Protoporphyrin IX Attenuates Liquid–Liquid Phase Separation of α-Synuclein by Inducing Its Compaction

α-Synuclein (αS), a neuronal intrinsically disordered protein, forms pathogenic amyloid fibrils, a hallmark of Parkinson's disease. Recent work shows that formation of liquid-like αS condensates can accelerate the generation of fibrils, and therefore, targeting the liquid-like phase separation process can provide a novel route to disrupt αS amyloid formation. Here, we report that endogenous small molecule protoporphyrin IX (PPIX) prevents αS droplet formation by shutting down early events in this process. Using ensemble and single-molecule fluorescence techniques, we show that binding of PPIX to αS results in a collapsed conformation. Live-cell imaging and single-cell point fluorescence correlation spectroscopy revealed PPIX mitigates αS condensate formation in cells. In addition, density functional theory calculations indicate the high dipole character of PPIX, highlighting a critical role of additional weak electrostatic interactions in αS binding. Our work provides the mechanistic underpinnings of PPIX-mediated suppression of αS LLPS and establishes PPIX's therapeutic potential for αS-related synucleinopathies.