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

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 Ile^L98 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 an

SUBMITTER: Henderson JN 

PROVIDER: S-EPMC7964926 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature