A Molecular Rotor-Based Halo-Tag Ligand Enables a Fluorogenic Proteome Stress Sensor to Detect Protein Misfolding in Mildly Stressed Proteome

Cellular stress leads to disruption of protein homeostasis (proteostasis) that is associated with global misfolding and aggregation of the endogenous proteome. Monitoring stress-induced proteostasis deficiency remains one of the major technical challenges facing established sensors of this process. Available sensors use solvatochromic fluorophores to detect protein aggregation in forms of soluble oligomers or insoluble aggregates when cells are subjected to severe stress conditions. Misfolded monomers induced by mild stresses, however, remain largely invisible to these sensors. Here, we describe a fluorogenic proteome stress sensor by conjugating a fluorescent molecular rotor with a metastable Halo-tag protein domain that contains a K73T mutation (named AgHalo hereinafter). In nonstressed cells, the AaHalo sensor remains largely folded and the AgHalo•ligand conjugate is fluorescent dark in the folded state. Under various stress conditions, the AgHalo sensor has been established to form both soluble and insoluble aggregates along with metastable proteins of the endogenous cellular proteome. Thus, the AgHalo•ligand conjugate fluoresces strongly when the sensor forms misfolded monomers (a 16-fold increase) or aggregates in both soluble and insoluble forms (a 20-fold increase). Compared to the solvatochromic fluorophore-based sensor, we demonstrate that the molecular rotor-based sensor not only is more effective in detecting mild proteome stress that induces primarily misfolding conformations, but also exhibits a higher fluorescence signal in detecting more severe proteome stress that involves protein aggregates. Thus, the conjugation of a fluorescent molecular rotor to AgHalo further improves the capacity of this sensor to detect conditions of proteome stress. This work highlights the utility of molecular rotor-based fluorophores in direct visualization of the protein aggregation cascade in live cells, providing new methodologies for real-time analyses of cellular proteostasis upon exposure to different types of stress conditions.