A dual-functional BODIPY-based molecular rotor probe reveals different viscosity of protein aggregates in live cells

Aberrant protein aggregation leads to various human diseases, but little is known about the physical chemical properties of these aggregated proteins in cells. Herein, we developed a boron-dipyrromethene (BODIPY)-based HaloTag probe, whose conjugation to HaloTag-fused proteins allows us to study protein aggregates using both fluorescence intensity and lifetime. Modulation of BODIPY fluorophore reveals key structural features to attain the dual function. The optimized probe exhibits increased fluorescence intensity and elongated fluorescence lifetime in protein aggregates. Fluorescence lifetime imaging using this probe indicates that protein aggregates afford different viscosity in the forms of soluble oligomers and insoluble aggregates in live cells. The strategy presented in this work can be extended to enable a wide class of HaloTag probes that can be used to study a variety of physical properties of protein aggregates, thus helping unravel the pathogenic mechanism and develop therapeutic strategy.