Journal
ANALYTICAL CHEMISTRY
Volume 92, Issue 18, Pages 12423-12428Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.0c02182
Keywords
-
Categories
Funding
- National Research, Development and Innovation Office [NKFIH-1157-8/2019-DT]
- NKFI [K124900, K119359]
- Deutsche Forschungsgemeinschaft (DFG) [LU 835/11-1, LU 835/131]
- HGF program BIFTM [47.02.04]
- CNRS
- CEA-Saclay
- French National Research Agency [ANR-14-ACHN0015]
Ask authors/readers for more resources
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 H-1-detection, while C-13-detection suffers from significantly reduced sensitivity. A favorable alternative, the conventional detection of nonexchangeable H-1 alpha, so far resulted in broad signals with insufficient resolution and sensitivity. To overcome this, we introduce here a selective H alpha,C alpha-correlating pure shift detection scheme, the selective H alpha,C alpha-HSQC (SHACA-HSQC), using extensive hetero- and homonuclear 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 fivefold increased S/N compared to the standard H-1,C-13-HSQC), as shown for resonance distinction and unambiguous assignment on the disordered transactivation domain of the tumor suppressor p53, a-synuclein, and folded ubiquitin. The detection scheme can be implemented in any H-1 alpha-detected triple resonance experiment and may also form the basis for the detection of isotope-labeled markers in biological studies or compound libraries.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available