Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water

Kurzbach, Dennis; Canet, Estel; Flamm, Andrea G.; Jhajharia, Aditya; Weber, Emmanuelle M. M.; Konrat, Robert; Bodenhausen, Geoffrey

doi:10.1002/anie.201608903

Cited by 60 publications

(71 citation statements)

References 35 publications

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“…Signal enhancement through hyperpolarized solvents has recently been illustrated for intrinsically disordered proteins (IDPs) by Szekely et al . and by Kurzbach et al., albeit with somewhat limited resolution. Here, we expand this approach to high‐resolution NMR of folded proteins, for which hyperpolarization is more challenging but also more informative, as—in contrast to IDPs—the solvent‐mediated enhancement factor is strongly dependent on the position of a residue in the secondary and tertiary structures of the protein.…”

Section: Figurementioning

confidence: 90%

“…Hyperpolarized exchange (HYPEX) NMR is possible thanks to the unique combination of an 800 MHz spectrometer and a D‐DNP prototype connected via a magnetic tunnel. Our experimental strategy is the following: H 2 O containing 17.5 m m TEMPOL (4‐hydroxy‐2,2,6,6‐tetramethylpiperidin‐1‐oxyl) radicals is (i) mixed with 15 % v/v glycerol, and (ii) hyperpolarized at T DNP =1.2 K in a magnetic field B DNP =6.7 T, yielding proton polarization levels P ( 1 H)>80 %. Subsequently, the sample is dissolved by a burst of superheated D 2 O (180 °C, 1 MPa) and propelled within 1.3 s by helium gas at 0.7 MPa through a ca.…”

Section: Figurementioning

confidence: 99%

“…A volume of 200 μL of a 15 m m TEMPOL solution in a 0:85:0.15 v/v mixture of water and glycerol was vitrified in liquid helium. The glassy sample was positively hyperpolarized for 3 h at 1.2 K in a magnetic field of 6.7 T by saturation of the EPR spectrum at 187.7 GHz . Dissolution was achieved with a burst of 5 mL D 2 O at 180 °C (453 K) under 1 MPa.…”

Section: Figurementioning

confidence: 99%

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High‐Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents

Kadeřávek

Ferrage

Bodenhausen

et al. 2018

Chemistry A European J

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Hyperpolarized 2D exchange spectroscopy (HYPEX) to obtain high-resolution nuclear magnetic resonance (NMR) spectra of folded proteins under near-physiological conditions is reported. The technique is based on hyperpolarized water, which is prepared by dissolution dynamic nuclear polarization and mixed in situ in an NMR spectrometer with a protein in a physiological saline buffer at body temperature. Rapid exchange of labile protons with the hyperpolarized solvent, combined with cross-relaxation effects (NOEs), leads to boosted signal intensities for many amide H- N correlations in the protein ubiquitin. As the introduction of hyperpolarization to the target protein is mediated via the solvent, the method is applicable to a broad spectrum of target molecules.

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Section: Figurementioning

confidence: 90%

Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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High‐Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents

Kadeřávek

Ferrage

Bodenhausen

et al. 2018

Chemistry A European J

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“…[27] The former has recently been used for measuring exchange kinetics from hyperpolarized water in an intrinsically disordered protein. [28] In the present work, 2D NMR would be interesting for further characterization of the directly hyperpolarized polypeptide conformation, since the overlap in the 13 C NMR spectra prevents residue-specific assignments and structure determination. A tradeoff in the application of 2D NMR in this context may be the loss of the directly observable time dependence of signal intensities.…”

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confidence: 99%

Real‐Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

et al. 2017

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The kinase inhibitory domain of the cell cycle regulatory protein p27Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structure, including α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of 13C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. Analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.

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“…When hyperpolarization can be performed for protons, whose detection is intrinsically more sensitive than that of other spins such as carbon‐13, the gain in signal affords improving the resolution of obtained images. Recent developments show that hyperpolarization can also be transferred from water to the detected biomarkers . The observation timescales of the method can be adapted for the durations needed for diagnostic by carefully selecting the molecular regions for storing magnetization.…”

Section: Molecular Imaging: Early Detection For High Dose‐rate Effectsmentioning

confidence: 99%

Laser‐driven radiation: Biomarkers for molecular imaging of high dose‐rate effects

et al. 2019

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Recently developed short‐pulsed laser sources garner high dose‐rate beams such as energetic ions and electrons, x rays, and gamma rays. The biological effects of laser‐generated ion beams observed in recent studies are different from those triggered by radiation generated using classical accelerators or sources, and this difference can be used to develop new strategies for cancer radiotherapy. High‐power lasers can now deliver particles in doses of up to several Gy within nanoseconds. The fast interaction of laser‐generated particles with cells alters cell viability via distinct molecular pathways compared to traditional, prolonged radiation exposure. The emerging consensus of recent literature is that the differences are due to the timescales on which reactive molecules are generated and persist, in various forms. Suitable molecular markers have to be adopted to monitor radiation effects, addressing relevant endogenous molecules that are accessible for investigation by noninvasive procedures and enable translation to clinical imaging. High sensitivity has to be attained for imaging molecular biomarkers in cells and in vivo to follow radiation‐induced functional changes. Signal‐enhanced MRI biomarkers enriched with stable magnetic nuclear isotopes can be used to monitor radiation effects, as demonstrated recently by the use of dynamic nuclear polarization (DNP) for biomolecular observations in vivo. In this context, nanoparticles can also be used as radiation enhancers or biomarker carriers. The radiobiology‐relevant features of high dose‐rate secondary radiation generated using high‐power lasers and the importance of noninvasive biomarkers for real‐time monitoring the biological effects of radiation early on during radiation pulse sequences are discussed.

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Investigation of Intrinsically Disordered Proteins through Exchange with Hyperpolarized Water

Cited by 60 publications

References 35 publications

High‐Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents

High‐Resolution NMR of Folded Proteins in Hyperpolarized Physiological Solvents

Real‐Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy

Laser‐driven radiation: Biomarkers for molecular imaging of high dose‐rate effects

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