Pulsed dipolar spectroscopy distance measurements in biomacromolecules labeled with Gd(III) markers

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Abstract

This work demonstrates the feasibility of using Gd(III) tags for long-range Double Electron Electron Resonance (DEER) distance measurements in biomacromolecules. Double-stranded 14- base pair Gd(III)-DNA conjugates were synthesized and investigated at Ka band. For the longest Gd(III) tag the average distance and average deviation between Gd(III) ions determined from the DEER time domains was about 59 ± 12 Å. This result demonstrates that DEER measurements with Gd(III) tags can be routinely carried out for distances of at least 60 Å, and analysis indicates that distance measurements up to 100 Å are possible. Compared with commonly used nitroxide labels, Gd(III)-based labels will be most beneficial for the detection of distance variations in large biomacromolecules, with an emphasis on large scale changes in shape or distance. Tracking the folding/unfolding and domain interactions of proteins and the conformational changes in DNA are examples of such applications.

Graphical abstract

Gd(III)-labeled DNA conjugates were synthesized and studied by DEER to measure the intramolecular distance between the Gd(III) labels. The advantages of using Gd(III) spin labels for long-distance DEER measurements are discussed in detail. The distance distribution between the labels is shown in the background.

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Research highlights

► Gd(III)-labeled DNA conjugates are synthesized and investigated by DEER. ► The advantages of Gd(III) tags for long-distance DEER measurements are discussed. ► Gd(III)-specific optimizations of the DEER measurement protocol are suggested.

Introduction

Due to recent advances in spin labeling and the increasingly common use of pulsed dipolar spectroscopy (PDS) [1], [2], measuring distances between strategic points in biomacromolecules has become routine [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31]. Mainstream PDS is based on the measurement of the static dipolar interaction between paramagnetic centers, typically spin labels, by either double electron–electron resonance (DEER) or double quantum coherence (DQC) techniques [32], [33]. Occasionally, a variety of other electron paramagnetic resonance (EPR) techniques, such as two-pulse electron spin echo (ESE), relaxation-induced dipolar modulation enhancement (RIDME), and “2 + 1”[34], [35], [36], [37], [38], [39] have also been employed. A complete description of PDS, including its practical application and limitations, can be found in the recent literature [2], [3], [10], [18], [38], [39]. Nitroxide radical based labels (LNO) that have been attached to the biomacromolecule of interest either by site-directed spin labeling (in the case of proteins) or by chemical modifications (in the case of DNA and RNA) serve as conventional spin labels [22], [30], [31], [40], [41], [42].

Since PDS as a technique for distance mapping in biomacromolecules is now well established [3], [11], [20], [43], [44], [45], the current direction in its development has become centered on increasing the range of measurable distances, while simultaneously decreasing the amount of required sample and making the acquisition itself more robust. Until recently, the maximum distances (dmax) accessible by PDS were around 50–60 Å [3], [23], [24], [25]. In most experiments, the minimum concentration of spin labeled molecules was ∼0.1–0.2 mM, corresponding to a LNO concentration of ∼0.2–0.4 mM, and the acquisition time required to obtain quality time domain patterns (TDP) with reasonable signal to noise (S/N) ratios was as long as 10–20 h. Increasing the maximum measurable distance (which has already been realized) [3], [11], [18], requires a simultaneous increase of the measurement time intervals and a decrease in the concentration of labeled molecules. Although the second requirement is relaxed when the distances between labels are relatively constrained, for less rigid cases, a decrease in concentration is necessary to unambiguously disentangle intra- and interpair TDPs. Even a modest increase in distance, necessitating a decrease in concentration, results in significant signal amplitude loss. Because the acquisition time cannot be unlimited, the problem of signal loss must be addressed in some other way in order to measure larger distances. Recently explored methods to accomplish this objective have been based on the use of more sophisticated pulse sequences [4], complete protein deuteration [25], and on performing measurements in Q and W microwave (mw) bands [46], [47] instead of the more commonly used X or Ku bands. Of these, the most significant progress has resulted from the construction of a new W-band instrument with a non-resonant cavity that allows oversized samples [48]. As a result, the absolute and concentration sensitivities become independent of each other, in contrast to the measurements with standard instrumentation. This instrument has allowed to decrease the concentration of LNO to as low as 1 μM, while preserving a high absolute sensitivity due to the higher operational frequency, superior pulse parameters, and an oversized sample. At the average concentration of 1 μM, the contribution of the interpair dipolar interactions becomes practically negligible, and the maximum potentially measurable distances increase from ∼60 Å to ∼100 Å. Therefore, there is little doubt that with the new pulsed techniques and instrumentation described above, dmax for the standard nitroxide spin labels can be extended to ∼100 Å.

In pursuit of the same goal of increasing dmax, we have been developing a different approach based on new Gd(III)-based spin tags (LGd) [49] that have magnetic resonance properties quite different from those of LNO. Although the details of these differences will be discussed later, it is important to note now that the PDS measurements using LGd must be performed in the high magnetic field/high frequency mw bands, e.g., in the Ka/W mw bands (mw frequency (νmw) ∼30–90 GHz) in order to avoid complications caused by crystal field interactions (cfi) [50], [51], [52], [53]. Previously, we already established that LGd can be used to measure shorter intrapair distances as compared to LNO [49], and the utility of LGd for the intermediate distance range (30–40 Å) was recently demonstrated in the first DEER measurements of Gd-labeled proteins [54].

The techniques for attaching Gd(III) tags to biomacromolecules, while not yet routine, are gradually becoming more so. The covalent attachment of Gd(III)-dipicolinic acid chelates to the cysteinyl sulfurs of two proteins (p75ICD and τC14) has been reported [54], and a number of new methods have been published for labeling biomacromolecules with lanthanide tags for both in vivo and ex vivo fluorescence imaging and paramagnetic NMR spectroscopy [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65]. Here we report the first synthesis of LGd-DNA conjugates, which are similar to LNO-DNA, a biomacromolecular model previously developed for the specific purpose of probing intrapair distances [30], [31]. Specifically, we have synthesized the LGd-DNA conjugates having 14 base pairs, which should result in a distance in the range of 50–70 Å between the labels. Finally, the results of PDS (DEER) measurements will be presented within the context of the general application of using Gd(III) as a long distance marker in biomolecules.

Section snippets

Gd(III) complexes and oligonucleotide conjugates

Two different complexes, Gd538 and Gd595 (Fig. 1), were chosen as potential Gd(III) tags for further routine investigations of oligonucleotide conjugates. The Gd595 and Gd538 tags were synthesized following reported and modified procedures. While Gd538 has a slightly shorter distance from the Gd(III) ion to the DNA 5′ attachment point (see Fig. S1 of Supporting Information, SI) than Gd595, the latter has substantially weaker cfi, which should make it better suited for PDS measurements. Gd(III)

Echo detected EPR and primary ESE measurements

The echo detected EPR spectra of the Gd595- and Gd538-DNA duplexes shown in Fig. 2a and b are similar to those recorded for nonconjugated Gd595 and Gd538 in our previous work [49]. The appearance of the spectra is similar for all known Gd(III) ions. Specifically, they consist of a central narrow line due to the −1/2  1/2 transition that is superimposed on a broad background due to all other transitions. A comprehensive description the Gd(III) EPR spectra would require numerous independent

Conclusion

This work describes the first synthesis of Gd(III) chelates conjugated to oligonucleotides and the utility of using Gd(III) tags to perform long-range distance measurements with DEER. Furthermore, this work has established that such measurements can be routinely carried out for distances of at least 60 Å. It was also estimated that by means of minor instrumental modifications this limit could be increased to 85 Å, and possibly to about 100 Å. As a result, reliable measurements of conformations of

Acknowledgment

This research was supported by the Binational Science Foundation (USA-Israel, BSF#2006179) NIH 1R01 EB005866-01, NSF DBI-0139459, DBI-9604939, BIR-922443 and NIH S10RR020959. A.R. is very thankful to Prof. D. Goldfarb for stimulating discussions.

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