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.
Original language | English (US) |
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Article number | 167455 |
Journal | Journal of molecular biology |
Volume | 434 |
Issue number | 8 |
DOIs | |
State | Published - Apr 30 2022 |
Keywords
- X-ray crystallography
- fluorescent proteins
- non-canonical amino acids
ASJC Scopus subject areas
- Biophysics
- Structural Biology
- Molecular Biology
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Dive into the research topics of 'Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent, Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment'. Together they form a unique fingerprint.Datasets
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Crystal structure of a Fab variant containing a fluorescent noncanonical amino acid with blocked excited state proton transfer and in complex with its antigen, CD40L
Henderson, J. N. (Contributor), Simmons, C. R. (Contributor) & Mills, J. (Contributor), Protein Data Bank (PDB), Feb 2 2022
DOI: 10.2210/pdb7SGM, https://www.wwpdb.org/pdb?id=pdb_00007sgm
Dataset
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Crystal structure of Fab containing a fluorescent noncanonical amino acid with blocked excited state proton transfer
Henderson, J. N. (Contributor), Simmons, C. R. (Contributor) & Mills, J. (Contributor), Protein Data Bank (PDB), Feb 2 2022
DOI: 10.2210/pdb7SEN, https://www.wwpdb.org/pdb?id=pdb_00007sen
Dataset