Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00159816%3A_____%2F22%3A00077616" target="_blank" >RIV/00159816:_____/22:00077616 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14310/22:00126373

Výsledek na webu

<a href="https://pubs.acs.org/doi/10.1021/jacsau.2c00002" target="_blank" >https://pubs.acs.org/doi/10.1021/jacsau.2c00002</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1021/jacsau.2c00002" target="_blank" >10.1021/jacsau.2c00002</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

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OECD FORD obor

10400 - Chemical sciences

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

JACS AU

ISSN

2691-3704

e-ISSN

2691-3704

Svazek periodika

2

Číslo periodika v rámci svazku

6

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

14

Strana od-do

1324-1337

Kód UT WoS článku

000819388500001

EID výsledku v databázi Scopus

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