Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling.
Journal
JACS Au
ISSN: 2691-3704
Titre abrégé: JACS Au
Pays: United States
ID NLM: 101775714
Informations de publication
Date de publication:
27 Jun 2022
27 Jun 2022
Historique:
received:
03
01
2022
revised:
08
04
2022
accepted:
08
04
2022
entrez:
5
7
2022
pubmed:
6
7
2022
medline:
6
7
2022
Statut:
epublish
Résumé
HaloTag labeling technology has introduced unrivaled potential in protein chemistry and molecular and cellular biology. A wide variety of ligands have been developed to meet the specific needs of diverse applications, but only a single protein tag, DhaAHT, is routinely used for their incorporation. Following a systematic kinetic and computational analysis of different reporters, a tetramethylrhodamine- and three 4-stilbazolium-based fluorescent ligands, we showed that the mechanism of incorporating different ligands depends both on the binding step and the efficiency of the chemical reaction. By studying the different haloalkane dehalogenases DhaA, LinB, and DmmA, we found that the architecture of the access tunnels is critical for the kinetics of both steps and the ligand specificity. We showed that highly efficient labeling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from the wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates the need for expensive and laborious protein engineering.
Identifiants
pubmed: 35783171
doi: 10.1021/jacsau.2c00002
pmc: PMC9241015
doi:
Types de publication
Journal Article
Langues
eng
Pagination
1324-1337Subventions
Organisme : NCI NIH HHS
ID : R01 CA217809
Pays : United States
Informations de copyright
© 2022 The Authors. Published by American Chemical Society.
Déclaration de conflit d'intérêts
The authors declare no competing financial interest.
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