Aggregation of the 140-residue protein α-synuclein (αSN) is a key factor in the etiology of Parkinson’s disease. Although the intensely anionic C-terminal domain (CTD) of αSN does not form part of the amyloid core region or affect membrane binding ability, truncation or reduction of charges in the CTD promotes fibrillation through as yet unknown mechanisms. Here, we study stepwise truncated CTDs and identify a threshold region around residue 121; constructs shorter than this dramatically increase their fibrillation tendency. Remarkably, these effects persist even when as little as 10% of the truncated variant is mixed with the full-length protein. Increased fibrillation can be explained by a substantial increase in self-replication, most likely via fragmentation. Paradoxically, truncation also suppresses toxic oligomer formation, and oligomers that can be formed by chemical modification show reduced membrane affinity and cytotoxicity. These remarkable changes correlate to the loss of negative electrostatic potential in the CTD and highlight a double-edged electrostatic safety guard.

Liquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP3. Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS.

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Nanoparticles (NPs) can modulate protein aggregation and fibril formation in the context of amyloid diseases. Understanding the mechanism of this action remains a critical next step in developing nanomedicines for the treatment or prevention of Parkinson's disease. α-Synuclein (α-Syn) can undergo interactions of different strength with nanoparticles, and these interactions can be prevented by the presence of a protein corona (PC) acquired during the exposure of NPs to serum proteins. Here, we develop a method to attach the PC irreversibly to the NPs, which enables us to study in detail the interaction of α-Syn and polyethylenimine-coated carboxylmodified polystyrene NPs (PsNPs-PEI) and the role of the dynamics of the interactions. Analysis of the kinetics of fibril formation reveals that the NPs surface promotes the primary nucleation step of amyloid fibril formation without significantly affecting the elongation and fragmentation steps or the final equilibrium. Furthermore, the results show that even though α-Syn can access the surface of NPs that are precoated with a PC, due to the dynamic nature of the PC proteins, the PC nevertheless reduces the acceleratoring effect of the NPs. This effect is likely to be caused by reducing the overall amount of weakly interacting α-Syn molecules on the NP surface and the access of further α-Syn required for fibril elongation. Our experimental approach provides microscopic insight into how serum proteins can modulate the complex interplay between NPs and amyloid proteins.

α-Synuclein (α-Syn) liquid-liquid phase separation (LLPS) leads to irreversible amyloid fibril formation associated with Parkinson's disease pathogenesis. Critical concentrations of α-Syn LLPS are relatively high under physiological solution conditions. Moreover, α-Syn exhibits delayed LLPS kinetics under certain conditions which deviates from the behaviour predicted by classical homogeneous nucleation theory. In the current body of work, using interferometric light scattering (iSCAT), also known as mass photometry, we experimentally probe that α-Syn can form nanoscale phase separated assemblies/clusters, containing tens to hundreds of moleculesboth above and below the critical LLPS concentration down to physiologically relevant scales. The formation of these clusters is instantaneous, even under conditions where the formation of microscopically visible droplets takes several days. However, they account for a very small volume fraction below saturation concentration. The slow growth of the nanoclusters can be attributed to a kinetic barrier which can be overcome by increasing the solution temperature to just below the droplet melting point. We provide reasons for caution in quantifying dilute phase concentrations for α-Syn LLPS samples containing nanoscale droplets-which can only be separated using ultracentrifugation. In addition, we also delineate that the presence of certain surfaces facilitates α-Syn droplet nucleation under conditions of delayed kinetics but is not a mandatory prerequisite for nanocluster formation. Taken together, our findings reveal that phase separation of α-Syn occurs at a wider range of solution conditions than predicted so far and provides an important step towards understanding α-Syn LLPS within physiological scales..

Functional amyloids are highly organized protein/peptide structures that inter alia promote biofilm formation in different bacteria. One such example is provided by a family of 20–45 residue-long peptides called phenol-soluble modulins (PSMs) from Staphylococcus aureus . External components such as eukaryotic host proteins, which alter self-assembly of bacterial amyloids, can affect the biofilm matrix. Here, we studied the effect of the highly prevalent human plasma protein fibrinogen (Fg) on fibrillation of PSMs. Fg inhibits or suppresses fibrillation of most PSMs tested (PSMα1, PSMβ1, and PSMβ2) except for PSMα3, whose already rapid aggregation is accelerated even further by Fg but leads to amorphous β-rich aggregates rather than fibrils. Fg also induces PSMβ2 to form amorphous aggregates and diverts PSMα1 into off-pathway oligomers which consist of both Fg and PSMα1 and cannot seed fibrillation. Peptide arrays showed that Fg bound to the N-terminus of PSMα1, while it bound to the entire length of PSMα3 (except the C terminus) and to the C-termini of PSMβ1 and PSMβ2. The latter peptides are all positively charged, while Fg is negatively charged at physiological pH. The positive charges complement Fg’s net negative charge of −7.6 at pH 7.4. Fg’s ability to inhibit PSM fibrillation reveals a potential host-defense mechanism to prevent bacterial biofilm growth and infections in the human body.

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