Giri, P.; Yarra, V.; Mathis, M.; Hurley, C.; Jones, C.; Eteme, O. N.; Hostetler, Z.; Cooley, R. B.; Kohli, R.; Mehl, R.; Petersson, E. J.

Precisely modifying proteins at multiple sites in their native, folded structures offers unique opportunities to answer molecular and cellular-level biological questions. Here, we present a genetic code expansion strategy for site-specific integration of a fluorophore-quencher pair comprising two non-canonical amino acids - acridonylalanine (Acd) and methyltetrazinyl phenylalanine (Tet) - into a protein expressed in E. coli. The Acd and Tet pair requires no post-translational labeling, and quenching can be switched off by biorthogonal or photochemical reactions of Tet for convenient internal control experiments. Mechanistic studies based on Stern-Volmer quenching, fluorescence lifetime measurements, and "proline ruler" peptides established the distance dependence of quenching. As proof-of-concept, we applied this strategy to study: 1) calmodulin, a calcium-sensing protein, 2) RecA, a DNA damage sensor in bacteria, and 3) LexA, a transcriptional repressor whose activation by RecA governs acquired antibiotic resistance in bacteria. Using these proteins, we demonstrate that dual Acd/Tet labeling provides molecular-level insights into protein dynamics, enables high-throughput drug screening, and advances tools for studying protein structure-function relationships.