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Double Electron-Electron Resonance Measured Between Gd3þ Ions and Nitroxide Radicals Petra Lueders, Gunnar Jeschke, and Maxim Yulikov* Laboratory of Physical Chemistry, ETH Zurich, Wolfgang-Pauli-Str. 10, 8093 Zurich, Switzerland

bS Supporting Information ABSTRACT: Double electron-electron resonance has attracted growing attention as a technique to study structure and conformational changes of biomacromolecules. Here, a new combination of paramagnetic labels is experimentally tested, one being a commonly used nitroxide radical, and the other being a Gd3þ ion. The Gd3þ-nitroxide spin pair can serve as a good substitute for the nitroxide-nitroxide pair of spin labels and potentially provides a link to other experimental approaches dealing with structural information.

SECTION: Macromolecules, Soft Matter

orientation selection.18 This may force one to measure a series of DEER traces in a range of different magnetic fields11 and to sum the obtained traces in order to get a reliable distance distribution. This increases the measurement time and thus reduces the sensitivity increase at higher bands. Therefore, despite the apparent success of nitroxide-label-based techniques, the search for new types of spin labels and improved experimental conditions is currently an active field of research. In particular, the availability of separately addressable labels would be of interest for studies of protein complexes, where different subunits of a complex under study could then be marked with different types of labels and observed independently. In the present Letter, we report the performance of the DEER experiment with one paramagnetic species being a chelate complex of Gd3þ instead of a nitroxide radical. Gd3þ centers were already reported as potential spin probes for high-frequency (Ka-band and W-band) DEER.19-21 In frozen solutions, such spin probes are expected to have much less pronounced orientation selection due to a stochastic distribution of the eigenframe orientations and magnitudes for the zero-field splitting (ZFS).22 Here, we show that the combination of one Gd3þ center and one nitroxide radical allows one to perform DEER measurements at the X and Q band with detection on Gd3þ with a sensitivity that should be sufficient to apply this technique to protein samples.

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ong range distance constraints in the range of 1-10 nm can be crucial for modeling structure and structural transitions of proteins and protein complexes. The recent development of EPR-based techniques,1-5 in particular, pulse double electronelectron resonance (DEER/PELDOR1,2,5), has made a significant contribution to the method arsenal for determining such long distances.6-10 In most cases, the DEER experiment is applied to macromolecules with two nitroxide spin labels.7 The upper detectable distance limit depends strongly on the type of system studied. For model systems and often for soluble proteins, it can be as long as 7.6 nm if the sample is prepared in deuterated solvent.11 This limit can reach beyond 11 nm if the macromolecule under study is completely deuterated.12 Nevertheless, in many cases, especially for membrane-incorporated proteins, the transverse relaxation times of nitroxide labels are in the range of 2-4 μs, rarely 5 μs,13 which restricts the detectable distance limit to not more than 5 nm even if deuterated solvents are used. The DEER experiment is also rather expensive in terms of the measurement time, with typical times for a single distance measurement of 12-24 h at protein concentrations of approximately 50-200 μM. The sensitivity of DEER increases significantly if the measurement is performed at Q-band frequency (∼34 GHz) instead of the commonly used X-band (∼9.5 GHz).14-16 The recent development of a high-power W-band spectrometer17 has yet more significantly increased the sensitivity of DEER measurements on nitroxides and added flexibility with respect to optimization of measurement conditions. Still, the sensitivity improvement comes at the expense of stronger r 2011 American Chemical Society

Received: January 17, 2011 Accepted: February 16, 2011 Published: February 28, 2011 604

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Figure 1. Selective measurement of the ED EPR spectra for the terpyridine complex of Gd3þ. (a) X-band and (b) Q-band ED EPR spectra with microwave pulses and repetition times optimized for the detection of the nitroxide radicals (shown in black; srt = 16000 μs) or Gd3þ centers (shown in red; srt = 337 μs). Broader range ED EPR spectra are shown in the insets of (a) and (b). The molecular structure of the Gd3þ-terpyridine complex is shown in the center of the figure.

We also show that the transverse relaxation time relevant for this experiment is comparable to the T2 times of nitroxide radicals attached to membrane proteins. This spin pair arrangement opens as well a possibility to combine DEER with relaxation-enhancement-based distance measurements.23 Furthermore, the use of a lanthanide label can provide a bridge between EPR-based and resonanceenergy-transfer-based24,25 methods for structure determination. Both nitroxide radicals and lanthanide complexes are also used as tags for paramagnetic NMR experiments.26 Such a possibility to combine different techniques could provide closer insight into the structure of a system of interest. In order to validate the prospect of the DEER experiment performed between a Gd3þ ion and a nitroxide spin label, we study a model system with well-defined distance between the two different paramagnetic centers, namely, a chelate complex of Gd3þ ion with a spin-labeled ligand (2.2,5-dihexyl-1-[2-(4-hydroxyphenyl)ethynyl]-4-[2-(2,20 :60 ,600 -terpyridin-40 -yl)ethynyl]benzene). The Gd3þ complex was prepared in deuterated solvent. For an analogous compound loaded with Cu2þ ion, the DEER-determined distance (2.38-2.46 nm) has been reported to agree with the theoretical prediction by density functional theory (DFT, 2.43 nm).27 The DEER experiments were carried out at X band (9.5 GHz) and Q band (34 GHz). A Bruker ElexSys II 580 combined X/Q EPR spectrometer was used for the DEER measurements. Additionally, the experiments at Q band with short (12 ns) pulses were performed at the home-built spectrometer described elsewhere28 with a rectangular resonator specially developed for oversized samples.29 This allows for using the same sample tube of 2.9 mm outer diameter for commercial Xand home-built Q-band spectrometers (for further experimental, details see Supporting Information). A control experiment performed on the model compound at X band with both pumping and observing frequencies on the nitroxide species showed a broad distance distribution with a very small modulation depth of not more than 0.06, which reveals no substantial formation of nitroxide radical pairs with welldefined mutual distance in the solution (see Supporting Information, Figure S1). Thus, we assume formation of mainly the 1:1 complex between Gd3þ and the terpyridine derivative. The |-1/2æ T |1/2æ transition of Gd3þ centers (S = 7/2) has a transition moment that is four times bigger than the one of nitroxide radicals (S = 1/2). In combination with the about 3 orders of magnitude ratio of the longitudinal relaxation times of the two paramagnetic species, this allows detection of Gd3þ

centers and nitroxide radicals almost independently from each other (Figure 1). Due to this behavior, the overlap of the nitroxide EPR spectrum with the central peak of the Gd3þ spectrum at X band (Figure 1a) is of no significant consequence for the DEER experiment. At Q-band frequencies, the nitroxide spectrum does not overlap with the center of the Gd3þ spectrum due to the increased resolution in the g-value scale. An observer frequency corresponding to the maximum absorption of Gd3þ is most advantageous, because the |-1/2æ T |1/2æ transition of Gd3þ corresponding to this position has the weakest anisotropy. This is advantageous if no orientation selection is desired. Furthermore, this detection position was chosen because transverse relaxation was detected to be slowest for this position in the Gd3þ spectrum (data not shown). The frequency for the pump pulse was set to the maximum absorption position of the nitroxide spectrum in order to provide maximum inversion efficiency and thus maximum modulation depth. This corresponds to a splitting between observer and pump frequencies of approximately 80 MHz at X band and 300 MHz at Q band. Data analysis was performed with the DeerAnalysis 2009 package,30 which was originally written for the detection on S = 1/2 species. This is permissible if the high-field approximation is valid and therefore the frequency change for each single-quantum transition of the S = 7/2 species upon the inversion of the nitroxide spin is the same as that for the S = 1/2 species. The high-field approximation is not generally applicable to the Gd3þ EPR spectrum; in particular, the |-1/2æ T |1/2æ states of Gd3þ are most sensitive to the values of ZFS parameters,31 which can lead to the distortions in the DEER experiment due to pseudosecular terms.20 The comparison of the echo-detected (ED) EPR spectrum of our sample to numerous EPR spectra reported for different Gd3þ centers (see, for instance, refs 19-22) suggests the presence of a big fraction of species with D in the range of 40-50 mT. This is close to the average value of D reported in ref 20, where the authors suspect effects of pseudosecular terms on the modulation depth in the Gd3þ—Gd3þ DEER experiment. However, when measuring the ED EPR spectrum with a long interpulse delay, which is comparable to the overall evolution time in the DEER experiment, a much narrower spectrum is obtained (for further details, see Supporting Information). This suggests that complexes with much smaller D dominate the response in the DEER measurement. In this situation, the highfield approximation is feasible, and it is adequate to use the analysis routines incorporated in the DeerAnalysis package. This 605

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Figure 2. DEER measurements of Gd3þ-nitroxide spin pairs (X band, cyan; Q band [short pulses], black; Q band [long pulses], green) performed at 10 K (srt = 357 μs) on a sample with a Gd3þ concentration of 600 μM. (a) Background-corrected experimental data F(t)/F(0) and best fits as calculated by DeerAnalysis 2009 (red dotted). (b) Corresponding distance distributions P(r) with artifacts due to admixing 2H nuclear modulation marked with (*). Spectra and distributions are shifted vertically for better visualization.

Figure 3. Optimization of measurement temperature for the DEER experiment in Gd3þ-nitroxide spin pairs. (a) T1 (blue) and T2 (red) values for Gd3þ centers versus measurement temperature; for T2, the slow component of the biexponential fit is shown. (b) Temperature dependence of the expected signal-to-noise ratio in Gd3þ-nitroxide DEER as calculated according to ref 7, with additional correction for the partial amplitude of the slow T2 component of Gd3þ for an experimental trace with tmax = 4 μs.

proposition is tested in the following by comparing results at Xand Q-band frequencies. The much higher transition moment for Gd3þ centers allows for a pulse scheme with a 10 ns pump pulse in the center of the resonator mode and all detection pulses on the Gd3þ of 12 ns at X band without any problems concerning the available power. The optimum settings for nitroxide-nitroxide DEER, where the splitting between pump and observer frequencies is about 65 MHz, are close to a 12 ns pump pulse and 32 ns observer pulses in order to avoid overlap of excitation bands. For Gd3þnitroxide DEER, a larger frequency splitting of 80 MHz is optimal so that stronger pulses with a larger excitation bandwidth can be used. No systematic optimization has been done in this respect. Fast longitudinal relaxation of Gd3þ allows for much shorter repetition times than for the detection on nitroxide species. In addition, the measurements can be performed at lower temperature, thus providing higher sensitivity due to the gain in the equilibrium magnetization of the sample. The DEER experiments (Figure 2) were performed at 10 K with a shot repetition time (srt) of 357 μs, which is still limited by the duty cycle of the microwave amplifier rather than by T1 of Gd3þ (see Figure 3). At Q band, a pump π-pulse length of 52 ns and a detection πpulse length of 128 ns were achieved with a 1.6 mm outer diameter sample tube on the commercial Bruker spectrometer equipped with a 1 W solid-state amplifier. On the home-built Q-band spectrometer, which is equipped with a 150 W TWT amplifier, a scheme with both pump and detection π- and π/2 pulses of 12 ns is possible, despite using an oversized sample.

At X band, a modulation depth of 0.4 was observed in the DEER experiment, which is comparable to nitroxide-nitroxide DEER where typical modulation depth in the case of 100% labeling efficiency is 0.5. At Q band, the achievable modulation depth strongly depends on the performance of the EPR spectrometer. For the commercial spectrometer (pump pulse of 52 ns), the modulation depth was measured to be approximately 0.06, whereas for the home-built spectrometer (pump pulse of 12 ns), a modulation depth of approximately 0.2 has been achieved. The baseline-corrected and normalized time domain DEER traces are presented in Figure 2. The distance distributions P(r) were obtained with DeerAnalysis 2009. The best fit was obtained with a Tikhonov regularization parameter of 1 for the X-band data and 0.01 for the Q-band data. The difference in the optimum regularization parameter is mainly due to the different noise level of the data. All distance distributions feature a main peak with a mean distance of 2.54 nm, which agrees with the Cu-nitroxide distance of approximately 2.43 nm reported for a related system.27 Compared to the copper(II) complex with two terpyridine ligands,27 the distance distribution is narrower, as is also apparent from the observation of many more oscillations in the DEER trace. A small increase of the measured distance is most probably related to the bigger ion radius of Gd3þ as compared to that of Cu2þ. Furthermore, probably more spin density is transferred to the directly coordinated nitrogen atoms in the copper case, and these are somewhat closer to the nitroxide. Beside the main peak, the distance distributions at both the X and Q bands also show some satellite features. The small peak that appears at around 2.8 nm at X band and shifts to approximately 1.8 nm at Q band can be unambiguously assigned to the 606

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At X band, the performance of Gd3þ-nitroxide DEER in terms of modulation depth is roughly comparable to the nitroxide-nitroxide DEER measurements. In the presented case, the obtained signal-to-noise ratio is worse than the typical performance of nitroxide-nitroxide DEER under similar experimental conditions (comparable T2 time, comparable length of the DEER trace, etc.). Apparently for Gd3þ-nitroxide DEER, the advantages of lower temperature and faster repetition are compensated for by the smaller fraction of observed spin pairs, which results from the larger width of the Gd3þ EPR spectrum compared to the nitroxide and from the spread of relaxation times for the Gd3þ complexes. Still, there is scope for sensitivity optimization, in particular, measurement temperature, ESEEM suppression, and the fraction of centers participating in the formation of the DEER echo. With the home-built Q-band spectrometer, the lengths of pump and detection pulses could be set to approximately the same values as those for the commercial X-band spectrometer. In this configuration, the signal-to-noise ratio increases by approximately a factor of 34 at Q band as compared to that at X band, which implies a speedup of the measurement by a square of this value, that is, by approximately a factor of 1156. It is known that the sensitivity of EPR measurements increases at Q band because of the larger equilibrium polarization of electron spins and because of the better sensitivity of the detection at higher frequency.15,16 In this case, the impressive increase is further aided by the narrowing of the central peak of the Gd3þ spectrum at higher frequencies19-22 as well as by the use of the same sample volume as that at X band.29 Another contribution comes from the fact that the deuterium ESEEM effect reduces the sensitivity at X band, while it does not strongly influence DEER measurement at Q band. Concerning our high-power Q-band setup, measurements of Gd3þ-nitroxide DEER on samples with spin concentrations of as low as 50 μM are feasible without further sensitivity optimization. A 1 h measurement for such a sample would result in a 3 μs long DEER trace with a signal-to-noise ratio normalized to the modulation depth of slightly more than 17 (see Table S1 in Supporting Information). The optimization of the measurement conditions can be done by measuring relaxation times of the sample at different temperatures (Figure 3) and analyzing them together with the echo amplitude, which follows Curie law. The signal-to-noise ratio depends on the length of the DEER experiment and can be computed as7
 sffiffiffiffiffiffiffiffiffiffiffiffi 1 -2tmax 1 S=NðTÞ  3 exp T T2 ðTÞ 3 T1 ðTÞ

incomplete suppression of the strong deuterium ESEEM oscillations (γ[2H] = 4.1  107 rad/T 3 s). The deuterium ESSEM oscillations also affect strongly the sensitivity of DEER at X band. As no significant increase of transverse relaxation time is observed in deuterated solvent compared to the Gd3þ complexes in nondeuterated solutions,19-21 the use of deuterated solvents in Gd3þ-nitroxide DEER at X band seems to be disadvantageous. At X band, an eight-step cycle of τ1 averages with increment Δτ1 = 56 ns was used to average out the deuterium ESEEM oscillations.32 At Q band, no such averaging was employed, but the oscillations were substantially suppressed due to a much bigger split between the detection and the pump frequencies and a lower nuclear modulation depth. Additionally, two more satellite peaks appear in the distance distribution roughly symmetrically around the main peak. If recomputed in the frequency domain, these two satellites correspond to frequencies slightly less than double and slightly more that half of the frequencies of the main oscillations. Currently, we can only speculate on the origin of these features and attribute them to violation of the high-field approximation and, for part of molecules, to effects arising from excitation of transitions other than the |-1/2æ T |1/2æ transition of Gd3þ. The effect of the pseudosecular term on the DEER modulation of Gd3þ has been analyzed in ref 20. It is expected to lead to an additional modulation frequency present in the DEER trace and to a suppression of the amplitude of the main modulation. In the cited work, the modulation on the second frequency might have been hidden by a broad distance distribution. In our case, the distance distribution is very narrow, and such an additional modulation should be detectable. Nevertheless, the behavior of the satellite lines that we observed does not fit the expected behavior of the second frequency component arising from the presence of non-negligible pseudosecular terms. First, instead of one additional frequency, we observe two. Second, the positions of these satellites do not change upon the change of the difference between pump and detection frequencies from 80 MHz at X band to 300 MHz at Q band, which would be expected for the pseudosecular contributions.20 In our sample, the presence of a fraction of different types of Gd-terpyridine complexes, for example, with more than one terpyridine ligand, cannot be excluded. Therefore, at present, we cannot decide whether the sidebands are due to the unusual effects in the spin dynamics of Gd3þ centers in the DEER experiment or to properties of the sample. The relative weight of the discussed satellites is apparently smaller at Q band than that at X band, which would favor the explanation based on the violation of the high-field approximation. The relative amplitude of satellite signals remained unchanged at different interpulse delays in the DEER sequence, which implies similar relaxation behavior for both main peak and satellite signals. If the excitation of different transitions of Gd3þ were the reason for these satellite features, then a different relaxation behavior would be expected. The satellite feature at 2 nm as well as the deuterium artifact do not appear in the distance distribution obtained from the Q-band DEER measurements on the commercial Bruker spectrometer. This establishes a lower limit of detectable distances with these pulse settings to somewhere between 2 and 2.5 nm. This lower limit results from suppression of DEER modulation when the excitation bandwidth of the pump or observer pulses is smaller than the dipole-dipole coupling.33,34

Here, tmax is the sum of the first and the second interpulse delays in the DEER pulse sequence, T is the measurement temperature, and T1(T) and T2(T) are, respectively, the temperature-dependent longitudinal and transverse relaxation times of paramagnetic centers. An additional correction is required if this formula is applied to the Gd3þ species. The transverse relaxation of Gd3þ was measured to be nonmonoexponential. It is known that a broad distribution of the values of ZFS parameters D and E between different Gd3þ complexes is present in shock-frozen solutions.22 This is expected to result in a distribution of relaxation times. The species with the slowest relaxation times are the ones with the smallest ZFS (normally the most symmetric arrangement of ligands). As is apparent from the ED EPR spectra 607

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detected with long interpulse delay (see Supporting Information), these slowly relaxing species are most relevant in the DEER experiment, and the above-presented expression should be corrected for this. We fitted the T2 relaxation curves for Gd3þ by biexponential decay functions and used the relative amplitude and decay time of the slower component of the fit to estimate the sensitivity of Gd3þ-nitroxide DEER in the temperature range between 5 and 40 K. Figure 3 shows that an additional improvement in the signal-to-noise ratio is expected if the measurements are performed at 5 K instead of 10 K, as has been done in the present work. The corresponding T2 times of Gd3þ range between 2 and 3 μs at the optimum temperatures. As a conclusion, the DEER experiment with detection on Gd3þ centers and pumping on nitroxide radicals shows promising performance and can become a valuable alternative to conventional nitroxide-nitroxide distance measurements, particularly at Q band. A routine use of such an experiment requires deeper insight into the spin dynamics of the high-spin Gd3þ centers during this experiment. At the present level of understanding, Gd3þ-nitroxide DEER is expected to provide a reliable average distance along with a distance distribution, which is possibly artificially broadened due to the presence of satellites. The nature of the satellites that we observed in the distance distribution of the Gd3þ-terpyridine system also has to be further clarified. Work along these lines is in progress.

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’ ASSOCIATED CONTENT

bS

Supporting Information. Experimental details; sample preparation details; calculation of the signal-to-noise ratio; nitroxide-nitroxide DEER control experiment; raw experimental time traces of the Gd3þ-nitroxide DEER experiments; and interpulse delay-dependent ED EPR spectra of Gd3þ centers. This material is available free of charge via the Internet at http:// pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail:[email protected].

’ ACKNOWLEDGMENT The authors thank an anonymous reviewer for valuable comments with respect to the conditions of the high-field approximation and on the effect of pseudosecular terms in high-spin systems and Rene Tschaggelar, Dr. Yevhen Polyhach, and Dr. Enrica Bordignon for technical support, numerous discussions on the EPR hardware and on the DEER experiment, and data processing. Dr. Evelyn Wessel (nee Narr) and Prof. Adelheid Godt are acknowledged for providing the terpyridine derivative. The work is funded by SNF (Grant 200021_121579). ’ REFERENCES (1) Milov, A. D.; Salikhov, K. M.; Shirov, M. D. Application of ELDOR in Electron-Spin Echo for Paramagnetic Center Space Distribution in Solids. Fiz. Tverd. Tela (Leningrad) 1981, 23, 957–982. (2) Milov, A. D.; Ponomarev, A. B.; Tsvetkov, Yu.D. Electron Electron Double Resonance in Electron-Spin Echo-Model Biradical Systems and the Sensitized Photolysis of Decalin. Chem. Phys. Lett. 1984, 110, 67–72. 608

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