Dual fluorine tag incorporation into SARS-CoV-2 NNTD Protein for 19F NMR spectroscopy
| Author(s) | Pedro de Jesus Pereira, João | |
| Date Accessioned | 2025-05-27T18:25:23Z | |
| Date Available | 2025-05-27T18:25:23Z | |
| Publication Date | 2025 | |
| SWORD Update | 2025-05-19T01:03:27Z | |
| 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. | |
| Advisor | Polenova, Tatyana | |
| Degree | M.S. | |
| Department | University of Delaware, Department of Chemistry and Biochemistry | |
| Unique Identifier | 1522124149 | |
| URL | https://udspace.udel.edu/handle/19716/36260 | |
| Language | en | |
| Publisher | University of Delaware | |
| URI | https://www.proquest.com/pqdtlocal1006271/dissertations-theses/dual-fluorine-tag-incorporation-into-sars-cov-2-n/docview/3205377932/sem-2?accountid=10457 | |
| Keywords | Fluorine labeling | |
| Keywords | Tryptophan residues | |
| Keywords | Fluorotryptophan | |
| Title | Dual fluorine tag incorporation into SARS-CoV-2 NNTD Protein for 19F NMR spectroscopy | |
| Type | Thesis |