Abstract
Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue IleL98 is replaced with the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent pKas of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pKa. Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.
Original language | English (US) |
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Pages (from-to) | 3401-3410 |
Number of pages | 10 |
Journal | Biochemistry |
Volume | 59 |
Issue number | 37 |
DOIs | |
State | Published - Sep 22 2020 |
ASJC Scopus subject areas
- Biochemistry
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Dive into the research topics of 'Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore'. Together they form a unique fingerprint.Datasets
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Crystal Structure of the Fab fragment of humanized 5c8 antibody containing the fluorescent non-canonical amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine in complex with CD40L at pH 6.8
Henderson, J. N. (Contributor), Simmons, C. R. (Contributor), Fahmi, N. E. (Contributor), Jeffs, J. W. (Contributor), Borges, C. (Contributor) & Mills, J. (Contributor), Protein Data Bank (PDB), Dec 23 2020
DOI: 10.2210/pdb6W9G, https://www.wwpdb.org/pdb?id=pdb_00006w9g
Dataset
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Crystal Structure of the Fab fragment of humanized 5c8 antibody containing the fluorescent non-canonical amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine at pH 9.7
Henderson, J. N. (Contributor), Simmons, C. R. (Contributor), Fahmi, N. E. (Contributor), Jeffs, J. W. (Contributor), Borges, C. (Contributor) & Mills, J. (Contributor), Protein Data Bank (PDB), Dec 23 2020
DOI: 10.2210/pdb6W5A, https://www.wwpdb.org/pdb?id=pdb_00006w5a
Dataset
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Crystal Structure of the Fab fragment of humanized 5c8 antibody
Henderson, J. N. (Contributor), Simmons, C. R. (Contributor), Fahmi, N. E. (Contributor), Jeffs, J. W. (Contributor), Borges, C. (Contributor) & Mills, J. (Contributor), Protein Data Bank (PDB), Dec 23 2020
DOI: 10.2210/pdb6W4W, https://www.wwpdb.org/pdb?id=pdb_00006w4w
Dataset