Genetic Encoding of Fluorinated Analogues of Phenylalanine for¹⁹F NMR Spectroscopy: Detection of Conformational Heterogeneity in Flaviviral NS2B-NS3 Proteases

Substituting a single hydrogen atom in a protein by fluorine provides a probe for site-specific sensing by ¹⁹F nuclear magnetic resonance (NMR) spectroscopy with minimal impact on the properties of the protein. Genetic encoding systems are presented for five different fluorinated analogues of phenylalanine: 2-, 3-, 4-fluorophenylalanine, 2,6-difluorophenylalanine, and 3,5-difluorophenylalanine. The systems allow the installation of each of these amino acids with high fidelity during in vivo bacterial protein synthesis in response to an amber stop codon. The respective target proteins are obtained in high yield. At the site of Phe116 in different constructs of the dengue virus and Zika virus NS2B-NS3 proteases, the fluorinated phenylalanine analogues reveal evidence of significant conformational heterogeneity in ¹⁹F NMR spectra and demonstrate conformational dynamics. The availability of different ¹⁹F NMR probes allows discriminating between impacts arising from the fluorine atoms and the properties intrinsic to the protein.