Louise Kjær Klausen

Phase separated condensates of α-synuclein (α-Syn) accelerate amyloid fibril formation, a process implicated in Parkinson's disease pathogenesis. Yet, the precise effects of pathologically relevant α-Syn sequence modifications on this process remain unclear. Here, we show that sequence truncations exert the strongest influence on condensate thermodynamics, material properties, and amyloid aggregation, whereas familial point mutations impart minimal effects. Among the tested familial variants (A30P, H50Q, G51D and A53T), only G51D forms condensates that show a reduced propensity for amyloid fibril formation. Truncated variants undergo rapid gelation and form amyloid fibrils almost immediately. Extending our study to multicomponent systems where α-Syn is a client, we show that α-Syn can dissolve DNA-peptide coacervates or assemble into Pickering clusters on condensate surfaces-regulating condensate fusion and nucleic acid partitioning. These functions depend on the acidic C-terminal domain of α-Syn. Together, our results show disease-relevant modifications can modulate α-Syn phase behavior, both in pathological and physiological contexts.

Amyloid fibrils are highly stable misfolded protein assemblies that play an important role in several neurodegenerative and systemic diseases. Although structural information of the amyloid state is now abundant, mechanistic details about the misfolding process remain elusive. Inspired by the Φ-value analysis of protein folding, we combined experiments and molecular simulations to resolve amino-acid contacts and determine the structure of the transition-state ensemble—the rate-limiting step—for fibril elongation of PI3K-SH3 amyloid fibrils. The ensemble was validated experimentally by Tanford β analysis and computationally by free energy calculations. Although protein folding proceeds on funnel-shaped landscapes, here we find that the energy landscape for the misfolding reaction consists of a large ‘golf course’ region, defined by a single energy barrier and transition state, accessing a sharply funnelled region. Thus, misfolding occurs by rare, successful monomer–fibril end collisions interspersed by numerous unsuccessful binding attempts. Taken together, these insights provide a quantitative and highly resolved description of a protein misfolding reaction.

Phase separated condensates can accelerate α-synuclein (α-Syn) amyloid fibril formation implicated in Parkinson’s disease pathogenesis. The effects of pathological modifications, i.e., truncations and familial mutations on the thermodynamics, material properties, and the extent of amyloid aggregation within α-Syn condensates remain elusive. Here, we quantitatively demonstrate that terminal truncations significantly alter α-Syn phase separation, while familial mutations impart minimal effects. Spontaneous sol-gel phase transitions of the truncated α-Syn variants could give rise to amyloid fibrils almost instantly within condensates, suggesting similarities between molecular interactions driving both processes. Extending our study to model coacervate and condensate systems where α-Syn acts as a client, we find α-Syn can dissolve coacervates and form Pickering clusters on condensate surfaces—regulating their size. Additionally, the C-terminal region of α-Syn modulates nucleic acid sequestration within condensates. Together, our findings reveal diverse effects of α-Syn modifications on phase separation, both in pathological and physiological contexts. GRAPHICAL