Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

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Publikace nespadá pod Lékařskou fakultu, ale pod Přírodovědeckou fakultu. Oficiální stránka publikace je na webu muni.cz.

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MARQUES Sérgio Manuel SLÁNSKÁ Michaela CHMELOVÁ Klaudia CHALOUPKOVÁ Radka MAREK Martin CLARK Spencer DAMBORSKÝ Jiří KOOL Eric T. BEDNÁŘ David PROKOP Zbyněk

Rok publikování 2022
Druh Článek v odborném periodiku
Časopis / Zdroj JACS AU
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Citace
www https://pubs.acs.org/doi/10.1021/jacsau.2c00002
Doi http://dx.doi.org/10.1021/jacsau.2c00002
Klíčová slova HaloTag; enzyme kinetics; molecular modeling; reaction mechanism; ligand binding; nucleophilic substitution; protein engineering; access tunnel; numerical integration
Přiložené soubory
Popis 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 tetramethyirhodamine- 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 Iigand 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.
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