At the core of the interaction: Probing charged side chains in flexible protein regions with simultaneous nuclear magnetic resonance experiments.

Charged amino acid side chains are crucial mediators of biomolecular recognition, but their characterization by nuclear magnetic resonance (NMR) is often hindered by conformational and solvent exchange, particularly for arginine guanidinium groups. We present two complementary 13C-detected NMR strategies that exploit multiple acquisition schemes to simultaneously monitor positively and negatively charged residues. A "NMR by Ordered Acquisition using 1H detection (NOAH)"-based experiment combines CζNη-HDQC and SC-CACO experiments, allowing the simultaneous detection of arginine, aspartate, and glutamate side chains resonances. In parallel, the Multiple-Receiver (MR) strategy integrates CP-HISQC and CζNε-HSQC, allowing full assignment of the arginine guanidinium group. We apply this approach to study the interaction between the SARS-CoV-2 nucleocapsid N-terminal domain and the negatively charged glycosaminoglycan enoxaparin. The experiments provide information about flexible, charged side chains at the protein ligand interface. Together, NOAH and MR approaches provide a powerful framework for the high-resolution characterization of charged side chains and electrostatically driven interactions.