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Double quantum coherence electron spin resonance on coupled Cu(II)–Cu(II) electron spins
Chemical Physics Letters 414 (2005) 248–252 www.elsevier.com/locate/cplett
Double quantum coherence electron spin resonance on coupled Cu(II)–Cu(II) electron spins James S. Becker, Sunil Saxena
*
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, United States Received 30 June 2005; in final form 6 August 2005 Available online 16 September 2005
Abstract We demonstrate for the first time the ability to generate double quantum coherences (DQCs) for the case of Cu(II). We show that small splittings (7 MHz) from the Cu(II)–Cu(II) electron–electron magnetic dipolar interaction can be reliably resolved even though the inhomogeneously broadened Cu(II) linewidth is 2 GHz. A Cu(II)–Cu(II) distance of 2.0 nm was measured on a model peptide system, thus, demonstrating that distances on the nanometer scale may be measured using DQC electron spin resonance (ESR). 2005 Elsevier B.V. All rights reserved.
1. Introduction The need for the determination of the structures of biomolecules and nanostructured materials has provided an impetus for development of new methods in electron spin resonance (ESR). Significant research involving concepts of double quantum coherences [1–5] or double resonance [6–11] have resulted in robust methods to directly measure the magnetic dipolar interaction between two electron spins on a macromolecule. This has made it possible to measure ˚ range between two interspin distances in the 16–75 A spin-labels in order to determine global folding patterns in proteins [12–16], nucleic acids [17–19], and ionic polymers [20–23], and conformational and aggregation states of polypeptides [24–27]. Thus far, the ESR method has largely been restricted to the use of nitroxides as spin labels. Recently, this methodology has been extended to the case of paramagnetic metal centers in metalloproteins using double electron–electron resonance (DEER) ESR [28,29]. In these experiments, the local dipolar field due to the coupled spin-partner is inverted using pulses of durations that are P10 ns. The primary echo is then modulated by the dipolar frequency providing sensitivity to distances. The *
Corresponding author. Fax: +1 412 624 8611. E-mail address: [email protected] (S. Saxena).
0009-2614/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2005.08.072
use of large pulse lengths necessarily leads to a reduced signal-to-noise ratio, and the orientational selectivity of the pulses can potentially complicate the analysis of the spectrum to measure distances. In addition, the use of selective pulses makes the measurement of small distances (ca. ˚ ) difficult. 8–15 A In principle, double quantum coherence (DQC)-ESR can circumvent these limitations. This has motivated us to explore the possibility of generating DQCs for the case Cu(II) binding systems. In DQC-ESR, spin–spin interactions generate the double quantum coherence. The rate of formation of the DQC directly reports on the strength of the dipolar interaction, providing sensitivity to distances. The generation of DQCs for the case of Cu(II) poses daunting challenges. The spectral extent of Cu(II) ESR can be as large as 2 GHz – whereas the dipolar interaction is weak (MHz to kHz) for distances of typical interest. On the other hand, DQC methodology optimally requires the use of non-selective pulses, which for the case of Cu(II) is prohibitively difficult. In addition, Cu(II) spin-echoes of biological systems typically experience deep electron spin echo envelope modulations (ESEEM) because of the electron–nuclear interaction with the nitrogen nuclei in the amino-acid coordination environment [30]. The resulting ESEEM peaks can interfere with DQC spectra.
J.S. Becker, S. Saxena / Chemical Physics Letters 414 (2005) 248–252
Despite these challenges, we have been successful in detecting a DQC-ESR spectrum for the case of Cu(II). In this work, we show that small splittings (7 MHz) from the Cu(II)–Cu(II) electron–electron magnetic dipolar interaction can be reliably resolved. 2. Experimental Two Cu(II) binding peptide samples, synthesized at the University of PittsburghÕs Molecular Medicine Institute, were used for the experiments, one a dimer and the other a Cu(II) complex. The Cu(II)–Cu(II) dimer, Ac-PPHGGGWPPPHGGGWPP-NH2 will be called Cu3P (Fig. 1a), and the control sample, designated as Pr, had the aminoacid sequence, Ac-PHGGGW-NH2. The HGGGW aminoacid sequence, displayed as a crystal structure in Fig. 1b, is a well-characterized Cu(II) binding unit of the prion protein [31–33]. A 3.5 mM solution of Cu3P was prepared by dissolving the peptide in a solvent consisting of 30% glycerol/30% 2,2,2-trifluoroethanol/40% water containing 150 mM NaCl and buffered with 25 mM N-ethylmorpholine to pH 7.35. Two mole equivalents of Cu(II) were added from an aqueous 1 M copper sulfate solution. For the control sample, a 4.0 mM solution was prepared with a solvent of 30% glycerol/70% water containing 150 mM NaCl and buffered with 25 mM N-ethylmorpholine to pH 7.74. One mole equivalent of Cu(II) was added from a 0.1 M aqueous copper sulfate solution. For the ESR experiments, the samples were bath sonicated for approximately 5 min before flash freezing in liquid nitrogen. After insertion into the ESR cavity, the temperature was stabilized to 8.0 ± 0.2 K using an Oxford ITC605 temperature controller and an Oxford ER 4118CF gas flow cryostat. Spectroscopic experiments were performed on a Bruker EleXsys E580 X-band CW/Pulse ESR spectrometer equipped with a Bruker ER4118X-MS2 split-ring resonator. The high output power of the ASE-TWTA (1 kW) combined with a quality factor much less than 100 provided a p/2 pulse with a length of 4 ns.
Fig. 1. (a) The dimer, Cu3P, is displayed from the CAChe model (cf. Section 3). Hydrogens are not shown in the model and the Cu(II) ions appear as large spheres connected by the interspin vector, r. (b) The crystal structure of the HGGGW copper binding unit reproduced with permission from http://chemistry.ucsc.edu/~glennm/ is shown [33].
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The six-pulse DQC-ESR sequence, shown in Fig. 2a, was implemented in the following manner. A 256 step phase cycle was used to select the correct coherence pathway [3] and the DQC-echo after the sixth pulse was integrated. The pulse lengths were 4 ns for p/2 pulses and 8 ns for p pulses. The initial delays of tp and t1 were 12 ns and the delay of t2 was 120 ns for Cu3P and 76 ns for Pr. The time, tp, was stepped by 10 ns for a total of 128 points. For each step 900 averages were collected at a repetition rate of 200 Hz for the dimer and 800 averages were collected at the same rate for the control. Three-pulse ESEEM experiments [30,34] were carried out with p/2 pulses of 16 ns length. The pulse sequence is displayed in Fig. 2b. The initial inter-pulse separations were s = 150 ns and T = 40 ns. The delay T was stepped out by 16 ns for a total of 256 data points. A total of 50 averages per point were collected with a repetition rate of 200 Hz. The ESR spectra were obtained from the time domain data as described previously [5]. 3. Results and discussion The field-swept echo-detected Cu(II) absorption spectrum is shown in Fig. 3a. The total width of the spectrum is approximately 750 G or about 2100 MHz. A magnetic field of 3250 G, corresponding to the g^ region of Cu(II) spectrum (shown by an arrow in Fig. 3a), was used for DQC experiments. The continuous wave ESR spectrum was simulated using the Bruker Simfonia program and the g-value was determined to be 2.1166. The creation of DQC depends on the coverage of the pulse, which can be limited by the pulse length as well as by the bandwidth of the resonator. To estimate the coverage, the echo signal from a nutation experiment on a standard nitroxide sample was collected at 8 K. The nutation frequency as a function of microwave frequency is shown in Fig. 3b. The nutation frequency is nearly constant over 250 MHz. Due to the large width of the nitroxide spectrum, this number is an overestimate of spectral coverage, but still provides a useful point of reference for the approximate coverage and center frequency. The maximal intensity of the Cu(II) spectrum resides in the g^ region of the absorption spectrum (cf. Fig. 3a). Thus, in terms of intensity a pulse with 250 MHz coverage applied on the g^ re-
Fig. 2. (a) The double quantum coherence six-pulse sequence is displayed. The pulse separation, tp, is stepped out in order to observe echo modulation in the DQC experiment. For the six-pulse ESEEM experiment tp is constant and t2 is stepped out. (b) The three-pulse ESEEM pulse sequence is shown.
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J.S. Becker, S. Saxena / Chemical Physics Letters 414 (2005) 248–252
Fig. 3. (a) The field-swept echo-detected spectrum of Cu3P is shown. An arrow denotes the magnetic field position for the DQC experiments, and the gi and g^ regions of the absorption spectrum are indicated for clarity. (b) The nutation frequency as a function of the spectrometer frequency is displayed. This experiment provides an estimate for the spectral coverage and center spectrometer frequency.
gion can irradiate a substantial part of the Cu(II) absorption spectrum. The DQC spectrum of the Cu(II)-dimer is displayed in Fig. 4a. The time domain DQC signal is given by Saxena and Freed [1] V ðtp ; t2 Þ ¼ sinð2pm1;2 tp Þ sinð2pm1;2 t2 Þektp ; ð1Þ where tp and t2 are the pulse delays described above (cf. Section 2.1), k is the relaxation rate of the DQC spin-echo, and m1,2 is m1;2 ¼
l0 g1 g2 b2 ð3cos2 h 1Þ þ 2J : 4phr3
ð2Þ
In Eq. (2), g1 and g2 are the g-factors of each electron, b is the Bohr magneton, h is PlanckÕs constant, r is the interspin vector, and h is the angle between magnetic field and the interspin vector. J is the exchange interaction, which for
Fig. 4. (a) The DQC spectrum of the dimer is shown with asterisks designating the 7.4 MHz electron–electron dipolar coupling peak. (b) The three-pulse ESEEM spectrum of Cu3P is displayed. (c) The control, Pr, DQC spectrum is displayed and the asterisks mark the absence of electron–electron dipolar splittings. (d) The spectrum of the six-pulse ESEEM experiment is shown with asterisks again marking the lack of peaks in the appropriate frequency range.
this sample is negligible due to the large separation between the Cu(II) centers. For frozen samples and for truly non-selective pulses a ÔPakeÕ pattern [3] is obtained in which the characteristic turning points corresponding to the parallel and perpendicular orientations (i.e., h = 0 and h = 90 in Eq. (2)) of the interspin vectors with respect to the dc-magnetic field are readily observable. The h = 0 turning point has much lower intensity and, therefore, for weaker pulses only the h = 90 turning point is obtained in the DQC spectrum [3]. In addition to this peak from the electron–electron dipolar interaction, ESEEM peaks due to hyperfine and nuclear quadrupolar interactions are also obtained. Many peaks are found upon immediate inspection of the Cu(II)-dimer DQC-spectrum (cf. Fig. 4a), several of which are ESEEM peaks. The most easily identified is at the proton Larmor frequency at ±14.0 MHz due to the coupling of the Cu(II) electron spin with the neighboring protons. Peaks found at frequencies less than 4.8 MHz (i.e., 4.8 and 2.0 MHz) are well characterized as ESEEM peaks due to hyperfine interactions with and quadrupolar interactions of 14N nuclei. Specifically, peaks arise from the remote, uncoordinated nitrogen of the imidazole ring and, also, from the uncoordinated amide nitrogen in the glycine backbone [31,32]. There is significant overlap between adjacent peaks because magnitude peaks decay slowly with frequency [35]. Furthermore, low frequency ESEEM peaks are not recorded because the decay of the DQC spin-echo was prohibitively fast (hundreds of ns). A single peak at ±7.4 MHz, denoted by an asterisk in Fig. 4a, remains unidentified in the DQC spectrum. We attribute the peak being due to the intramolecular electron–electron dipolar interaction between the two Cu(II)– Cu(II) electron spins This peak assignment is supported by data from three control experiments. Firstly, a three-pulse ESEEM experiment (Fig. 4b) was performed on the dimer as a control experiment to further support our conclusions. The three-pulse ESEEM spectrum clearly displays the effects of electron–nuclear couplings between the Cu(II) electron spin and neighboring
J.S. Becker, S. Saxena / Chemical Physics Letters 414 (2005) 248–252
nuclei (i.e., nitrogen and hydrogen). The data serve two important purposes for this work. There is a conspicuous lack of peaks between the proton modulation occurring at ±14.2 MHz, and the 14N nuclear double quantum transition at ±4.6 MHz, thus, supporting our assignment of the ±7.4 MHz peak in the DQC spectrum (cf. Fig. 4a) as being due to the electron–electron dipolar interaction. It also proves that the Cu(II) is coordinated in the same environment as determined in the literature [31–33]. It must also be clarified that the proton modulation peak at ±14.0 MHz is greatly attenuated because of the proton blind-spot at this s value [34]. Secondly, in Fig. 4c, we show the DQC spectrum of the Cu(II) complex, Pr. The DQC signal of Pr originates from intermolecular dipolar interaction and leads to a decay modulated by ESEEM [3]. A peak at ±7.4 MHz is not obtained, thus, eliminating uncertainty in the nature and origin of the ±7.4 MHz peak in the Cu(II)-dimer DQC spectrum. Comparison of the two spectra offers direct evidence supporting the assignment of dipole–dipole coupling between the two electron spins. Finally, to be certain the ±7.4 MHz peak is not an artifact of unknown origin; a six-pulse control experiment was created in order to generate only an ESEEM pattern from this sequence. In this experiment, the pulse sequence in Fig. 2a was used, but tp was held constant at 12 ns, and only the last pulse was stepped out by 10 ns. In this way, the p/2–p–p/2 DQC generator sequence remains active to form the DQC, but the electron–electron dipolar coupling is not resolved because the pulse separation, tp, is constant in time. A spectrum is obtained that has similar signal-tonoise ratio and the same nuclear modulations, but lacks the intramolecular dipole–dipole coupling modulation inherent to the proper DQC time-domain signal. The FT spectrum of the signal obtained from the Cu(II)-dimer is shown in Fig. 4d. The spectrum contains the expected ESEEM peaks (cf. Fig. 4a), but is devoid of peaks between ±14.2 and ±4.8 MHz. We, therefore, justify our assignment of the ±7.4 MHz to an intramolecular electron–electron dipolar interaction. By using a calculated g-value of 2.1166 and assuming h = p/2 in the point dipole approximation, a frequency of 7.4 MHz yields an experimental distance of 2.0 nm. The Cu3P molecule was modeled using CAChe Software (Fujitsu) (cf. Fig. 1a) by first creating a representation of the HGGGW crystal structure determined by Millhauser and coworkers [32]. The segment was reproduced to make two copies and the proline residues were covalently attached to the copper binding units. Energy minimization was performed using the MM3 force field and this yielded a Cu(II)–Cu(II) distance of 1.97 nm. The experimental distance is very reasonable when compared to the model given the uncertainty when calculating polypeptide structures. DQC-ESR is a rapidly evolving technique and its application to paramagnetic metals centers requires further improvements in technology. This would include the use
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of shorter pulses to improve bandwidth. In addition, characteristic ESEEM signals interfere with the clean observation of peaks from the electron–electron dipolar interactions, especially for larger distances. Methods of ESEEM suppression must be implemented to reduce such interferences. We are, however, encouraged by two observations. Despite the large spectral width of the Cu(II) spectrum, we demonstrate that DQC can be generated and distances in the nm lengthscale directly measured. Secondly, the DQC spectrum with a high SNR (80) can be routinely measured. 4. Conclusion Double quantum coherences can be generated in Cu(II)– Cu(II) systems despite the large spectral extent of the Cu(II)-ESR spectrum. Using a single Cu(II) binding unit and a six-pulse experiment to selectively measure ESEEM as controls, the peak due to the electron–electron dipolar frequency can be identified in the DQC spectrum. The measured distance of 2.0 nm is in good agreement with molecular models for this peptide. We thus demonstrate the ability to measure Cu(II)–Cu(II) distances on the nanometer scale using double quantum coherence ESR. We anticipate that DQC-ESR will be an important tool for the determination of structure–function relationships in metalloproteins. Acknowledgments We acknowledge support for this work from a University of Pittsburgh start-up grant and the NSF CAREER Award (MCB 0346898). References [1] S. Saxena, J.H. Freed, J. Chem. Phys. 107 (1997) 1317. [2] P.P. Borbat, J.H. Freed, Chem. Phys. Lett. 313 (1999) 145. [3] P.P. Borbat, J.H. Freed, Double-Quantum ESR and Distance Measurement, Biological Magnetic Resonance, Kluwer Academics/ Plenum Publishers, New York, 2000. [4] P.P. Borbat, A.J. Costa-Filho, K.A. Earle, J.K. Moscicki, J.H. Freed, Science 291 (2001) 266. [5] M. Bonora, J. Becker, S. Saxena, J. Magn. Reson. 170 (2004) 278. [6] A.D. Milov, A.B. Ponomerev, Y.D. Tsvetkov, Chem. Phys. Lett. 110 (1984) 67. [7] R.G. Larson, D.J. Singel, J. Chem. Phys. 98 (1993) 5134. [8] A.D. Milov, A.G. Maryasev, Y.D. Tsvetkov, Appl. Magn. Reson. 15 (1998) 107. [9] M. Pannier, S. Veit, A. Godt, G. Jeschke, H.W. Spiess, J. Magn. Reson. 142 (2000) 331. [10] G. Jeschke, ChemPhysChem 3 (2002) 927. [11] G. Jeschke, A. Bender, H. Paulsen, H. Zimmermann, A. Godt, J. Magn. Reson. 169 (2004) 1. [12] M. Persson, J.R. Harbridge, P. Hammarstrom, R. Mitri, L.G. Martensson, U. Carlsson, G.R. Eaton, S.S. Eaton, Biophys. J. 80 (2001) 2886. [13] P.P. Borbat, H.S. Mchourab, J.H. Freed, J. Am. Chem. Soc. 124 (2002) 5304. [14] M. Bennati, A. Weber, J. Antonic, D.L. Perlstein, J. Robblee, J. Stubbe, J. Am. Chem. Soc. 125 (2003) 14988.
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[15] G. Jeschke, C. Wegener, M. Nietschke, H. Jung, H.-J. Steinhoff, Biophys. J. 86 (2004) 2551. [16] G. Jeschke, A. Bender, T. Schweikardt, G. Panek, H. Decker, H. Paulsen, J. Biol. Chem. 280 (2005) 18623. [17] O. Schiemann, A. Weber, T.E. Edwards, T.F. Prisner, S.T. Sigurdsson, J. Am. Chem. Soc. 125 (2003) 3434. [18] P.P. Borbat, J.H. Davis, S.E. Butcher, J.H. Freed, J. Am. Chem. Soc. 126 (2004) 7746. [19] O. Schiemann, N. Piton, Y. Mu, G. Stock, J.W. Engels, T.F. Prisner, J. Am. Chem. Soc. 126 (2004) 5722. [20] M. Pannier, V. Schadler, M. Schops, U. Wiesner, G. Jeschke, H.W. Spiess, Macromolecules 33 (2000) 7812. [21] G. Jeschke, Macromol. Rapid Commun. 23 (2002) 227. [22] D. Hinderberger, O. Schmelz, M. Rehahn, G. Jeschke, Angew. Chem. Intl. Ed. 43 (2004) 4616. [23] D. Hinderberger, H.W. Spiess, G. Jeschke, J. Phys. Chem. B 108 (2004) 3698. [24] A.D. Milov, A.G. Maryasov, Y.D. Tsvetkov, J. Raap, Chem. Phys. Lett. 303 (1999) 135. [25] A.D. Milov, Y.D. Tsvetkov, F. Formaggio, M. Crisma, C. Toniolo, J. Raap, J. Am. Chem. Soc. 123 (2001) 3784.
[26] A.D. Milov, Y.D. Tsvetkov, F. Formaggio, S. Oancea, C. Toniolo, J. Raap, J. Phys. Chem. A 107 (2003) 13719. [27] B.G. Dzikovski, P.P. Borbat, J.H. Freed, Biophys. J. 87 (2004) 3504. [28] C. Elsasser, M. Brecht, R. Bittl, J. Am. Chem. Soc. 124 (2002) 12606. [29] I.M.C.v. Amsterdam, M. Ubbink, G.W. Canters, M. Huber, Angew. Chem. Int. Ed. 42 (2003) 62. [30] S.A. Dikanov, Y.D. Tsvetkov, Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy, CRC Press, Boca Raton, FL, 1992. [31] E. Aronoff-Spencer, C.S. Burns, N.I. Avdievich, G.J. Gerfen, J. Peisach, W.E. Antholine, H.L. Ball, F.E. Cohen, S.B. Prusiner, G.L. Millhauser, Biochemistry 39 (2000) 13760. [32] C.S. Burns, E. Aronoff-Spencer, C.M. Dunham, P. Lario, N.I. Avdievich, W.E. Antholine, M.M. Olmstead, A. Vrielink, G.J. Gerfen, J. Peisach, W.G. Scott, G.L. Millhauser, Biochemistry 41 (2002) 3991. [33] G.L. Millhauser, Acc. Chem. Res. 37 (2004) 79. [34] A. Schweiger, G. Jeschke, Principles of Pulse Electron Paramagnetic Resonance, Oxford University Press, New York, 2001. [35] S. Saxena, J.H. Freed, J. Magn. Reson. 124 (1997) 439.
Double quantum coherence electron spin resonance on coupled Cu(II)–Cu(II) electron spins James S. Becker, Sunil Saxena
*
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, United States Received 30 June 2005; in final form 6 August 2005 Available online 16 September 2005
Abstract We demonstrate for the first time the ability to generate double quantum coherences (DQCs) for the case of Cu(II). We show that small splittings (7 MHz) from the Cu(II)–Cu(II) electron–electron magnetic dipolar interaction can be reliably resolved even though the inhomogeneously broadened Cu(II) linewidth is 2 GHz. A Cu(II)–Cu(II) distance of 2.0 nm was measured on a model peptide system, thus, demonstrating that distances on the nanometer scale may be measured using DQC electron spin resonance (ESR). 2005 Elsevier B.V. All rights reserved.
1. Introduction The need for the determination of the structures of biomolecules and nanostructured materials has provided an impetus for development of new methods in electron spin resonance (ESR). Significant research involving concepts of double quantum coherences [1–5] or double resonance [6–11] have resulted in robust methods to directly measure the magnetic dipolar interaction between two electron spins on a macromolecule. This has made it possible to measure ˚ range between two interspin distances in the 16–75 A spin-labels in order to determine global folding patterns in proteins [12–16], nucleic acids [17–19], and ionic polymers [20–23], and conformational and aggregation states of polypeptides [24–27]. Thus far, the ESR method has largely been restricted to the use of nitroxides as spin labels. Recently, this methodology has been extended to the case of paramagnetic metal centers in metalloproteins using double electron–electron resonance (DEER) ESR [28,29]. In these experiments, the local dipolar field due to the coupled spin-partner is inverted using pulses of durations that are P10 ns. The primary echo is then modulated by the dipolar frequency providing sensitivity to distances. The *
Corresponding author. Fax: +1 412 624 8611. E-mail address: [email protected] (S. Saxena).
0009-2614/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2005.08.072
use of large pulse lengths necessarily leads to a reduced signal-to-noise ratio, and the orientational selectivity of the pulses can potentially complicate the analysis of the spectrum to measure distances. In addition, the use of selective pulses makes the measurement of small distances (ca. ˚ ) difficult. 8–15 A In principle, double quantum coherence (DQC)-ESR can circumvent these limitations. This has motivated us to explore the possibility of generating DQCs for the case Cu(II) binding systems. In DQC-ESR, spin–spin interactions generate the double quantum coherence. The rate of formation of the DQC directly reports on the strength of the dipolar interaction, providing sensitivity to distances. The generation of DQCs for the case of Cu(II) poses daunting challenges. The spectral extent of Cu(II) ESR can be as large as 2 GHz – whereas the dipolar interaction is weak (MHz to kHz) for distances of typical interest. On the other hand, DQC methodology optimally requires the use of non-selective pulses, which for the case of Cu(II) is prohibitively difficult. In addition, Cu(II) spin-echoes of biological systems typically experience deep electron spin echo envelope modulations (ESEEM) because of the electron–nuclear interaction with the nitrogen nuclei in the amino-acid coordination environment [30]. The resulting ESEEM peaks can interfere with DQC spectra.
J.S. Becker, S. Saxena / Chemical Physics Letters 414 (2005) 248–252
Despite these challenges, we have been successful in detecting a DQC-ESR spectrum for the case of Cu(II). In this work, we show that small splittings (7 MHz) from the Cu(II)–Cu(II) electron–electron magnetic dipolar interaction can be reliably resolved. 2. Experimental Two Cu(II) binding peptide samples, synthesized at the University of PittsburghÕs Molecular Medicine Institute, were used for the experiments, one a dimer and the other a Cu(II) complex. The Cu(II)–Cu(II) dimer, Ac-PPHGGGWPPPHGGGWPP-NH2 will be called Cu3P (Fig. 1a), and the control sample, designated as Pr, had the aminoacid sequence, Ac-PHGGGW-NH2. The HGGGW aminoacid sequence, displayed as a crystal structure in Fig. 1b, is a well-characterized Cu(II) binding unit of the prion protein [31–33]. A 3.5 mM solution of Cu3P was prepared by dissolving the peptide in a solvent consisting of 30% glycerol/30% 2,2,2-trifluoroethanol/40% water containing 150 mM NaCl and buffered with 25 mM N-ethylmorpholine to pH 7.35. Two mole equivalents of Cu(II) were added from an aqueous 1 M copper sulfate solution. For the control sample, a 4.0 mM solution was prepared with a solvent of 30% glycerol/70% water containing 150 mM NaCl and buffered with 25 mM N-ethylmorpholine to pH 7.74. One mole equivalent of Cu(II) was added from a 0.1 M aqueous copper sulfate solution. For the ESR experiments, the samples were bath sonicated for approximately 5 min before flash freezing in liquid nitrogen. After insertion into the ESR cavity, the temperature was stabilized to 8.0 ± 0.2 K using an Oxford ITC605 temperature controller and an Oxford ER 4118CF gas flow cryostat. Spectroscopic experiments were performed on a Bruker EleXsys E580 X-band CW/Pulse ESR spectrometer equipped with a Bruker ER4118X-MS2 split-ring resonator. The high output power of the ASE-TWTA (1 kW) combined with a quality factor much less than 100 provided a p/2 pulse with a length of 4 ns.
Fig. 1. (a) The dimer, Cu3P, is displayed from the CAChe model (cf. Section 3). Hydrogens are not shown in the model and the Cu(II) ions appear as large spheres connected by the interspin vector, r. (b) The crystal structure of the HGGGW copper binding unit reproduced with permission from http://chemistry.ucsc.edu/~glennm/ is shown [33].
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The six-pulse DQC-ESR sequence, shown in Fig. 2a, was implemented in the following manner. A 256 step phase cycle was used to select the correct coherence pathway [3] and the DQC-echo after the sixth pulse was integrated. The pulse lengths were 4 ns for p/2 pulses and 8 ns for p pulses. The initial delays of tp and t1 were 12 ns and the delay of t2 was 120 ns for Cu3P and 76 ns for Pr. The time, tp, was stepped by 10 ns for a total of 128 points. For each step 900 averages were collected at a repetition rate of 200 Hz for the dimer and 800 averages were collected at the same rate for the control. Three-pulse ESEEM experiments [30,34] were carried out with p/2 pulses of 16 ns length. The pulse sequence is displayed in Fig. 2b. The initial inter-pulse separations were s = 150 ns and T = 40 ns. The delay T was stepped out by 16 ns for a total of 256 data points. A total of 50 averages per point were collected with a repetition rate of 200 Hz. The ESR spectra were obtained from the time domain data as described previously [5]. 3. Results and discussion The field-swept echo-detected Cu(II) absorption spectrum is shown in Fig. 3a. The total width of the spectrum is approximately 750 G or about 2100 MHz. A magnetic field of 3250 G, corresponding to the g^ region of Cu(II) spectrum (shown by an arrow in Fig. 3a), was used for DQC experiments. The continuous wave ESR spectrum was simulated using the Bruker Simfonia program and the g-value was determined to be 2.1166. The creation of DQC depends on the coverage of the pulse, which can be limited by the pulse length as well as by the bandwidth of the resonator. To estimate the coverage, the echo signal from a nutation experiment on a standard nitroxide sample was collected at 8 K. The nutation frequency as a function of microwave frequency is shown in Fig. 3b. The nutation frequency is nearly constant over 250 MHz. Due to the large width of the nitroxide spectrum, this number is an overestimate of spectral coverage, but still provides a useful point of reference for the approximate coverage and center frequency. The maximal intensity of the Cu(II) spectrum resides in the g^ region of the absorption spectrum (cf. Fig. 3a). Thus, in terms of intensity a pulse with 250 MHz coverage applied on the g^ re-
Fig. 2. (a) The double quantum coherence six-pulse sequence is displayed. The pulse separation, tp, is stepped out in order to observe echo modulation in the DQC experiment. For the six-pulse ESEEM experiment tp is constant and t2 is stepped out. (b) The three-pulse ESEEM pulse sequence is shown.
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Fig. 3. (a) The field-swept echo-detected spectrum of Cu3P is shown. An arrow denotes the magnetic field position for the DQC experiments, and the gi and g^ regions of the absorption spectrum are indicated for clarity. (b) The nutation frequency as a function of the spectrometer frequency is displayed. This experiment provides an estimate for the spectral coverage and center spectrometer frequency.
gion can irradiate a substantial part of the Cu(II) absorption spectrum. The DQC spectrum of the Cu(II)-dimer is displayed in Fig. 4a. The time domain DQC signal is given by Saxena and Freed [1] V ðtp ; t2 Þ ¼ sinð2pm1;2 tp Þ sinð2pm1;2 t2 Þektp ; ð1Þ where tp and t2 are the pulse delays described above (cf. Section 2.1), k is the relaxation rate of the DQC spin-echo, and m1,2 is m1;2 ¼
l0 g1 g2 b2 ð3cos2 h 1Þ þ 2J : 4phr3
ð2Þ
In Eq. (2), g1 and g2 are the g-factors of each electron, b is the Bohr magneton, h is PlanckÕs constant, r is the interspin vector, and h is the angle between magnetic field and the interspin vector. J is the exchange interaction, which for
Fig. 4. (a) The DQC spectrum of the dimer is shown with asterisks designating the 7.4 MHz electron–electron dipolar coupling peak. (b) The three-pulse ESEEM spectrum of Cu3P is displayed. (c) The control, Pr, DQC spectrum is displayed and the asterisks mark the absence of electron–electron dipolar splittings. (d) The spectrum of the six-pulse ESEEM experiment is shown with asterisks again marking the lack of peaks in the appropriate frequency range.
this sample is negligible due to the large separation between the Cu(II) centers. For frozen samples and for truly non-selective pulses a ÔPakeÕ pattern [3] is obtained in which the characteristic turning points corresponding to the parallel and perpendicular orientations (i.e., h = 0 and h = 90 in Eq. (2)) of the interspin vectors with respect to the dc-magnetic field are readily observable. The h = 0 turning point has much lower intensity and, therefore, for weaker pulses only the h = 90 turning point is obtained in the DQC spectrum [3]. In addition to this peak from the electron–electron dipolar interaction, ESEEM peaks due to hyperfine and nuclear quadrupolar interactions are also obtained. Many peaks are found upon immediate inspection of the Cu(II)-dimer DQC-spectrum (cf. Fig. 4a), several of which are ESEEM peaks. The most easily identified is at the proton Larmor frequency at ±14.0 MHz due to the coupling of the Cu(II) electron spin with the neighboring protons. Peaks found at frequencies less than 4.8 MHz (i.e., 4.8 and 2.0 MHz) are well characterized as ESEEM peaks due to hyperfine interactions with and quadrupolar interactions of 14N nuclei. Specifically, peaks arise from the remote, uncoordinated nitrogen of the imidazole ring and, also, from the uncoordinated amide nitrogen in the glycine backbone [31,32]. There is significant overlap between adjacent peaks because magnitude peaks decay slowly with frequency [35]. Furthermore, low frequency ESEEM peaks are not recorded because the decay of the DQC spin-echo was prohibitively fast (hundreds of ns). A single peak at ±7.4 MHz, denoted by an asterisk in Fig. 4a, remains unidentified in the DQC spectrum. We attribute the peak being due to the intramolecular electron–electron dipolar interaction between the two Cu(II)– Cu(II) electron spins This peak assignment is supported by data from three control experiments. Firstly, a three-pulse ESEEM experiment (Fig. 4b) was performed on the dimer as a control experiment to further support our conclusions. The three-pulse ESEEM spectrum clearly displays the effects of electron–nuclear couplings between the Cu(II) electron spin and neighboring
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nuclei (i.e., nitrogen and hydrogen). The data serve two important purposes for this work. There is a conspicuous lack of peaks between the proton modulation occurring at ±14.2 MHz, and the 14N nuclear double quantum transition at ±4.6 MHz, thus, supporting our assignment of the ±7.4 MHz peak in the DQC spectrum (cf. Fig. 4a) as being due to the electron–electron dipolar interaction. It also proves that the Cu(II) is coordinated in the same environment as determined in the literature [31–33]. It must also be clarified that the proton modulation peak at ±14.0 MHz is greatly attenuated because of the proton blind-spot at this s value [34]. Secondly, in Fig. 4c, we show the DQC spectrum of the Cu(II) complex, Pr. The DQC signal of Pr originates from intermolecular dipolar interaction and leads to a decay modulated by ESEEM [3]. A peak at ±7.4 MHz is not obtained, thus, eliminating uncertainty in the nature and origin of the ±7.4 MHz peak in the Cu(II)-dimer DQC spectrum. Comparison of the two spectra offers direct evidence supporting the assignment of dipole–dipole coupling between the two electron spins. Finally, to be certain the ±7.4 MHz peak is not an artifact of unknown origin; a six-pulse control experiment was created in order to generate only an ESEEM pattern from this sequence. In this experiment, the pulse sequence in Fig. 2a was used, but tp was held constant at 12 ns, and only the last pulse was stepped out by 10 ns. In this way, the p/2–p–p/2 DQC generator sequence remains active to form the DQC, but the electron–electron dipolar coupling is not resolved because the pulse separation, tp, is constant in time. A spectrum is obtained that has similar signal-tonoise ratio and the same nuclear modulations, but lacks the intramolecular dipole–dipole coupling modulation inherent to the proper DQC time-domain signal. The FT spectrum of the signal obtained from the Cu(II)-dimer is shown in Fig. 4d. The spectrum contains the expected ESEEM peaks (cf. Fig. 4a), but is devoid of peaks between ±14.2 and ±4.8 MHz. We, therefore, justify our assignment of the ±7.4 MHz to an intramolecular electron–electron dipolar interaction. By using a calculated g-value of 2.1166 and assuming h = p/2 in the point dipole approximation, a frequency of 7.4 MHz yields an experimental distance of 2.0 nm. The Cu3P molecule was modeled using CAChe Software (Fujitsu) (cf. Fig. 1a) by first creating a representation of the HGGGW crystal structure determined by Millhauser and coworkers [32]. The segment was reproduced to make two copies and the proline residues were covalently attached to the copper binding units. Energy minimization was performed using the MM3 force field and this yielded a Cu(II)–Cu(II) distance of 1.97 nm. The experimental distance is very reasonable when compared to the model given the uncertainty when calculating polypeptide structures. DQC-ESR is a rapidly evolving technique and its application to paramagnetic metals centers requires further improvements in technology. This would include the use
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of shorter pulses to improve bandwidth. In addition, characteristic ESEEM signals interfere with the clean observation of peaks from the electron–electron dipolar interactions, especially for larger distances. Methods of ESEEM suppression must be implemented to reduce such interferences. We are, however, encouraged by two observations. Despite the large spectral width of the Cu(II) spectrum, we demonstrate that DQC can be generated and distances in the nm lengthscale directly measured. Secondly, the DQC spectrum with a high SNR (80) can be routinely measured. 4. Conclusion Double quantum coherences can be generated in Cu(II)– Cu(II) systems despite the large spectral extent of the Cu(II)-ESR spectrum. Using a single Cu(II) binding unit and a six-pulse experiment to selectively measure ESEEM as controls, the peak due to the electron–electron dipolar frequency can be identified in the DQC spectrum. The measured distance of 2.0 nm is in good agreement with molecular models for this peptide. We thus demonstrate the ability to measure Cu(II)–Cu(II) distances on the nanometer scale using double quantum coherence ESR. We anticipate that DQC-ESR will be an important tool for the determination of structure–function relationships in metalloproteins. Acknowledgments We acknowledge support for this work from a University of Pittsburgh start-up grant and the NSF CAREER Award (MCB 0346898). References [1] S. Saxena, J.H. Freed, J. Chem. Phys. 107 (1997) 1317. [2] P.P. Borbat, J.H. Freed, Chem. Phys. Lett. 313 (1999) 145. [3] P.P. Borbat, J.H. Freed, Double-Quantum ESR and Distance Measurement, Biological Magnetic Resonance, Kluwer Academics/ Plenum Publishers, New York, 2000. [4] P.P. Borbat, A.J. Costa-Filho, K.A. Earle, J.K. Moscicki, J.H. Freed, Science 291 (2001) 266. [5] M. Bonora, J. Becker, S. Saxena, J. Magn. Reson. 170 (2004) 278. [6] A.D. Milov, A.B. Ponomerev, Y.D. Tsvetkov, Chem. Phys. Lett. 110 (1984) 67. [7] R.G. Larson, D.J. Singel, J. Chem. Phys. 98 (1993) 5134. [8] A.D. Milov, A.G. Maryasev, Y.D. Tsvetkov, Appl. Magn. Reson. 15 (1998) 107. [9] M. Pannier, S. Veit, A. Godt, G. Jeschke, H.W. Spiess, J. Magn. Reson. 142 (2000) 331. [10] G. Jeschke, ChemPhysChem 3 (2002) 927. [11] G. Jeschke, A. Bender, H. Paulsen, H. Zimmermann, A. Godt, J. Magn. Reson. 169 (2004) 1. [12] M. Persson, J.R. Harbridge, P. Hammarstrom, R. Mitri, L.G. Martensson, U. Carlsson, G.R. Eaton, S.S. Eaton, Biophys. J. 80 (2001) 2886. [13] P.P. Borbat, H.S. Mchourab, J.H. Freed, J. Am. Chem. Soc. 124 (2002) 5304. [14] M. Bennati, A. Weber, J. Antonic, D.L. Perlstein, J. Robblee, J. Stubbe, J. Am. Chem. Soc. 125 (2003) 14988.
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[15] G. Jeschke, C. Wegener, M. Nietschke, H. Jung, H.-J. Steinhoff, Biophys. J. 86 (2004) 2551. [16] G. Jeschke, A. Bender, T. Schweikardt, G. Panek, H. Decker, H. Paulsen, J. Biol. Chem. 280 (2005) 18623. [17] O. Schiemann, A. Weber, T.E. Edwards, T.F. Prisner, S.T. Sigurdsson, J. Am. Chem. Soc. 125 (2003) 3434. [18] P.P. Borbat, J.H. Davis, S.E. Butcher, J.H. Freed, J. Am. Chem. Soc. 126 (2004) 7746. [19] O. Schiemann, N. Piton, Y. Mu, G. Stock, J.W. Engels, T.F. Prisner, J. Am. Chem. Soc. 126 (2004) 5722. [20] M. Pannier, V. Schadler, M. Schops, U. Wiesner, G. Jeschke, H.W. Spiess, Macromolecules 33 (2000) 7812. [21] G. Jeschke, Macromol. Rapid Commun. 23 (2002) 227. [22] D. Hinderberger, O. Schmelz, M. Rehahn, G. Jeschke, Angew. Chem. Intl. Ed. 43 (2004) 4616. [23] D. Hinderberger, H.W. Spiess, G. Jeschke, J. Phys. Chem. B 108 (2004) 3698. [24] A.D. Milov, A.G. Maryasov, Y.D. Tsvetkov, J. Raap, Chem. Phys. Lett. 303 (1999) 135. [25] A.D. Milov, Y.D. Tsvetkov, F. Formaggio, M. Crisma, C. Toniolo, J. Raap, J. Am. Chem. Soc. 123 (2001) 3784.
[26] A.D. Milov, Y.D. Tsvetkov, F. Formaggio, S. Oancea, C. Toniolo, J. Raap, J. Phys. Chem. A 107 (2003) 13719. [27] B.G. Dzikovski, P.P. Borbat, J.H. Freed, Biophys. J. 87 (2004) 3504. [28] C. Elsasser, M. Brecht, R. Bittl, J. Am. Chem. Soc. 124 (2002) 12606. [29] I.M.C.v. Amsterdam, M. Ubbink, G.W. Canters, M. Huber, Angew. Chem. Int. Ed. 42 (2003) 62. [30] S.A. Dikanov, Y.D. Tsvetkov, Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy, CRC Press, Boca Raton, FL, 1992. [31] E. Aronoff-Spencer, C.S. Burns, N.I. Avdievich, G.J. Gerfen, J. Peisach, W.E. Antholine, H.L. Ball, F.E. Cohen, S.B. Prusiner, G.L. Millhauser, Biochemistry 39 (2000) 13760. [32] C.S. Burns, E. Aronoff-Spencer, C.M. Dunham, P. Lario, N.I. Avdievich, W.E. Antholine, M.M. Olmstead, A. Vrielink, G.J. Gerfen, J. Peisach, W.G. Scott, G.L. Millhauser, Biochemistry 41 (2002) 3991. [33] G.L. Millhauser, Acc. Chem. Res. 37 (2004) 79. [34] A. Schweiger, G. Jeschke, Principles of Pulse Electron Paramagnetic Resonance, Oxford University Press, New York, 2001. [35] S. Saxena, J.H. Freed, J. Magn. Reson. 124 (1997) 439.