Crowding-Induced Liquid–Liquid Phase Separation in the ATP-Binding ACC 1–13 K 8 Peptide Leads to a Distinct Amyloid Variant

Liquid–liquid phase separation (LLPS) is involved in both the self-assembly of vital cellular organelles and the disease-associated protein misfolding, where LLPS precedes a liquid–solid phase transition (LSPT) leading to amyloid aggregates. Chimeric ACC 1–13 K n peptides are insightful models to study coupled LLPS/LSPT processes triggered by ATP-binding. Here, we investigated the impact of macromolecular crowding on the selection of the aggregation pathway in the ACC 1–13 K 8 -ATP system. While it has been previously shown that peptides with relatively short oligolysine segments (K 16 and shorter) skip the LLPS stage on their pathway to amyloid fibrils, we show here that concentrated polyethylene glycol (PEG), mimicking intracellular crowding conditions, induces prior formation of liquid droplets that subsequently facilitate fibril formation. The influence of PEG contrasts with the behavior of other types of macromolecular crowding agents, Dextran and Ficoll, which accelerate aggregation without a detectable LLPS phase, and that of serum albumin, which prolongs the nucleation phase. In the presence of PEG-induced macromolecular crowding, the fibrillization in the ACC 1–13 K 8 -ATP system appears to reach a maximal rate limited by diffusion coupled to the conformational dynamics of the polypeptide chains within the droplets. Importantly, the ACC 1–13 K 8 -ATP fibrils formed in the presence of PEG are distinct from those of the ACC 1–13 K 8 -ATP amyloid formed in the absence of crowding in terms of their infrared characteristics, morphological features, and overall stability. Our findings suggest that macromolecular crowding can switch between kinetically and thermodynamically favored amyloid polymorphs and that the chemical properties of the crowding agents are key factors in their impact on protein aggregation processes. The results are discussed in the context of the mechanisms of LLPS-dependent protein misfolding and amyloid formation.