Genetic code expansion for site-specific encoding of a switchable, intrinsic fluorophore–quencher pair to monitor protein dynamics
6 affiliations
- 1Department of Chemistry, School of Arts and Sciences, University of Pennsylvania 231 South 34th Street Philadelphia PA 19104 USA
- 2Department of Biochemistry & Biophysics, Oregon State University Corvallis OR 97331 USA
- 3Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania 3620 Hamilton Walk Philadelphia PA 19104 USA
- 4Department of Organic Chemistry, Faculty of Science, University of Yaoundé I Yaoundé Cameroon
- 5Department of Medicine, Perelman School of Medicine, University of Pennsylvania 3400 Civic Center Boulevard Philadelphia PA 19104 USA
- 6Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania 421 Curie Boulevard Philadelphia PA 19104 USA
Abstract
Precisely modifying proteins at multiple sites in their native, folded structures offers unique opportunities to answer molecular and cellular-level biological questions. Here, we present a genetic code expansion strategy for site-specific integration of a fluorophore–quencher pair comprising two non-canonical amino acids—acridonylalanine (Acd) and methyltetrazinyl phenylalanine (Tet)—into a protein expressed in E. coli. The Acd and Tet pair requires no post-translational labeling, and quenching can be switched off by bioorthogonal or photochemical reactions of Tet for convenient internal control experiments. Mechanistic studies based on Stern–Volmer quenching, fluorescence lifetime measurements, and “proline ruler” peptides established the distance dependence of quenching. As proof-of-concept, we applied this strategy to study: (1) calmodulin, a calcium-sensing protein, (2) RecA, a DNA damage sensor in bacteria, and (3) LexA, a transcriptional repressor whose activation by RecA governs acquired antibiotic resistance in bacteria. Using these proteins, we demonstrate that dual Acd/Tet labeling provides molecular-level insights into protein dynamics, shows potential for adaptation to high-throughput drug screening, and advances tools for studying protein structure–function relationships.
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BibTeX
@article{giri2026genetic,
title = {{Genetic code expansion for site-specific encoding of a switchable, intrinsic fluorophore–quencher pair to monitor protein dynamics}},
author = {Priyanda Giri and Yarra Venkatesh and Moriah H. Mathis and Christina M. Hurley and Chloe M. Jones and Olivier Ndogo Eteme and Zachary M. Hostetler and Richard B. Cooley and Rahul M. Kohli and Ryan A. Mehl and E. James Petersson},
journal = {Chemical Science},
year = {2026},
month = nov,
doi = {10.1039/d6sc06723a},
url = {https://doi.org/10.1039/d6sc06723a},
note = {CC BY 3.0},
}
RIS
TY - JOUR
TI - Genetic code expansion for site-specific encoding of a switchable, intrinsic fluorophore–quencher pair to monitor protein dynamics
AU - Priyanda Giri
AU - Yarra Venkatesh
AU - Moriah H. Mathis
AU - Christina M. Hurley
AU - Chloe M. Jones
AU - Olivier Ndogo Eteme
AU - Zachary M. Hostetler
AU - Richard B. Cooley
AU - Rahul M. Kohli
AU - Ryan A. Mehl
AU - E. James Petersson
PY - 2026
DA - 2026/11/04
JO - Chemical Science
DO - 10.1039/d6sc06723a
UR - https://doi.org/10.1039/d6sc06723a
AB - Precisely modifying proteins at multiple sites in their native, folded structures offers unique opportunities to answer molecular and cellular-level biological questions. Here, we present a genetic code expansion strategy for site-specific integration of a fluorophore–quencher pair comprising two non-canonical amino acids—acridonylalanine (Acd) and methyltetrazinyl phenylalanine (Tet)—into a protein expressed in E. coli. The Acd and Tet pair requires no post-translational labeling, and quenching can be switched off by bioorthogonal or photochemical reactions of Tet for convenient internal control experiments. Mechanistic studies based on Stern–Volmer quenching, fluorescence lifetime measurements, and “proline ruler” peptides established the distance dependence of quenching. As proof-of-concept, we applied this strategy to study: (1) calmodulin, a calcium-sensing protein, (2) RecA, a DNA damage sensor in bacteria, and (3) LexA, a transcriptional repressor whose activation by RecA governs acquired antibiotic resistance in bacteria. Using these proteins, we demonstrate that dual Acd/Tet labeling provides molecular-level insights into protein dynamics, shows potential for adaptation to high-throughput drug screening, and advances tools for studying protein structure–function relationships.
SN - 2041-6520
SN - 2041-6539
ER -