Mechanism-Based Strategy for Optimizing HaloTag Protein Labeling

HaloTag labeling technology has introduced unrivaled potential in protein chemistry, 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, 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 show that highly efficient labelling with specific ligands is achievable with natural dehalogenases. We propose a simple protocol for selecting the optimal protein tag for a specific ligand from a wide pool of available enzymes with diverse access tunnel architectures. The application of this protocol eliminates a need for expensive and laborious protein engineering.