7SEN

Crystal structure of Fab containing a fluorescent noncanonical amino acid with blocked excited state proton transfer

Summary for 7SEN

• Entry DOI: 10.2210/pdb7sen/pdb
• Descriptor: 5c8* Fab heavy chain, 5c8* Fab light chain (3 entities in total)
• Functional Keywords: fragment antigen binding (fab), noncanonical amino acid, 7-hydroxycoumarin, immune system
• Biological source: Homo sapiens; More
• Total number of polymer chains: 2
• Total formula weight: 48002.38

Authors

Henderson, J.N.,Mills, J.H.,Simmons, C.R. (deposition date: 2021-09-30, release date: 2022-02-02, Last modification date: 2024-10-16)

Primary citation

Henderson, J.N.,Simmons, C.R.,Mills, J.H.
Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent, Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment.
J.Mol.Biol., 434:167455-167455, 2022

PubMed Abstract: 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.

PubMed: 35033559
DOI: 10.1016/j.jmb.2022.167455

Experimental method

X-RAY DIFFRACTION (2.09 Å)