Pulsed dipolar spectroscopy distance measurements in biomacromolecules labeled with Gd(III) markers
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.
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.
References (86)
- et al.
Two-component signaling systems, Part B. Measuring distances by pulsed dipolar ESR spectroscopy: spin-labeled histidine kinases
Methods Enzymol.
(2007) - et al.
Distance measurements in the borderline region of applicability of CW EPR and DEER: a model study on a homologous series of spin-labelled peptides
J. Magn. Reson.
(2008) - et al.
Sensitivity enhancement in pulse EPR distance measurements
J. Magn. Reson.
(2004) - et al.
Spin pair geometry revealed by high-field DEER in the presence of conformational distributions
J. Magn. Reson.
(2007) - et al.
EPR Distance measurements in deuterated proteins
J. Magn. Reson.
(2010) - et al.
Distance between a native cofactor and a spin label in the reaction centre of Rhodobacter sphaeroides by a two-frequency pulsed electron paramagnetic resonance method and molecular dynamics simulations
J. Magn. Reson.
(2006) - et al.
Nanometer distance measurements in RNA using site-directed spin labeling
Biophys. J.
(2007) - et al.
Multiple-quantum ESR and distance measurements
Chem. Phys. Lett.
(1999) - et al.
Electron dipole–dipole interaction in ESEEM of nitroxide biradicals
Chem. Phys. Lett.
(2001) - et al.
A pulsed EPR method to determine distances between paramagnetic centers with strong spectral anisotropy and radicals: the dead-time free RIDME sequence
J. Magn. Reson.
(2009)
Dipolar spectroscopy and spin alignment in electron paramagnetic resonance
Chem. Phys. Lett.
Electron–electron double resonance in electron spin echo: model biradical systems and the sensitized photolysis of decalin
Chem. Phys. Lett.
Increased sensitivity and extended range of distance measurements in spin-labeled membrane proteins: Q-band double electron–electron resonance and nanoscale bilayers
Biophys. J.
HYSCORE and DEER with an upgraded 95 GHz pulse EPR spectrometer
J. Magn. Reson.
Distance measurements in model bis-Gd(III) complexes with flexible “bridge”. Emulation of biological molecules having flexible structure with Gd(III) labels attached
J. Magn. Reson.
Phase memory relaxation times of spin labels in human carbonic anhydrase II: pulsed EPR to determine spin label location
Biophys. Chem.
Electron spin echo decay kinetics of an ion track in β-irradiated frozen solution of sulfuric acid. Numerical simulation by the Monte Carlo method and experiment
Chem. Phys.
The determination of pair distance distributions by pulsed ESR using Tikhonov regularization
J. Magn. Reson.
Maximum entropy: a complement to Tikhonov regularization for determination of pair distance distributions by pulsed ESR
J. Magn. Reson.
Refocused primary echo: a zero dead time detection of the electron spin echo envelope modulation
J. Magn. Reson.
Pros and Cons of Pulse Dipolar ESR
EPR News Lett.
Measurement of large distances in biomolecules using double-quantum-filtered refocused electron-spin-echoes
J. Am. Chem. Soc.
Characterizing the structure and dynamics of folded oligomers: pulsed ESR studies of peptoid helices
Chem. Commun.
A scissors mechanism for stimulation of SNARE-mediated lipid mixing by cholesterol
PNAS
Protein structure determination using long-distance constraints from double-quantum coherence ESR: study of T4 lysozyme
J. Am. Chem. Soc.
Membrane-bound α-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles
J. Am. Chem. Soc.
Determination of the oligomeric states of human and rat monoamine oxidases in the outer mitochondrial membrane and octyl D-glucopyranoside micelles using pulsed dipolar electron spin resonance spectroscopy
Biochemistry
Distance measurements in the nanometer range by pulse EPR
ChemPhysChem
Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonane
PhysChemChemPhys
Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR
PNAS
Assessing oligomerization of membrane proteins by four-pulse DEER: pH-dependent dimerization of NhaA Na1/H1 antiporter of E. coli
Biophys. J.
Counting the monomers in nanometer-sized oligomers by pulsed electron–electron double resonance
J. Am. Chem. Soc.
Long-range distance determinations in biomacromolecules by EPR spectroscopy
Q. Rev. Biophys.
Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances
Nat. Protocols
A PELDOR-based nanometer distance ruler for oligonucleotides
J. Am. Chem. Soc.
Relative orientation of rigid nitroxides by PELDOR: beyond distance measurements in nucleic acids
Angew. Chem., Int. Ed.
Nanometer distance measurements on RNA using PELDOR
J. Am. Chem. Soc.
Long distance PELDOR measurements on the histone core
J. Am. Chem. Soc.
Probing the (H3–H4)2 histone tetramer structure using pulsed EPR spectroscopy combined with site directed spin labeling
Nucleic Acids Res.
PELDOR study of conformations of double-spin-labeled single- and double-stranded DNA with non-nucleotide inserts
Phys. Chem. Chem. Phys.
Double electron–electron resonance (DEER): a convenient method to probe DNA conformational changes
Angew. Chem., Int. Ed.
Distance distributions of end-labeled curved bispeptide oligomers by electron spin resonance
ACS Nano
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2017, Journal of Magnetic ResonanceCitation Excerpt :The most common spin labels for proteins are nitroxides, especially the methylthiosulfonate MTSSL. Additionally, DEER measurements have been demonstrated on molecules labeled with triarylmethyl-radicals [5] or with complexes of metal ions like Gd(III), Mn(II) and Cu(II) [6–15]. Gd(III)-based spin labels are of increasing importance due to their stability in reducing environments like the cell cytosol, holding promise towards in cell DEER measurements on proteins [16–19].