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

• Published in: Journal of molecular biology 2022-04, Vol.434 (8), p.167455-167455, Article 167455
• Main Authors: Henderson, J.Nathan, Simmons, Chad R., Mills, Jeremy H.
• Format: Article
• Language: English
• Subjects: Biosensing Techniques; Crystallography, X-Ray; Fluorescent Dyes - chemistry; fluorescent proteins; Glycine - analogs & derivatives; Glycine - chemistry; Glycine - genetics; Immunoglobulin Fragments - chemistry; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; non-canonical amino acids; Protons; Spectrometry, Fluorescence; Umbelliferones - chemistry; X-ray crystallography
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2022.167455

[Display omitted] •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. The fluorescent non-canonical amino acid (fNCAA) L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) contains a photoacidic 7-hydroxycoumarin (7-HC) side chain whose fluorescence properties can be tuned by its environment. In proteins, many alterations to 7-HCAA’s fluorescence spectra have been reported including increases and decreases in intensity and red- and blue-shifted emission maxima. The ability to rationally design protein environments that alter 7-HCAA’s fluorescence properties in predictable ways could lead to novel protein-based sensors of biological function. However, these efforts are likely limited by a lack of structural characterization of 7-HCAA-containing proteins. Here, we report the steady-state spectroscopic and x-ray crystallographic characterization of a 7-HCAA-containing antibody fragment (in the apo and antigen-bound forms) in which a substantially blue-shifted 7-HCAA emission maximum (∼70 nm) is observed relative to the free amino acid. Our structural characterization of these proteins provides evidence that the blue shift is a consequence of the fact that excited state proton transfer (ESPT) from the 7-HC phenol has been almost completely blocked by interactions with the protein backbone. Furthermore, a direct interaction between a residue in the antigen and the fluorophore served to further block proton transfer relative to the apoprotein. The structural basis of the unprecedented blue shift in 7-HCAA emission reported here provides a framework for the development of new fluorescent protein-based sensors.