Four-dimensional NOE-NOE spectroscopy of SARS-CoV-2 Main Protease to facilitate resonance assignment and structural analysis

Abstract. Resonance assignment and structural studies of larger proteins by nuclear magnetic resonance (NMR) can be challenging when exchange broadening, multiple stable conformations, 1H back-exchange the fully deuterated chain pose problems. These difficulties arise for SARS-CoV-2 Main Protease, a homodimer 2 × 306 residues. We demonstrate that combination four-dimensional (4D) TROSY-NOESY-TROSY spectroscopy 4D NOESY-NOESY-TROSY provides an effective tool delineating 1H–1H dipolar relaxation network. In with detailed information obtained from prior X-ray crystallography work, such data are particularly useful extending validating assignments as well probing features.