@article{7e2c034ec72d499e99d1a7829c0ef292,
title = "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 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. ",
author = "Henderson, {J. Nathan} and Simmons, {Chad R.} and Fahmi, {Nour Eddine} and Jeffs, {Joshua W.} and Borges, {Chad R.} and Mills, {Jeremy H.}",
note = "Funding Information: The authors thank Peter Schultz for the generous gift of the pEVOL-CouRS synthetase plasmid. The BCSB at the Advanced Light Source is supported in part by the National Institutes of Health (NIH), the National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the U.S. Department of Energy (DOE) under Contract DE-AC02-05CH11231. The SBC (beamline 19-ID) of the Advanced Photon Source is a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the DOE, Office of Science, Office of Basic Energy Sciences, under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institutes of General Medical Sciences (Grant P41GM103393). Funding Information: The authors thank Peter Schultz for the generous gift of the pEVOL-CouRS synthetase plasmid. The BCSB at the Advanced Light Source is supported in part by the National Institutes of Health (NIH), the National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported by the U.S. Department of Energy (DOE) under Contract DE-AC02-05CH11231. The SBC (beamline 19-ID) of the Advanced Photon Source is a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the DOE, Office of Science, Office of Basic Energy Sciences, under Contract DEAC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institutes of General Medical Sciences (Grant P41GM103393). Funding Information: Research reported in this publication was also supported by the National Institutes of General Medical Sciences of the Arizona State University and the National Institutes of Health under Grant 1 R01 GM136996 01 to J.H.M. Publisher Copyright: Copyright {\textcopyright} 2020 American Chemical Society.",
year = "2020",
month = sep,
day = "22",
doi = "10.1021/acs.biochem.0c00474",
language = "English (US)",
volume = "59",
pages = "3401--3410",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "37",
}