Genetic Code Expansion for Site-Specific Encoding of a Switchable, Intrinsic Fluorophore-Quencher Pair to Monitor Protein Dynamics

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 measu