Application of W-band<sup>19</sup>F electron nuclear double resonance (ENDOR) spectroscopy to distance measurement using a trityl spin probe and a fluorine label

Asanbaeva, Nargiz B.; Суханов, А. А.; Diveikina, Anastasiya A.; Rogozhnikova, Olga Yu.; Trukhin, Dmitry V.; Tormyshev, Victor M.; Chubarov, Alexey S.; Maryasov, Alexander G.; Genaev, A. M.; Shernyukov, Аndrey V.; Salnikov, Georgy E.; Lomzov, Alexander A.; Пышный, Д. В.; Bagryanskaya, Elena G.

doi:10.1039/d1cp05445g

Cited by 18 publications

(61 citation statements)

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“…6b are also partially suppressed and do not resolve the T > feature as would be expected for a single distance associated with a purely through-space dipolar coupling, again pointing to a significant isotropic contribution to the hyperfine interaction. This parallels the problems encountered by the authors of the recent publication on 19 F-ENDOR with a trityl radical paramagnetic spin tag, 21 which required more complicated computational modelling to validate the observed splittings. Whether interactions other than conjugation contribute to the splitting observed in the 19 F-ENDOR spectrum of the Gd 3+ - 19 F ruler is open to further investigation.…”

Section: F-endor Distance Measurementsmentioning

confidence: 62%

“…To date, reliable 19 F-ENDOR distance measurements with trityl tags have only been achieved in the range of 8-12 Å with model compounds. 21 As trityl radicals are large and feature significant delocalization of the spin density, this introduces considerable uncertainty into the distance measurement, in particular if the trityl radical is attached to the target molecule via a flexible tether. Furthermore, for a tether containing conjugated linkers, a significant isotropic (fermi contact) hyperfine interaction at inter-spin distances shorter than about 8 Å is observed, 21 which complicates distance determination.…”

Section: Introductionmentioning

confidence: 99%

“…400 mW, Dn = 60 MHz). In addition, the unpaired electrons in a Gd 3+ ion relax faster (their T 1 is up to 5-10 times shorter than for nitroxide 15,16 and trityl radicals 21 ), allowing data accumulation at lower temperatures than with nitroxides (10 K vs. 50 K), which further improves sensitivity. Like trityl labels, Gd 3+ tags are also free from orientation selection artefacts, as shown earlier for DEER spectroscopy, 22,23 i.e.…”

Section: Introductionmentioning

confidence: 99%

“…Bagryanskaya and coworkers investigated the use of triarylmethyl (trityl) radicals as alternative tags. 21 Trityl radicals possess narrow EPR lines due to low g -anisotropy, and thus the ENDOR spectrum can be collected at a single magnetic field position without needing to account for orientation selectivity. They also possess relatively long electronic phase-memory ( T M ) relaxation times (4–5 μs at 80 K), allowing the use of long delays and long RF pulses ( t RF ), as required to properly resolve small line splittings arising from longer distances.…”

Section: Introductionmentioning

confidence: 99%

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Short-range ENDOR distance measurements between Gd(iii) and trifluoromethyl labels in proteins

Judd

Abdelkader

et al. 2022

Phys. Chem. Chem. Phys.

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Section: F-endor Distance Measurementsmentioning

confidence: 62%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Short-range ENDOR distance measurements between Gd(iii) and trifluoromethyl labels in proteins

Judd

Abdelkader

et al. 2022

Phys. Chem. Chem. Phys.

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“…This means, that small hyperfine couplings may be investigated by the choice of very long preparation intervals (up to 4 µs). [14,60] The limiting factor for the choice of τ is however the phase memory time of the investigated PC, as the overall ENDOR sensitivity S ENDOR is a product of the ENDOR efficiency and the echo intensity determined by relaxation:…”

Section: Endormentioning

confidence: 99%

<sup>17</sup>O hyperfine spectroscopy to investigate water binding to organic radicals

Hecker¹

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Hyperfine spectroscopy experiments detect nuclear spins around paramagnetic centers. They reveal magnetic interactions that contain valuable structural information on distance and orientation of the coupling partners. One can therefore use them, to study the magnetic nuclei in water molecules around organic radicals. Such radicals occur as important intermediates in enzymatic reactions. 17 O is a particularly interesting target nucleus to detect water molecules, since it does not exchange with other oxygen moieties in protein environments, allowing for unambiguous assignment of spectral signatures to H 2 17 O. It has already been used to study water coordination around transition metal ions. The low gyromagnetic ratio and high nuclear spin have, however, discouraged the use of 17 O hyperfine spectroscopy to study water around organic radicals so far. This thesis shows the application of 17 O hyperfine spectroscopy to organic nitroxides and tyrosyl radicals. The Mims ENDOR experiment is used at 94 and 263 GHz to detect small, isotropic hyperfine couplings in a range of 0.5 − 0.7 MHz at three trapped tyrosyl intermediates in "active" complexes of E. coli ribonucleotide reductase. The sharp spectral features give the first direct experimental evidence of hydrogen-bound water molecules at the radical intermediates, which are part of a long range proton-coupled electron transfer chain across different subunits of the enzyme. Small theoretical models are used to link the observed hyperfine couplings to a well defined, in-plane coordination of the water molecules. Very small amounts of spin density (∼ 0.01 %) on the oxygen nucleus, facilitated by the hydrogen-bond, are enough to cause detectable hyperfine splitting in the spectra. The exquisite capability of very high-field ENDOR spectroscopy to produce narrow spectral lines is shown and rationalized, allowing a reevaluation of our previous models of the tyrosyl radical intermediate Y • 356 . The thesis then answers the previously open question: Which hyperfine spectroscopy experiment is best suited to study 17 O water around organic radicals ? The performance of three different types of hyperfine spectroscopy, recorded at 34 and 94 GHz EPR frequency, is compared for two nitroxide radicals as well as one tyrosyl radical. While all techniques iii detect 17 O signals, the HYSCORE experiments at 34 GHz best show the presence of large hyperfine couplings in the range of 1 − 8 MHz for the two nitroxide radicals. Mims ENDOR experiments at 94 GHz best reveal small, isotropic couplings of 0.6 − 0.8 MHz for the nitroxide radical with a five-membered ring as well as the tyrosyl radical. Theoretical models and molecular dynamics simulations are used to show that large, anisotropic 17 O couplings correspond to out-of-plane coordination while small isotropic couplings indicate in-plane coordination for all three radicals.All experiments performed in this thesis show that 17 O hyperfine spectroscopy is a well suited method to detect water molecules at organic radicals. The strong dep...

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Gd^III‐¹⁹F Distance Measurements for Proteins in Cells by Electron‐Nuclear Double Resonance

et al. 2023

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Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron‐electron resonance (DEER) technique can track proteins’ conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII‐19F Mims electron‐nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in‐cell ENDOR measurements, complemented with room temperature solution and in‐cell GdIII‐19F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin‐labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in‐cell derived GdIII‐19F distances were essentially identical and lie in the 1–1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIII and 19F regions in the cell.

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Application of W-band¹⁹F electron nuclear double resonance (ENDOR) spectroscopy to distance measurement using a trityl spin probe and a fluorine label

Abstract: Recently, Marina Bennati and coworkers (Angew. Chemie - Int. Ed., 2020, 59, 373–379., A. J. Magn. Reson., 2021, 333, 107091) proposed to use electron nuclear double resonance (ENDOR) spectroscopy in...

Cited by 18 publications

References 67 publications

Short-range ENDOR distance measurements between Gd(iii) and trifluoromethyl labels in proteins

Short-range ENDOR distance measurements between Gd(iii) and trifluoromethyl labels in proteins

<sup>17</sup>O hyperfine spectroscopy to investigate water binding to organic radicals

Gd^III‐¹⁹F Distance Measurements for Proteins in Cells by Electron‐Nuclear Double Resonance

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Application of W-band19F electron nuclear double resonance (ENDOR) spectroscopy to distance measurement using a trityl spin probe and a fluorine label

Abstract: Recently, Marina Bennati and coworkers (Angew. Chemie - Int. Ed., 2020, 59, 373–379., A. J. Magn. Reson., 2021, 333, 107091) proposed to use electron nuclear double resonance (ENDOR) spectroscopy in...

Cited by 18 publications

References 67 publications

Short-range ENDOR distance measurements between Gd(iii) and trifluoromethyl labels in proteins

Short-range ENDOR distance measurements between Gd(iii) and trifluoromethyl labels in proteins

<sup>17</sup>O hyperfine spectroscopy to investigate water binding to organic radicals

GdIII‐19F Distance Measurements for Proteins in Cells by Electron‐Nuclear Double Resonance

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Application of W-band¹⁹F electron nuclear double resonance (ENDOR) spectroscopy to distance measurement using a trityl spin probe and a fluorine label

Gd^III‐¹⁹F Distance Measurements for Proteins in Cells by Electron‐Nuclear Double Resonance