Steric confinement-induced emission probe for monitoring protein conformations in live cells.

Protein conformational changes drive signal transduction to regulate cellular activities, yet monitoring of these changes in living cells remains challenging. Here, we introduce BIOSCE (BIOprobe based on Steric Confinement-induced Emission), a technique that enables tracking of individual protein conformations in living cells across millisecond-to-minute timescales. BIOSCE reports protein conformational changes via steric confinement-induced luminescence switching from non-luminescent to luminescent states. We demonstrate that BIOSCE rapidly senses calmodulin conformational changes triggered by intracellular calcium fluctuations. The BIOSCE platform achieved millisecond-resolution monitoring of single-protein conformations within cellular signaling pathways, as evidenced by its sensitive detection of rapamycin-dependent FKBP (FK506-binding protein)-FRB (FKBP-rapamycin binding) interactions regardless of the labeled partner. Furthermore, we applied BIOSCE to track the spatial distribution of SNAP25 (25 kDa synaptosomal nerve-associated protein) during botulinum neurotoxin A (BoNT/A) intoxication, revealing differential catalytic processing of its cleavage fragments. This generalizable approach provides a robust platform for investigating single-molecule conformational changes with high spatiotemporal resolution and enables direct evaluation of transient cellular events.