Qianzhu, HaochengAbdelkader, Elwy H.Otting, GottfriedHuber, Thomas2024-12-122024-12-120002-7863PubMed:38687675WOS:001227574300001ORCID:/0000-0002-5388-3949/work/206890848ORCID:/0000-0002-0563-0146/work/206892042ORCID:/0000-0002-7546-4411/work/206893824https://hdl.handle.net/1885/733730043The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by 19F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The 19F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.The authors thank Dr. Harpreet Vohra and Michael Devoy at the John Curtin School of Medical Research, Australian National University, for technical support on FACS experiments. Financial support by the Australian Research Council, including the Centre of Excellence for Innovations in Peptide and Protein Science (CE200100012) and a Discovery Project (DP230100079) is gratefully acknowledged.10en© 2024 American Chemical SocietyRelaxationFluorotryptophan incorporationNs2b-ns3 protease5-fluorotryptophanUnnatural amino-acidsRotational mobilityFluorescenceF-19 nmrEscherichia-coliProbe2024-04-3010.1021/jacs.4c0374385192077671