Double quantum coherence electron spin resonance on coupled Cu(II)–Cu(II) electron spins
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], [2], [3], [4], [5] or double resonance [6], [7], [8], [9], [10], [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 interspin distances in the ∼16–75 Å range between two spin-labels in order to determine global folding patterns in proteins [12], [13], [14], [15], [16], nucleic acids [17], [18], [19], and ionic polymers [20], [21], [22], [23], and conformational and aggregation states of polypeptides [24], [25], [26], [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 ⩾10 ns. The primary echo is then modulated by the dipolar frequency providing sensitivity to distances. The 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. 8–15 Å) difficult.
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.
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.
Section snippets
Experimental
Two Cu(II) binding peptide samples, synthesized at the University of Pittsburgh’s 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 amino-acid sequence, Ac-PHGGGW-NH2. The HGGGW amino-acid sequence, displayed as a crystal structure in Fig. 1b, is a well-characterized Cu(II) binding unit of the prion protein [31]
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
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
Acknowledgments
We acknowledge support for this work from a University of Pittsburgh start-up grant and the NSF CAREER Award (MCB 0346898).
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