Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore

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...

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Bibliographic Details
Published in:Biochemistry (Easton) 2020-09, Vol.59 (37), p.3401-3410
Main Authors: Henderson, J. Nathan, Simmons, Chad R, Fahmi, Nour Eddine, Jeffs, Joshua W, Borges, Chad R, Mills, Jeremy H
Format: Article
Language:English
Subjects:
Amino Acids - chemistry
Amino Acids - metabolism
Binding Sites, Antibody
CD40 Ligand - chemistry
CD40 Ligand - metabolism
Crystallography, X-Ray
Fluorescence
Fluorescent Dyes - chemistry
Humans
Immunoglobulin Fab Fragments - chemistry
Immunoglobulin Fab Fragments - metabolism
Models, Molecular
Protein Conformation
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Summary: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 pK as of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pK a. 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.
ISSN:0006-2960
1520-4995
DOI:10.1021/acs.biochem.0c00474