Dual fluorine tag incorporation into SARS-CoV-2 NNTD Protein for 19F NMR spectroscopy

Abstract

This master’s thesis describes efforts towards the development and application of a dual fluorine labeling strategy for 19F nuclear magnetic resonance (NMR) spectroscopy. Using the SARS-CoV-2 nucleocapsid N-terminal domain (NNTD) as a model, we combined two labeling techniques: site-specific incorporation of trifluoromethyl-phenylalanine (tfmF) at position Phe-28 and biosynthetic incorporation of 7-fluorotryptophan (7F-Trp) at all native tryptophan residues (Trp-14, Trp-70, Trp-94). High-efficiency labeling (>90%) was achieved in E. coli Rosetta DL3 using orthogonal tRNA/synthetase systems, confirmed by mass spectrometry and SDS-PAGE. ☐ Solution-state ¹⁹F NMR showed distinct and well-resolved signals for both tfmF (−14.6 ppm) and 7F-Trp (−52 to −57 ppm), indicating successful site-specific incorporation with minimal impact on protein structure. Moreover, the CF₃- tag provided approximately 9-fold greater signal sensitivity and can serve as an independent reporter on local structure in 19F NMR experiments. Crystallization and solid-state NMR data confirmed that the dual-labeled NNTD maintained its structural integrity, supporting its use for NMR experiments. ☐ This work lays the foundation for future NMR studies. The methodology is broadly applicable to other systems, offering a versatile framework for atomic-resolution structural biology in physiological contexts.