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Here, we present a genetic code expansion strategy for site-specific integration of a fluorophore-quencher pair comprising two non-canonical amino acids\u2014acridonylalanine (Acd) and methyltetrazinyl phenylalanine (Tet) \u2014 into a protein expressed in\n                  <jats:italic>E. coli<\/jats:italic>\n                  . 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\u2013Volmer quenching, fluorescence lifetime measurements, and \u201cproline ruler\u201d 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. 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