Multisite Labeling of Proteins Using the Ligand-Directed Reactivity of Triggerable Michael Acceptors

Targetedmodification of endogenous proteins withoutgenetic manipulationof protein expression machinery has a range of applications from chemicalbiology to drug discovery. Despite being demonstrated to be effectivein various applications, target-specific protein labeling using ligand-directedstrategies is limited by stringent amino acid selectivity. Here, wepresent highly reactive ligand-directed triggerable Michael acceptors(LD-TMAcs) that feature rapid protein labeling. Unlike previous approaches,the unique reactivity of LD-TMAcs enables multiple modifications ona single target protein, effectively mapping the ligand binding site.This capability is attributed to the tunable reactivity of TMAcs thatenable the labeling of several amino acid functionalities via a binding-inducedincrease in local concentration while remaining fully dormant in theabsence of protein binding. We demonstrate the target selectivityof these molecules in cell lysates using carbonic anhydrase as themodel protein. Furthermore, we demonstrate the utility of this methodby selectively labeling membrane-bound carbonic anhydrase XII in livecells. We envision that the unique features of LD-TMAcs will finduse in target identification, investigation of binding/allostericsites, and studying membrane proteins.

Automated summary

Targeted modification of endogenous proteins without genetic manipulation has wide applications. This study presents ligand-directed triggerable Michael acceptors (LD-TMAcs) for rapid protein labeling. LD-TMAcs possess unique reactivity allowing multiple modifications on a single target protein, enabling the mapping of ligand binding site. The selectivity and utility of LD-TMAcs are demonstrated in cell lysates and live cells, suggesting their potential in target identification and studying membrane proteins.