Power of Pure Shift HαCα Correlations: A Way to Characterize Biomolecules under Physiological Conditions

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.