Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of alpha-synuclein, the amyloidogenic protein in Parkinson's disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for alpha-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during alpha-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall alpha-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down alpha-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in alpha-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.

Automated summary

The presence of macromolecular crowding agent Ficoll70 accelerates primary nucleation and fiber elongation steps during alpha-synuclein amyloid formation, favoring secondary nucleation and resulting in faster overall reaction. In contrast, sucrose slows down alpha-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics, such as accelerating individual steps in alpha-synuclein amyloid formation, is a crucial consideration for biochemical reactions in living systems.