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Article Dans Une Revue Analytical Chemistry Année : 2020

The power of pure shift HαCα correlations: a way to characterize biomolecules under physiological conditions

Andrea Bodor
  • Fonction : Auteur
Jens D. Haller
  • Fonction : Auteur
Chafiaa Bouguechtouli
  • Fonction : Auteur
László Nyitray
Burkhard Luy
  • Fonction : Auteur

Résumé

Intrinsically disordered proteins (IDPs) constitute an important class of biomolecules with high flexibility. Atomic resolution studies for these molecules are essentially limited to NMR spectroscopy, which should be performed under physiological pH and temperature to populate relevant conformational ensembles. In this context, however, fundamental problems arise with established triple resonance NMR experiments: high solvent accessibility of IDPs promotes water-exchange, which disfavors classical amide 1H-detection, while 13C-detection suffers from significantly reduced sensitivity. A favorable alternative, the conventional detection of non-exchangeable 1Hα so far resulted in broad signals with insufficient resolution and sensitivity. To overcome this we introduce here a selective Hα,Cα-correlating pure shift detection scheme, the SHACA-HSQC, using extensive hetero- and homo-nuclear decoupling applicable to aqueous samples (≥ 90% H2O) and tested on small molecules and proteins. SHACA-HSQC spectra acquired on IDPs provide uncompromised resolution and sensitivity (up to 5-fold increased S/N compared to the standard 1H,13C-HSQC), as shown for resonance distinction and unambiguous assignment on the disordered transactivation domain of the tumorsuppressor p53, α-synuclein, and folded ubiquitin. The detection scheme can be implemented in any 1Hα-detected triple resonance experiment, but may also form the basis for the detection of isotope-labeled markers in biological studies or compound libraries.
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Dates et versions

hal-02928175 , version 1 (08-12-2020)

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Andrea Bodor, Jens D. Haller, Chafiaa Bouguechtouli, Francois-Xavier Theillet, László Nyitray, et al.. The power of pure shift HαCα correlations: a way to characterize biomolecules under physiological conditions. Analytical Chemistry, 2020, 92 (18), pp.12423-12428. ⟨10.1021/acs.analchem.0c02182⟩. ⟨hal-02928175⟩
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