Maria Rodriguez-Rios, Conner Craigon, Mark A. Nakasone, Gajanan Sathe, Adam G. Bond, Mark Dorward, Anthony K. Edmonds, Mark C. Norley, Robert E. Arnold, Paul M. Wood, Stephen J. Reynolds, Joel O. Cresser-Brown, Graham P. Marsh, Hannah J. Maple, Alessio Ciulli
Visualizing and manipulating proteins in live cells is crucial for studying complex biological processes. Self-labelling protein (SLP) tags such as HaloTag and SNAP-tag are widely used for protein labelling, and new systems are needed to expand multiplexing capabilities and broaden the scope of applications. Here we present BromoCatch, a small ~13 kDa bromodomain (BD)-based SLP platform, engineered with a nucleophilic cysteine for covalent ligand engagement. A structure-based designed library of electrophilic ligands was screened against two cysteine-containing mutants using differential scanning fluorimetry and intact protein mass spectrometry to assess covalent complex formation. We identified a para-acrylamide bumped derivative MR116 and the Brd4-BD2 double mutant L387A,E438C as the optimal protein-ligand pair, and reveal the binding mode through an X-ray co-crystal structure solved to 1.3 Å resolution. BromoCatch demonstrated potent and irreversible cellular target engagement in NanoBRET and residence-time assays. Its versatility was demonstrated through the design of a biotinylated conjugate, PROTAC-based degraders, and fluorescent full-on and "switch-on" probes for ex-cellulo and live-cell imaging, including side-by-side comparison and orthogonality with HaloTag. Together, these results establish BromoCatch as a robust, modular, and orthogonal SLP tool with broad potential for multiplexed labelling and targeted protein manipulation.