Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent, Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment.
J Mol Biol
; 434(8): 167455, 2022 04 30.
Article
en En
| MEDLINE
| ID: mdl-35033559
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 (â¼70 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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Protones
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Umbeliferonas
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Fragmentos de Inmunoglobulinas
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Técnicas Biosensibles
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Glicina
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Proteínas Luminiscentes
Idioma:
En
Revista:
J Mol Biol
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
HOLANDA
/
HOLLAND
/
NETHERLANDS
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NL
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PAISES BAJOS
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THE NETHERLANDS