Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent, Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment

•Structure of an Fab with a 7-hydroxycoumarin-containing non-canonical amino acid.•A protein environment blocks excited state proton transfer from 7-hydroxycoumarin.•Blue shifted emission from blocked proton transfer is hardly detectable by eye.•Structural results provide a framework for development of new protein-based sensors.