Liquid-liquid phase separation of α-synuclein is highly sensitive to sequence complexity

The Parkinson's-associated protein alpha-synuclein (alpha-syn) can undergo liquid-liquid phase separation (LLPS), which typically leads to the formation of amyloid fibrils. The coincidence of LLPS and amyloid formation has complicated the identification of the molecular determinants unique to LLPS of alpha-syn. Moreover, the lack of strategies to selectively perturb LLPS makes it difficult to dissect the biological roles specific to alpha-syn LLPS, independent of fibrillation. Herein, using a combination of subtle missense mutations, we show that LLPS of alpha-syn is highly sensitive to its sequence complexity. In fact, we find that even a highly conservative mutation (V16I) that increases sequence complexity without perturbing physicochemical and structural properties, is sufficient to reduce LLPS by 75%; this effect can be reversed by an adjacent V-to-I mutation (V15I) that restores the original sequence complexity. A18T, a complexity-enhancing PD-associated mutation, was likewise found to reduce LLPS, implicating sequence complexity in alpha-syn pathogenicity. Furthermore, leveraging the differences in LLPS propensities among different alpha-syn variants, we demonstrate that fibrillation of alpha-syn does not necessarily correlate with its LLPS. In fact, we identify mutations that selectively perturb LLPS or fibrillation of alpha-syn, unlike previously studied mutations. The variants and design principles reported herein should therefore empower future studies to disentangle these two phenomena and distinguish their (patho)biological roles.