¹⁹F NMR study of proteins with parallel incorporation of multiple fluorinated aromatic amino acids

Although the parallel incorporation of fluorinated aromatic amino acids (FAAAs) into proteins has been explored since 2010, the corresponding ¹⁹F NMR investigation has been lacking to date. To assess the potential of the parallel incorporation of FAAAs in studies of protein structure, dynamics and interactions, we examine the protein yields, fluorine incorporation efficiencies, and ¹⁹F NMR spectra upon simultaneously incorporating FAAAs including 3-fluorotyrosine (3FY), 4-fluorophenylalanine (4FF) and 5-fluorotryptophan (5FW) into two model proteins: the structured protein GB1 domain (GB1) and the intrinsically disordered protein α-synuclein (α-syn). We found that the simultaneous incorporation of 3FY and 5FW achieved higher efficiency than combinations of 4FF and 5FW, 4FF and 3FY, or 3FY, 4FF and 5FW. Moreover, incorporating more types of FAAAs leads to a reduction in overall yield. The ¹⁹F spectra of 3FY, 4FF and 5FW residues in α-syn and GB1 exhibited good dispersion of chemical shifts. However, the GB1 spectrum showed complexity due to incomplete fluorination and interactions within aromatic cluster. This complexity could be significantly reduced by supplementing the medium with natural aromatic amino acids. To validate the advantage of ¹⁹F NMR spectrum, we use 3FY-4FF-5FW-F94W-α-syn to investigate the interaction between α-syn and sodium dodecyl sulphate (SDS) micelle. Our study demonstrates that ¹⁹F NMR spectroscopy of proteins with parallel incorporation of FAAAs provides a valuable tool for investigating protein conformation, dynamics and interactions.