it from other electron spin interactions. From this dipolar interaction, distance distributions in the range of approximately 1.5−8 nm or even up to 12 nm30 can be evaluated and transferred into information about the nanostructure of interest. Recent developments comprise new experimental techniques,31 using higher fields and frequencies,32−34 broad-band excitation by arbitrary waveforms,35,36 or combinations thereof.37−41 They strive to broaden the field of applications of pulsed EPR dipolar spectroscopy, increase the sensitivity of the method, and overcome present distance limitations. In this regard, we set out to introduce and implement a new technique in pulsed EPR dipolar spectroscopy maintaining compatibility to all aforementioned developments, laser-induced magnetic dipole (LaserIMD) spectroscopy. The corresponding pulse sequence is shown in Figure 1 and compared to four-pulse DEER. In DEER, two spin labels with, © 2016 American Chemical Society

in most cases, SD = 2 are addressed by two microwave frequencies (pump and observer frequencies). A pump pulse is traversed over a primary echo, generated by a Hahn echo sequence. The intensity of the refocused echo is detected as a function of the position t in time of the pump pulse. For LaserIMD, a conventional spin label with (but not limited to) 1 spin SD = 2 is used as the observed species, whereas a chromophore replaces the pumped spin label species. LaserIMD utilizes switching of the dipolar coupling by optical excitation of the chromophore into its triplet state with spin ST = 1 instead of pumping a spin species by addressing it with a second microwave frequency. Accordingly, the pump pulse is replaced by a laser flash. Because it does not interfere with microwave pulses, it can be applied simultaneously with the microwave pulses and is traversed over the complete echo sequence. Thus, a simple two-pulse Hahn echo sequence, akin to three-pulse DEER,42 is sufficient to obtain dead time free data. To derive an expression for the LaserIMD signal of a spin 1 pair consisting of a persistent observer spin SD = 2 and a transiently generated triplet spin ST = 1, we first consider the case where the laser pulse occurs after the π/2 pulse that generates SD spin coherence but before the π pulse that refocuses this coherence. Before the laser flash at time t′− (Figure 1), the photoexcitable label is in an EPR-silent singlet state and does not significantly influence the spin Hamiltonian or relaxation of the observer spin SD, which acquires phase ΩS,D Received: April 11, 2016 Accepted: May 10, 2016 Published: May 10, 2016 2204

DOI: 10.1021/acs.jpclett.6b00765 J. Phys. Chem. Lett. 2016, 7, 2204−2209

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The Journal of Physical Chemistry Letters

Figure 1. (A) Four pulse DEER pulse sequence. (B) LaserIMD pulse sequence. Figure 2. LaserIMD experiment on a 0.1 mM solution of TPP-Ala(Aib-Ala)4-TOAC-(Aib-Ala)2-OH in methanol-d4 with 2 vol % D2O performed in the Q band at 10 K. Light excitation is carried out at 351 nm with pulse energies of 3 mJ. (A) Raw data of the time domain signal with the background contribution determined experimentally. The top and bottom traces correspond to the real and imaginary parts of the quadrature signal, respectively. (B) Comparison of both branches (blue and green) of the LaserIMD raw data. (C) Corresponding form factor after experimental background correction with a model free fit obtained by Tikhonov regularization. (D) Fourier-transformed dipolar coupling spectra with fit. (E) Corresponding distance distribution with a mean distance of ⟨r⟩± = 2.12 nm and a width of s±(r) = 0.06 nm.

t′−. At time t′−, the triplet is generated with triplet quantum yield ΦT. Assuming that the electron Zeeman interaction of the triplet spin is much larger than its zero-field splitting, the magnetic quantum number of the triplet spin is a good quantum number and assumes the values mS,T = −1, 0, +1 with probabilities p−1, p0, and p+1, respectively. Fractions ΦT pmS,T of all spin pairs gain additional phase (ΩS,D + mS,Tωdd)(τ − t′−) before the refocusing pulse, where ωdd is the coupling between the two spins. The refocusing pulse inverts phase, resulting in total acquired phase −ΩS,Dτ + mS,Tωdd(t′− − τ). Between the refocusing pulse and echo refocusing, the respective spin pairs gain phase (ΩS,D + mS,Tωdd)τ, resulting in a phase offset at echo formation of mS,Tωddt′−. This phase offset is manifest in a cosine modulation of the echo signal with respect to time t′−. Because the cosine is an even function, pairs with mS,T = −1 and mS,T = +1 contribute the same modulation factor cos(ωddt′−), whereas the pairs with mS,T = 0 do not contribute to dipolar modulation. If the signal is detected in quadrature, the contributions to the imaginary part have the form ±p±1 sin(ωddt′−). In cases where p−1 and p+1 differ significantly from each other, a significant out-of-phase component of the dipolar evolution function would thus be observed, akin to the situation in DEER when the high-temperature approximation does not apply.43 In the following, we neglect this component as it is insignificant in our experimental data (see below). Thus, the signal contribution of an isolated spin pair can be written as V (t ′− ) = 1 − λ + λ cos(ωddt ′− )

to the external magnetic field as well as on the populations px, py, and pz of the zero-field triplet sublevels after intersystem crossing. The relative populations can be obtained by transient EPR.44 Equation 1 has the same form as the expression for dipolar modulation of an isolated spin pair in the DEER experiment. Hence, all further considerations on the DEER signal4 and all established procedures for data processing apply to LaserIMD as well. For the case where the laser pulse occurs after the refocusing microwave pulse, at a time t′+ before echo formation, the observer spins have acquired phase ΩS,D[−τ + (τ − t′+)] at the instant of the laser pulse. Between the laser pulse and echo refocusing, they gain phase (ΩS,D + mS,Tωdd)t′+. As a result, the echo phase is ωddt′+, and the signal is given by

(1)

where the modulation depth λ is given by λ = ΦT(p−1 + p+1 )

V (t ′+ ) = 1 − λ + λ cos(ωddt ′+ ) (2)

(3)

These expressions for the signal are valid if both longitudinal relaxation between triplet sublevels and triplet decay to the singlet ground state are slow on the time scale of the

The populations p−1 and p+1 of the triplet sublevels at high field depend on the orientation of the molecular frame with respect 2205


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Figure 3. Cytochrome C iso-1 (PDB 1YCC) singly labeled with MTSSL near the C-terminus on cysteine, residue number 102. The prosthetic heme group is used as the triplet probe.

experiment. Otherwise, the dipolar modulation would be damped. LaserIMD has some potential advantages compared to DEER: • In contrast to bandwidth-limited microwave pump pulses, the laser flash is capable of exciting chromophores with all possible orientations with respect to the external magnetic field, corresponding to excitation of the whole triplet EPR spectrum, thus avoiding orientation selection for the pumped spin and potentially increasing the modulation depth. • Other than DEER, LaserIMD does not require a second microwave excitation with frequency offset. Therefore, the quality factor Q of the resonator can be increased; therefore, more incident microwave power enables shorter pulse lengths, and detection sensitivity is increased. • The lower limit of distances accessible by DEER (approximately 1.5 nm) is determined by the effect of limited excitation bandwidth.45,46 For LaserIMD with its virtually infinite pump excitation bandwidth, the lowest detectable distance is given solely by the observer pulse lengths,47 allowing the measurement of shorter distances. • In LaserIMD, the observation can be performed at the maximum of the spectrum without compromising modulation depth, which increases sensitivity compared to DEER. • The laser flash in LaserIMD does not interfere with microwave pulses; thus, observer echo refocusing is not required for avoiding dead time. • No nuclear modulation effects, which are present in DEER/PELDOR48 as well as DQC,49 exist in LaserIMD. These advantages potentially lead to significantly shorter measurement times or can be used to extend the distance ranges accessible with dipolar spectroscopy in EPR. In this work, we show first LaserIMD experiments and estimate how much sensitivity could be gained by optimization of triplet excitation. We also show that this experiment can utilize

Figure 4. LaserIMD distance measurements on a 0.1 mM solution of singly nitroxide labeled CytC in methanol-d4 performed in the Q band at 10 K. Light excitation is carried out at 351 nm with pulse energies of 3 mJ. (A) Raw data of the time domain signal with three-dimensional homogeneous background contribution. (B) Corresponding form factor after background correction with the fit obtained by Tikhonov regularization. (C) Corresponding distance distribution with a mean distance of ⟨r⟩ = 1.96 nm and a width of s(r) = 0.35 nm.

endogenous prosthetic groups like the heme, without modifying them chemically. For a proof of concept experiment, we use the peptide Ala(Aib-Ala)4-TOAC-(Aib-Ala)2-OH labeled with 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (TPP) at the N-terminus (cf. Figure S1 for the chemical structure; predicted mean interspin distance ⟨r⟩ = 2.2 nm29), which is a suitable model system for measuring nitroxide−porphyrin distances.29 The porphyrin is excited into its triplet state with laser pulses with 3 mJ of energy in the UV (351 nm wavelength) coupled into the resonator via a quartz glass fiber. In Figure 2A, the time domain signal obtained with LaserIMD in the Q band is shown as raw data. The dipolar modulation stemming from the dipolar interaction between the triplet and nitroxide spin is readily observed. Furthermore, 2206


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of ⟨r⟩± = 2.12 nm and width of the distribution s±(r) = 0.06 nm. A similar result is obtained with triplet-DEER as well (cf. Figure S4 and the SI of the paper that introduced tripletDEER29). The spin density distribution over the triplet-state molecule can potentially complicate interpretation of the mean distance and distance distribution. For the analogous case of trityl radical labels, it has been shown, however, that good agreement between theory and experiment is obtained when the spin density distribution is taken into account.52 Comparing LaserIMD to a DEER measurement on the Ala(Aib-Ala)4-TOAC-(Aib-Ala)2-OH labeled with 1-oxyl-2,2,5,5tetramethylpyrroline-3-carboxylic acid (TEMPYO) instead of TPP, the modulation-to-noise ratio (MNR) of both measurements performed on the same spectrometer under identical conditions (temperature, SRT, and concentration) is comMNR parable at the current state ( ρ = MNRLaserIMD = 1.0; cf. Figure

intensities before and after the dipolar evolution are different, which can be attributed to a reduced phase memory time Tm of the nitroxide spins in the presence of the TPP triplet (laser flash before the Hahn echo pulse sequence) compared to the absence of the triplet (laser flash after the Hahn echo sequence; cf. Figure S2 for an equivalent control experiment with anthracene triplets). During the dipolar evolution time, the influence of the reduced phase memory time Tm decreases, leading to an additional contribution to the background factor obtained by a separate measurement on a homogeneous solution of Ala-(Aib-Ala)4-TOAC-(Aib-Ala)2-OH and TPP. The logarithm of such experimental background functions can be fitted by a low-order polynomial function,50 in our case by a polynomial function of degree 5, which was then used to correct the time domain signal. Relying on a homogeneous three-dimensional background instead yields identical results. A low-amplitude out-of-phase component of the signal (bottom traces in 2A) is detected. However, we cannot safely conclude that this component stems from a difference between p−1 and p+1 and refrain from any further interpretation. The symmetry between both branches of the time domain signal at around t′±,max can be used to determine the correct zero time similar to the way it is done with DEER data, where the symmetry is with respect to the unobserved primary echo at time 2τ1 (see section 4 of the SI for details). If the raw data is processed this way, two nearly identical time traces are obtained (Figure 2B). Both time domain signals can be corrected by their background contribution, as discussed before, which yields pure dipolar modulations (Figure 2C). We investigated the influence of the shot repetition rate (SRT) on the experiment. For different SRTs from 4 to 1000 ms, the temperature (10−50 K) was chosen to not suppress the nitroxide signal due to its longitudinal relaxation time T1. At these SRTs, the modulation depth is maintained, excluding triplet-state saturation. Therefore, the triplet lifetime has to be considerably shorter than 4 ms. For the proof of concept experiment shown in Figure 2, a high SRT of 400 ms was chosen. Varying the concentration of the sample, we did not observe effects of insufficient triplet excitation at concentrations typical for distance measurements (200 μM or lower). Modulation depths of λLaserIMD ≈ 9% are obtained. Because insufficient triplet excitation due to a too fast repetition of the experiment or due to a too high concentration was excluded, we conclude that the modulation depth is limited due to the intrinsic triplet quantum yield of the chromophore. The intrinsic quantum yield is the ratio between the number of chromophores that end up in the triplet state and the number of chromophores that absorbed a photon. Assuming an optical spin polarization featuring approximately one-third of triplets residing in the mS = 0 state (not contributing to the modulation depth in LaserIMD), our experiments suggest a triplet quantum yield of ΦT ≈ 0.13. This low ΦT is due to the excitation of the second excited singlet state S2 in our current experimental setup because we excite the porphyrin in its Soret band (see the SI for UV/vis spectra). The triplet quantum yield can probably be increased by excitation of the first excited singlet state S1 by an excitation wavelength in the porphyrin’s Q bands between λexc. = 450 and 600 nm to ΦT ≈ 0.9.51 The Fourier transforms of the form factors lead to the dipolar coupling spectra in frequency space (Figure 2D) and can be fit via Tikhonov regularization to yield the distance distribution shown in Figure 2E with a mean interspin distance

DEER

S5). The full potential of LaserIMD could be unlocked if a triplet quantum yield ΦT = 1 could be reached. This would lead to a LaserIMD MNR enhancement factor up to ρ ≈ 6 (cf. the SI for details), cutting down measurement times by ρ2 compared to DEER. These results show that LaserIMD is a viable and attractive technique for EPR distance measurements if one label is replaced by TPP. Next we show that LaserIMD can directly exploit endogenous groups present in proteins. One important example is the heme prosthetic group.53,54 In order to show the applicability to directly utilize this group as a probe in distance measurements with LaserIMD, we chose to investigate cytochrome C. Cytochromes are iron-containing heme proteins that rely in their function on the heme. Here we used cytochrome C iso-1 isolated from Saccharomyces cerevisiae (CytC) that contains a low-spin ferric heme and can be spinlabeled with MTSSL on the free cysteine 102.55 For MTSSL attached to Cys102, a theoretical prediction of the distance distribution56 from the crystal structure with PDB ID 1YCC57 returns only one possible rotamer due to steric clashes of other rotamers with the neighboring loop 15−49 (Figure 3). The distance between the heme iron and the oxygen of the attached spin label in this structure is 1.8 nm, in broad agreement with an earlier line broadening analysis.55 Because the labeled site does not exhibit defined secondary structure and is next to the C-terminal residue Glu103, it is rather likely that it can avoid interaction with the loop 15−49 by a conformational change of the backbone, which would broaden the distance distribution. We want to point out that we were not able to detect a spin echo from the heme triplet state in CytC because its Tm is reduced significantly by the iron (cf. Figure S6). Utilizing electron spin echos of ferric heme centers in nanometer range distance measurements is still challenging,41,58 and LaserIMD does not rely on the presence of paramagnetic metal centers like the closely related relaxation-induced dipolar modulation enhancement (RIDME) technique.59,60 The short Tm prevents utilizing the triplet state as the observer species for DEER distance measurements29 and, if Tm is too short for applying an inversion pulse, also utilizing it as a spin pumped by a microwave pulse. However, LaserIMD only requires that the spin can be generated by photoexcitation and that its state survives for the duration of the echo experiment, thus posing the less strict requirement T1 > τLaserIMD. 2207


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Flexibility in Porphyrin-Based Molecular Wires Using Double Electron Electron Resonance. J. Am. Chem. Soc. 2009, 131, 13852−13859. (8) Jeschke, G.; Sajid, M.; Schulte, M.; Ramezanian, N.; Volkov, A.; Zimmermann, H.; Godt, A. Flexibility of Shape-Persistent Molecular Building Blocks Composed of p-Phenylene and Ethynylene Units. J. Am. Chem. Soc. 2010, 132, 10107−10117. (9) Kurzbach, D.; Kattnig, D. R.; Zhang, B.; Schlüter, A. D.; Hinderberger, D. Loading and Release Capabilities of Charged Dendronized Polymers Revealed by EPR Spectroscopy. Chem. Sci. 2012, 3, 2550−2558. (10) Hintze, C.; Schütze, F.; Drescher, M.; Mecking, S. Probing of Chain Conformations in Conjugated Polymer Nanoparticles by Electron Spin Resonance Spectroscopy. Phys. Chem. Chem. Phys. 2015, 17, 32289−32296. (11) Borbat, P. P.; Freed, J. H. Multiple-Quantum ESR and Distance Measurements. Chem. Phys. Lett. 1999, 313, 145−154. (12) Borbat, P. P.; Freed, J. H. Biological Magnetic Resonance 19. In Distance Measurements in Biological Systems by EPR; Berliner, L. J., Eaton, G. R., Eaton, S. S., Eds.; Springer: New York, 2002; pp 383− 459. (13) Borbat, P. P.; Freed, J. H. Pros and Cons of Pulse Dipolar ESR: DQC and DEER. EPR Newsletter 2007, 17, 21−33. (14) Hubbell, W. L.; Altenbach, C. Investigation of Structure and Dynamics in Membrane Proteins Using Site-Directed Spin Labeling. Curr. Opin. Struct. Biol. 1994, 4, 566−573. (15) Hubbell, W. L.; Mchaourab, H. S.; Altenbach, C.; Lietzow, M. A. Watching Proteins Move Using Site-Directed Spin Labeling. Structure (Oxford, U. K.) 1996, 4, 779−783. (16) Hemminga, M. A.; Berliner, L. J. ESR Spectroscopy in Membrane Biophysics; Springer: New York, 2007. (17) Klug, C. S.; Feix, J. B. Biophysical Tools for Biologists, Vol. One: In Vitro Techniques. Methods and Applications of Site-Directed Spin Labeling EPR Spectroscopy; Academic Press, 2008; Vol. 84; pp 617− 658. (18) Klare, J. P.; Steinhoff, H.-J. Spin Labeling EPR. Photosynth. Res. 2009, 102, 377−390. (19) Drescher, M. EPR in Protein Science. In EPR Spectroscopy; Drescher, M., Jeschke, G., Eds.; Springer: Berlin/Heidelberg, Germany, 2012; Vol. 321; pp 91−119. (20) Likhtenshtein, G. I.; Yamauchi, J.; Nakatsuji, S.; Smirnov, A. I.; Tamura, R. Nitroxides: Applications in Chemistry, Biomedicine, and Materials Science; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2008. (21) Hubbell, W. L.; López, C. J.; Altenbach, C.; Yang, Z. Technological advances in site-directed spin labeling of proteins. Curr. Opin. Struct. Biol. 2013, 23, 725−733. (22) Fielding, A. J.; Concilio, M. G.; Heaven, G.; Hollas, M. A. New Developments in Spin Labels for Pulsed Dipolar EPR. Molecules 2014, 19, 16998−17025. (23) Martorana, A.; Bellapadrona, G.; Feintuch, A.; Di Gregorio, E.; Aime, S.; Goldfarb, D. Probing Protein Conformation in Cells by EPR Distance Measurements using Gd3+ Spin Labeling. J. Am. Chem. Soc. 2014, 136, 13458−13465. (24) Qi, M.; Groß, A.; Jeschke, G.; Godt, A.; Drescher, M. Gd(III)PyMTA Label Is Suitable for In-Cell EPR. J. Am. Chem. Soc. 2014, 136, 15366−15378. (25) Jagtap, A. P.; Krstic, I.; Kunjir, N. C.; Hänsel, R.; Prisner, T. F.; Sigurdsson, S. T. Sterically Shielded Spin Labels for In-Cell EPR Spectroscopy: Analysis of Stability in Reducing Environment. Free Radical Res. 2015, 49, 78−85. (26) van Amsterdam, I. M. C.; Ubbink, M.; Canters, G. W.; Huber, M. Measurement of a Cu-Cu Distance of 26 by a Pulsed EPR Method. Angew. Chem., Int. Ed. 2003, 42, 62−64. (27) Lyubenova, S.; Maly, T.; Zwicker, K.; Brandt, U.; Ludwig, B.; Prisner, T. Multifrequency Pulsed Electron Paramagnetic Resonance on Metalloproteins. Acc. Chem. Res. 2010, 43, 181−189. (28) Ruthstein, S.; Ji, M.; Mehta, P.; Jen-Jacobson, L.; Saxena, S. Sensitive Cu2+-Cu2+ Distance Measurements in a Protein-DNA

LaserIMD measurements between the MTSSL and the heme group of CytC were successful (Figure 4). The resulting distance distribution yields a mean distance of ⟨r⟩ = 1.96 nm, in good agreement with the theoretically predicted distance. The LaserIMD experiment gives access to distances as low as 1.2 nm. This lower limit is defined by the jitter of the laser used (fwhm ≤ 20 ns; cf. Figure S10) and the excitation bandwidth of the observer π pulse length of 20 ns.47 Laser jitter could be reduced by a different triggering scheme, and excitation bandwidth correction46 could be applied, which could enable detection of even shorter distances down to 1 nm. In summary, we have shown that LaserIMD allows for measurement of distance distributions in the nanometer range, both when using TPP as a spin label and when exploiting an endogenous low-spin heme as the photoexcitable species. In the latter case, the technique poses lesser requirements on relaxation times of the triplet spin than are posed by the triplet-DEER experiment. If the triplet quantum yield can be improved, LaserIMD has the potential to access longer distances or decrease the measurement time or measure at lower concentration than is possible with DEER techniques.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.6b00765. Characterization of Ala-(Aib-Ala)4-TOAC-(Aib-Ala)2OH and cytochrome C; sample preparation; EPR experiments and analysis; and additional EPR measurements (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ¶

C.H. and D.B. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the DFG (DR 743/2-1 and DR 743/10-1). REFERENCES

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