Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling.

JACS Au

Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, 625 00 Brno, Czech Republic.

Published: June 2022

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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9241015PMC
http://dx.doi.org/10.1021/jacsau.2c00002DOI Listing

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